Document KR3LvkDO81QMDy2nB4LZRyp10

Reprinted from the h{. A. Archives e( Industrial Health September 19S5, VoL 12, pp. Copyright 29$$, by American Medical Association :t of<3nlialed (Commercial Sdi^dt rou6 2 Slnima.1 Ol6Sue5 (Calcium Silicate < )ujt on A* it*4j a. W. H. M.DS DSa. T. M. DUJtfcAH, MX t+4 A. I. &ELAWAHT, $r**c UU, N. T, iraiwir~raiymfci~i i~~ Commercial hydrous calcitfn silicate is one of the products that have been studied by long-term inhalation experiments at The Saranac Laboratory. These studies were commenced tn 1943 under the direction of Dr. L. U. Gardner and with the assistance of two of us (T. M. D. and A. B. D.) who ear ned the work to its completion after Dr. Gardner's death. Guinea pigs, rats, and ham sters were used, and the experiments were designed to reveal the nature of the pulmo nary tissue reaction to inhaled dust of the material in normal animals and in those har boring an experimentally induced tuberculous infection. From experience with other siliceous materials one would expect to find that a product composed only of calcium silicate would be relatively inert in its effect on tissue. Extensive clinical studies of industrial workers exposed to cement dust in high concentration have, for instance, shown that the effect of the inhaled dust of that material on the lungs is insignificant. Portland cement is composed principally of two calcium sili cates, namely, dicalcium silicate and tri calcium silicate. Recognition must be given, however, to the possibility that any poten- Recorded for publication July 15, 19:5. Director, (Dr. Schepers), Associate Director (Mr. Durkin), and Research Associate (Mr. Deiahint), The Saranac Laboratory- tialiy hazardous raw- materials used to make a product might not be entirely convened to a nonhaaardous form during the manu facturing process and, therefore, might appear to a greater or less extent in an unchanged condition in the final product. The clay and shale used in making cement often have a quartz content of 20 to 307f or more, but chemical reactions, occurring when the raw mixture, is heated, convert practically all the,hazardous free-silica min eral to a relatively harmless silicate. In most samples of cement the amount of quartz that has come through the manufacturing process unchanged is Jess than 0.1 The hydrous calcium silicate product used tn The Saranac Laboratory studies was mad; from calcium hydroxide and silica plus a moderate amount of asbestos, which was incorporated in the mixture to impart cer tain desirable physical properties to the fin ished product. Analysis of one sample of this finished product disclosed that approximate!;. 8O'yc of the raw mixture had been converted to hydrous calcium silicate and that about 15/c was magnesium silicate (Table 1). About 1 `yc quart2 persisted in the final prod uct. Probably the greater part of the mag nesium silicate was present as the fibrous mineral chrysotiie, although a portion may have been in the form of serpentine, a min eral similar to chrysotiie in chemical compo sition but one which is nonnbrous . EXPERIMENTAL METHOD To determine the effect of inhaled dust of the product on pulmonary tissue, in inhalation experi ment was conducted. In that experiment froups of guinea pifs. rats, and hamsters ere exposed in a cubical dust room, 8 ft. in dimension., in wmrr -f 1 2 TaLI . 1.--Composition of Commercial Hyd'ovj Calcium Silicate Compo&cflt Aaalyili IO, ...................................... F.O., AJ.Oa, TtOi................. C*0 ...................................... M*0 ...................................... Iraiuoo ioas ......................... Ptr Cut 4 an atmospheric suspension of the hydrous calcium silicate product was created by the action of a paddle which routed inside a hopper containing the material in finely divided form. The dust cloud generated in this manner floated out into the.room where it was maintained for eight hours on five days of the wk and for four hours on Saturday. Some of the animals were exposed to the dust for periods as long as three years. At regular intervals during the experiment a few animals, were killed, and the organs examined grossly,! and micro scopically to determine the nature and the extett of the tissue reaction to the calcium silicate product. The tissue was also analyzed chemically to estimate the amount of the inhaled dust that was retained in the lungs of animals exposed for definite periods of time. Only guinea pigs were used in the studies dealing with infected animals. The R, low-virulence strain of the tubercle bacilli was introduced intra tracheal ly by means of the insufflation technique. Dust counts of atmospheric samples collected in the dust room were made regularly by means of the midget impingef. The concentration of the hydrous calcium silicate aerosol to which the uninfected animals were exposed was generally within the range of 100,000,000 to 125,000.000 particles per cubic foot of air by light-field count, and the over all average was 115,000,000. In the studies on in fected animals the concentration was higher, the average being 205.000,000. Ooapeaad AnaJyait Per Cut (LpotoO Hydros* eaJoJoa tOkU.................. COdam exrfrooau .......................... 4 Marasjua tllieau .......................... 14 -- ICO EXPOSURE or uninfected ANIMALS Three species--guinea pig, rat, and ham ster--were employed in this phase of the investigation. Summaries of the findings 2re given in Tables 2, 3, and 4. Attention should first be drawn to the relatively high mortality rate reflected in Table 2. That these deaths were due to imercurrent epizootic infection was almost certain in the case of the guinea pigs and is emphasized the more forcefully by the fact that the animals died not only of pneumonia but also of pericarditis, peri tonitis, and cervical adenitis with abscess fonn2tion. That all the hamsters which died did so within the first year may also have some bearing on the question. In the case of the rats, on the contrary, no animals died within the first year, and the rate a: which animals succumbed increased with the pas sage of time. In view of the progressive nature of the pulmonary disease which en sued as a result of the -exposure to the dust of the hydrous calcium silicate product, there is some presumptive evidence that the deaths and dust exposures bore some relation tc each other in this instance. Table 2.--Biological Action of Commercial Hxdrous Calcium: Silicate Dust Guinea Pigs, Hamsters, and Rats Were Exposed by Inhalation to an Aerosol c: Commerc.al Hydrous Calcium Silicate Dust Until Death Record of Animais, Exposed to the Dust Alone, Which Died Spontaneously PneuBoalt fipweft* OulDM Pi*.......................... Biaiiu................... .......................... No. 51* 10* to: Per Cut *0.9 4O.0 too Cu*e o: PencarCstU Per No. Out T 4a .. ... .. ... Pf rti ooJiif No. 11 ' .. .. Per Cut 9.6 ... ... Cmf: per No. Cut J * ,, ... .. ... Nc T3 )-j o,. C*ci Ki fc. < je< * tea*1!* died vtt&Ja Sr*-, yea.: o' txotnsioi t loccr* vitals art: ytar of arpertsost. 10e*i dM ntbtB Qrc year Of erpenoieat I T*0 death* from other riuwi 4 The three species of experimental animals reacted somewhat differently to the dust, though the difference was more one of degree than of quality. The most marked lesions were provoked in the guinea pigs, but this was probably largely due to the fact that dusting was carried on for a total of 56 months in the case of this species, while the procedure was terminated at the end of the 18th month in the case of the rats and hamsters. When cognizance is taken of this fact, it appears that, stage for stage, the latter rwo animal groups actually suffered greater pulmonary damage sooner than did the guinea pigs. This difference is brought out by a compari son of Tables 3 and 4, in the construction of which the same scales of values were used. Pigmentation of the pulmonary tissue or of pulmonary lymph nodes never became prominent features in these animals. It in creased diffusely as a light brown discolora tion which was most marked along the anterior margins of the lungs in the guinea pigs. As it became macroseopically detectable at the 12th month of exposure only, its absence in the case of the rats or hamsters before the 18th month may have no true significance. It would seem that the pigment was almost entirely due to hemosiderin, as shown by Prussian blue staining. Pigmented koniophores and giant cells became a promi nent feature toward the terminal phase of the experiment on guinea pigs, and clusters of such cells could even be found.in rat lungs where they were grouped around the smaller blood vessels. Lymphoid hyperplasia was a prominent and persistent feature of the reaction in the guinea pigs. There was mild hyperplasia about the main bronchi in some of the rats, but this was not an impressive observation for this species. No lymphoid hyperpiasia was seen in the hamsters. The hyperplasia consisted almost entirely of lymphoid cell proliferation without any evidence of central macrophage accumulation such as may occur as a result of exposure to certain amorphous siliceous dusts. Mostly the lymph deposits remained spherical in shape and were thus well defined from sur rounding structures. Occasionally, however, there was some lymphoid invasion of the walls of adjacent alveoli. At a later stage macrophages tended to accumulate around these lymph foci. In spite of this tendency toward intrapulmonary lymphoid hyperplasia, the pulmo nary lymph nodes showed no consistent corresponding change. In some of the an imals there was early lymphoid hyperplasia, but in the majority the nodes enlarged slightly or moderately only at the star.. After the animals had been in the dust atmosphere (or two years, the nodes became more con sistently enlarged, and in the instances in which this occurred the cause was almost always infiltration of the medullary 2ones by macrophages, which no doubt migrated thence from the pulmonary tissues. The cor tical follicles did not enlarge and often showed signs of atrophy. In both the rats and hamsters the reaction in the pulmonary lymph nodes was even less marked. Undoubtedly the most prominent ksion discovered in this series of animals was pres ent as chronic bronchiolitis and bronchial ulceration, with emphasis on the former. The disease was progressive in nature and de structive in its effects, although the tendency to epithelial desquamation, so characteristic of the reaction to quartz dust, was not present. The bronchiolar lesions commenced early in the guinea pigs and hamsters but were delayed in the rats. In neither of the latter groups were the severe grades of obliterative bronchiolitis or bronchiolitis deformans, which characterized the guinea pig response, observed. In the rat the stress was rather on bronchiolar ulceration and peribronchi olitis. As the dusting proceeded beyond the firs: year of experiment, the infiammatcrv re action in the bronchioles gave way to pro gressive distention and distortion This change was brought into sharp focus by the increasing cellular deposits around the air passages. Toward the end of the second yea: the crenated outlines of the brcnchioia* lumma were thrown into sharp reiie: by a marked tendency to peribronchiolar atelec tasis, with epitheliaiizatioo of the walls of the shrinking' alveoli and cellular infiltration among these minute cystic spaces. This change produced a microscopic sectional effect resembling multiple adenomatosis, but as there is no lack of differentiation of the cellular components, no neoplastic change could be postulated. This adenomatoid pat tern was seen only in the guinea pigs, but as its evolution was in the nature of a Re layed phenomenon, it is .possible that it would also have appeared in the rats and hamsters had exposure been continued in their ases beyond the 18th month. Phagocytosis of the inhaled dust could be demonstrated in all animals from the com mencement of the experiment. It was most marked in the case of the guinea pigs and least prominent as a feature iij the case of the hamsters. The phagocyte^ were domi nantly mononuclear macrophages until about the end of the first year of the exposure when multinucleated giant cells commenced to be substituted in progressively greater numbers. At the same time these giant cells grew in size so that they often filled the alveoli completely, and toward the end of the third year of exposure the giant cells replaced the mononuclear cells almost completely, especially around the distorted bronchioles. As time passed, their cytoplasm became in creasingly more acidophilic and their centers more charged with ingested particles. There was no evidence of necrosis of these cells, such as occurs in experiments with finely divided quanta dust and silica fume, which provoke similar giant cells. Interstitial cellular proliferation and infil tration are a late sign in all three species. It commences first toward the end of the ninth month of exposure, and for the suc ceeding six months it is almost entirely lim ited to macrophage accumulations around smaller blood vessels and bronchioles, at the angles between adjacent alveoli, and in the interlobular septa. Among these macrophages may be found isolated eosinophiles and plasmacyies. Fibrocytes first make an appearance between the 15th and the 18th month of dust inhalation, and soon after this strands of collagen may be perceived among tine cells. Fibrosis proceeds slowly, however, before the end of the 30th month of dust exposure. Thereafter it is detectable in increasing amounts, particularly in relation to the ade nomatoid areas around the bronchioles. Fibrosis also appears prominently in local areas of consolidation, which become pro gressively commoner toward the end of the third year of exposure. In animals killed, respectively, at 33 and 36 months from the start of the dusting, extensive areas of lobu lar consolidation could be found, and similar consolidation associated with cystic disten tion of trapped bronchi could be seen in sev eral of the rats killed at the 15- and lS-mor.th periods. No acute inflammatory process was demonstrable to account for these results. Asbestos bodies of an elongated, slender, tapering, minute variety were demonstabie by oil-immersion microscopy from about the 15th month onward in rats and hamsters and from the 18th month onward in the guinea pig. Occasionally opaque dubbed bodies could be found, but in most instances the bodies were difficult to bring into focus and tended to be curved and spindle-shaped. Often they were intracellular, being partly engulfed by one or more macrophages. None were found lying free within alveoli. They were so constantly found in association with the areas of fibrosis that the conclusion of a causal relationship between these phenomena is almost inescapable. Emphysema of the hypertrophic variety, appeared as an early sign in the hamsters, in which it was present to a quite marked and widely distributed degree at the end of the IS months o: dust exposure. In the rats the lesions were also consistently present from an early stage but to a lower degree than in the hamsters. In the guinea pigs on the contrary, hypertrophic emphysema was contrastingly absent during the first twe years of the experiment. In the final 12 months, compensatory emphysema was man ifested as a prominent lesion silhouetted against the foci of cellular proliferation and peribronchiolar atelectasis. 6 Unlike in many other dust experiments, culous disease and the tissue reaction to the there was no marked tendency toward sub- inhaled dust will develop simultaneously. pleural congregation of the hydrous taldum In the reactivatioa-phase experiment, the silicate dust. Consequently the reaction at animals, aiter being infected with the tubercle this site was minimal and delayed. The most bacilli, are aJiowed to live in a normal at. conspicuous changes occurred in the rats mosphert for several months before being where there were occasional subpleural cellu exposed to dust. During this period the lar condensations and a recurrent tendency tuberculous lesions generally regress, and to the formation of interlobular adhesions. in some instances complete healing by reso In some of the guinea pigs which outlasted lution may take place. Foliowing this inter three years of exposure to the dust, foci of val, the exposure of the animals to some subpleural cellular proliferation attended by dusts has no significant effect oo the usual giant-cell accumulation were demonstrable. course of the tuberculous process, and the The relatively marked macrophage infil lesions continue to TegTess, while exposure tration of the pulmonary lymph nodes con to other dusts may cause the tuberculous trasted sharply with the paucity of giant cells process to become active and to spread. A among them, with the minimal amount of interstitial fibrosis which ensued, and with the absence of asbestos bodies. Periadenitis of a mild degree became a consistent finding in a majority of animals beyond the end of the first year of dust inhalation. Deposition predisposition-phase experiment is one in which animals are exposed to dust for several months, are then infected with the tubercle bacilli, and are immediately returned to the dust room where their exposure to dust is continued. In this phase the effect of a pre of fibrous strands around the lymph nodes vious dust exposure on the early course of followed successive phases of afferent lym a 'tuberculous process can be studied. phangiectasia and macrophage transportation along these channels, with littoral arrest of increasing numbers of these cells. The lack of fibrosis, except at sites where asbestos bodies occur with greatest preva lence, is indeed remarkable, the more so when regard is had to the dense cellular in filtrations w-hich ultimately ensue. One is led to the conclusion, therefore, that the hydrous calcium silicate dust alone, except for its chrysotile component, is oonnbrogenic though it provokes a cellular reaction. SivtriTAXxous Pbasz: Reaction rs Guinea Pjcs Infected with Tuiexcee Bac:el: at Onset or Dost Inbauatiox In the first experiment in which animals were infected with tubercle bacilli a: the time that their exposure to the dust was started. 25 guinea pigs were used, bu: an epidemic of pneumonia during the first 10 months re duced to 16 the number of animals available for study. Two of these died at fourteen months, apparently irom progressive tubercu losis, and two others died from undetermined EXfOiVUZ OF TVSEJtCULOUS AXIKALS There are three types, or phases, of in vestigation that have been found invaluable for studying the effect of inhaled dust on the course of experimentally induced tuberculosis in animals. These three types of experiment are sometimes called the simultaneous phase, the reactivation phase, and the predisposi tion phase. Id the simultaneous phase, nor ma! animals are infected with attenuated causes bu: not from tuberculosis. Ir. the re maining 12 animals the course of the tubercu lous disease was followed by killing 1 c: 2 animals at 1, 6, 9, 12, IS, 18. and 2* months after infection. In 9 of the 25 animals the le sions were multiple, isolated, and healed, often w-ith central calcification. In six ci the remaining animals the lesions were still circumscribed but showed neither a tendmr;to heal nor to spread even at the end c: 18 tubercle bacilli of the Ri strain by an inhala months. In six other animals there was 3 tion method and then are immediately moderate tendency to local spread oi the transferred to the dust room. Thus the tuber Tuberculous process, while in the remaining 'Ktf / Table 5.---Cawo of Death in Guinea Pigt Exposed by Inhalation to Commercial Hydro*: Calcium Silicate Dujt and Also Injected uifn Tubercle Bacilli of the P> Strain T7p* of Expertwat ErC Asiaik. >0. SlBUJtAMOUl P6K ......... fc*afUr*U08 pbtae........... .. ts Fraditpoeiuoa pbtM ....... Oootroi: Group A ........... Group B ........... .. U pvffloai Per So. Out t a.6 1 i.o > Xs < U.T i u.o C*um o! Dtiib PtnetrdUi pwiieoJU* Owb*r C*a** Per beat**, Per DatUtf, Per Xo. Get So. Oast Xe. Cest 1 10.0 1 11 X 6.E 1 4.0 1 4.0 1 4.0 t s.o Tru: Pt* Xo. Cait !< M S 4 HO 1 u.t ( U.T 6 u.o animal, which was killed at 15 months, the lesions were widespread throughout the iur.g but fibrosis had been produced. In an at tempt to develop a strain of guinea pig re sistant to the infection that had caused the pneumonia, some of the animals were used for breeding during this experiment. The two animals that died from progressive tu berculosis (at 14 months) and the single animal (killed at 15 months) that exhibited widespread lesions were all used as breeders during the experiment, as were also four other animals in the experiment, which had failed to show extensive disease. Owing to the element of uncertainty introduced by this factor of breeding, and also because of the high mortality from pneumonia and to verify the suggestive evidence of stimulating action of the inha3ed dust upon tuberculosis, the experiment was repeated. TaBLE 6.--Course oj Tuberculosis Induced in Guinea Pigs by Injection uhlh Tubercle Bacilli oj the Ri Strain Control Study: Infection Only; No Dust Exposure Guises Pij.Xo. .. it] a: t0*0 \ M U n. r. 6016 1 BE ! ti C BP * T OS TO n TS T77? n sc n 82 $4 IS K Afur IslKtioe. D*7* .. 4S .. ir .. ISO .. 140 .. e&j .. rr. .. ns .. ass .. 450 .. 4K> .. MO .. rar Pate DeC Li jtC ElbeC EibeC ESb*C Eliied Dice DleC EEJibDeedtf DleO e:uj Eibed D>e ElUed EJbeC El DeC EibeC EilteC EJDeC Elbe* ElbeC EEUlbieeCd EE:ib)*eCC Lliled EU*C LibtC EliC LibeC EEllbbeeCd ?*\Uaoeir7 Loaion* BoeUot is F-.Usost77 Lxnpb XoOn ArreataC Spreads* To&ereie*. Tv&erruXo. ten* COaOaPet- * .. IS .... 7 .. 4 .. X .. t> .. .. i j ; -r-r : .. 4 .. 11 .. TVbeetle Oetauleoffst- Ealartt- Tors* Pteurtfl* seat tio: -fc. .. -4-- T Pttx--!atia Un: Spices T- 7* T- 7- 7-- --- .. 7-- -- -- 7-- -- --- 7------ 7---- -- 7_ ---- --- o4 .... ---- -- 5 .. 7 7--- -- __ :t .. f --T 4 .. b .. - S. 3. 1 .. --- X .. ---- ! .. S . -- 16 ..... 6 .. 6 .... 4I .... Pi 0 .. 7-f era&ou: alifbt reaetion; 4-f. zx>c-3t.-u rtirooc: -t-t-p. rtKOoc: T. ectxrei* forsauoa. 8 Taiue 70/ lrJ*aled Commercial Hydrovj Calcium Silicate Dust an the Course oi Experimentally Induced Tuberculosu in Guinea Pigs Simultaneous Phase: Aiunuis Received Their Tsbercolocs In/ectioo and Started 7'neir Period of Dost Exposure Simuiuaeoosly fctboiort Guloss Pi*. No. u>DDjull, 4 10 +i e 3 t U1 J 12b i 120 U 12S ti 140 10 m 110 110 to IU u X* s B0 e UO is 240 a SIS u Si IS ns 17 CIS 2D 120 IS *93 ) 440 *7 07 a UO is no n flBO 19 720 a TtC a 710 Tt W Vud xmta DMd Die El 1*4 Dirt Vl*3 DkO EilkC xuke Due Dktf D*4 KUkO Eilkfi DWfi Eilkd DmO DeC Eilke EU44 WeO D*C D*a D4 euw EU4 EUk4 rv.---- - Pa l0-8iJ7 LmJo&--, ... - .... LrFaUpBftOXaotdrri TAubrmem1o*4t, N'o. Spm4tn* eTaulboeiur- CtfiuUos Pioar.ai ' SMalBtirle- Teoolboturl- ' I -74- .. * 4- 4- -7 10 2 -4 4 4-7 > r T .. - C -- * 4-1--j. -r-4* r4 4--r 4 4 t 4-r4 4-- 4-4 4--7 4- 7 1 .. 44-4-7 4-7 * j 44 4* 4* 4-r + +*r 4- t -r-r 4- "J 4-444-H* 9 r + ,t 4- 4 14 -7 4--7 4-7 4-4-7 S *74-7 4-4--7 i 4---4 4 4-4 4-7 4-r-t --*> .. + 4 2 4--7 4-- 4-7 i 4-r 4-7 4 Utu Tt-7 Tt Sp*j X-- r-- r--- ermbU: 4. *Uri; euoe; -r--r. aoeciu KhcOoB; t--4. astride rtieiios; F. fibrosa: N. oeerocvi, T. ;: euloiU. When conducted a second time, 30 guinea pigs, instead of 25, were used in this simul taneous infection experiment. The animals were killed in pairs for study at 2, 4, 6, 8, 12, 15, and 24 months after infection. Again the incidence of pneumonia was unusually high, six of the animals dying from that cause dur ing the first 8 months of exposure and two more during the following 16 months. An additional three died of pericarditis, one of peritonitis, and two more of other causes (Table 5). This left 2 balance of 16 animals which were killed as planned. The results are assembled in Table 7 which should be compared with the control study recorded in Table 6, in connection with which the same culture batch of tubercle bacilli was used but the animals were not exposed to dust. As may be seen in Table 5, the mortality rate among the control group was but 11.7 5c as against the high rate of 46.6'yc found in the animals which were caused to inhale dust. As these experiments were conducted simultaneously and ia the same iaberator;. except that the animals receiving cus: ex posure were even better protected from out side contacts, the different death rates ma;have some significance after a!! The numbers of tubercles which developed in the control series did not differ materially from those which were discovered in the animals exposed to dust after infection In one control animal there w-as a marked spread which caused the death of the guinea 9 pig. Massive caseating lesions were found in the lungs, the liver, and the spleen. It was suspected that this animal may have been accidentally infected with virulent human tubercle bacilli. Consequently, bacilli were recovered, cultured, and subinoculated into the gToins of four healthy guinea pigs..These animals developed local abortive lesions char acteristic of the Ri bacillus, and no systemic tuberculosis ensued. By this study it was fairly satisfactorily demonstrated that Guinea Pig 72 was unduly susceptible to tuberculfc- sis. - Id the guinea pigs which were caused to breathe the hydrous calcium silicate dust after infection, localized spreads of the tu berculosis occurred in 13 out of the 30 cases. In four of these the tuberculous process was quite advanced, and in four others it was moderately marked at the time of autopsy. It should be noted too that f^-e of these spreads occurred during the second year of the experiment, i. e., long after the tubercles should have been arrested and healed. At tention should also be drawn to the presence of cavitation within two tuberculous pneu monic areas in Guinea Pig 25. Pleural adhesions of a chronic tough va riety were also quite common in the animals receiving the dust exposure. The pulmonary lymph nodes showed a greater prevalence of late active tuberculous foci, such lesions in the control animals having usually disap peared from the nodes before the end of the first year. ` From this simultaneous phase experiment it would seem, therefore, that the hydrous calcium silicate had a mild to moderately ad verse effect on the course of the Bj tubercle infection. Reactivation Phase. Course or Tu*t*cvurs rx CurxtA Pics Wbice Were Exposed to Hydrous Calcium Silicate Durr Several Wox7Hs After Infection with Ro Tusercli Bacilli Infected guinea pigs were placed in the dust chambers at intervals of two months. L e., 10 at two months, 8 at four months, and 8 more at six months after infection. Ir, order to follow the course of the tissue reac tion, a pair of animals of the first subgroup (two months in normal air) was killed after only 2 months of dust exposure, and, in ad dition, animals from all groups were kiiied in pairs after 4, 8, and 12 months of exposure to the dust. Examination of the tissue of the dusted animals failed to reveal a significant reactivation of the tuberculous disease by the inhaled dust (Table 8). Only five animals showed evidence of spreading pulmonary tu berculosis. In three the disease was minima; in extent, and in two others there was moderately extensive local spread. Casea tion persisted in one animal to the end of a year, and slight to moderate foci of fibrosis could be discerned in a few animals, suggest ing that not all the tubercles healed by resolu tion. Chronic pleuritis and pleural adhesions were present in 10 of the guinea pigs, which was considerably in excess of what is cus tomarily found in a typical reaction to the introduction of R tubercle bacilli. Dissemin ation of the tuberculous process to abdominal organs and to the pulmonary lymph nodes did not occur in any but exceptional cases. Guinea Pig 52 was probably one of these "sports." The term "sport" is given to a guinea pig in which the inhalation infection with the attenuated bacilli is not confined principally to the lungs and pulmonary lymph nodes but extends also to other organs and produces in them tuberculous changes of sufficient extent to be recognized macroscopically, Since "sports" represent a de parture from the norma! partem of tissue reaction to attenuated rubercle bacilli, whether the infection is combined with Gust exposure or not, such animals must be ex cluded in assaying the effect of an inhaiec dust uocr, a tuberculous infection. It is be lieved that "sports" are animals whose na tive resistance to the attenuated Ri organ ism is unusually low-. Support ior this belie: is given by experience wj*h quartz dust, a definitely hazardous materia! which reacti vates an inhalation infection produced by attenuated Rj bacilli but ordinarily is not as sociated with tuberculous extension to organs other than the lungs and pulmonary lymph nodes. 10 T*JU 8.--Jnfinmee of Inkcitd Ccm*-rrc\2l Hy4*cr*j Cilcvtm SU\&lt Dus! an lA< Co*rtt af Experimentally Induced T*br'cLix\i C**nta Pigi Reactjvxtioo Phase: AnkiuJi, After Receiving Their TubercoJcui in/ectios Were Allowed to Live is * Sorval Environment for 2 Period of Two to Six Month* bdort They Were Expoted to Dust ?vtooarT L*tonj iprt4' Gcisei lo Dull. fit. be. D*ji TVtXfTj**. Tutxr- Coet- Am be. cuJom Uso Erporort LO Dull l4 Itimd T-* Mosul A!ur LaiUoo <7 9 pwo fi U rx2 U *0 D*4n 0 xa*4 c 90 11*4 as 120 1C 120 11*4 n 240 11*4 at 240 11*4 b i t ,1 , 1 < ? 2 *3 26S 0*4 1 s 2SS 0*4 IS -- 40 ICS 11*4 9 + 4-h ipo<urr la Z>ut w* Sur! Tear Motion xsut lo laeuos ii 120 11*4 44 120 11*4 <1 240 11*4 5 4 <2 240 11*4 t 42 26S 11*4 1 44 264 11*4 t rpoiun ID Dull Wm Sund Six Moduli 49 XU D*4 t to 120 11*4 2 43 ISO li*4 T 43 240 11*4 1 *4 240 U*4 9 46 264 11*4 46 264 11*4 b Dug'a.'atuoa nlo*C*T? tyttpa So*%* naiifT4AcDl T\:x f euioiit Ue: f + ++ 4-t- 4. * m-M- -- * * 6p*i 7., Syoboli: -f. ti*&i; -t-K as404.-*u; r , *4r*ue4; T, n;Krei)o*iA PxCDisrosinok Prasi : Reaction :k Guisu Pics Ixfecteo wxtb TmttCLr Bacc^i Taxts Moxtas Arrzx Ts'zit Ezrosvix to Dvs7 Was Staxtxd An experiment of this type is a severe (est, because a dust that is only very slightly toxic may produce tissue changes which, though minor in character, may be sufficient to alter profoundly the development of a fresh tuberculous infection. In this experi ment, which was designed to study the ef fect of the dust accumulated in the lung upon a newly developing tuberculous disease, 30 guinea pigs were exposed to the dust for three months and then were infected with at tenuated tubercle bacilli. The dust exposure was immediately resumed and earned on for another 21 months. The over-all death .rate in this series of animals was relatively low (Table 5). It may be of some significance that the majority 0: the eight animals that died from pncu^icrji did so during the latter half of the dusting phase. It is also significant perhaps that local and diffuse spreads of the tuberculous process had occurred most commonly m. these animals prior to their terminal fataillness. The prevalence of such spreads in this ser.es of guinea pigs does indeed appear tc be significant (Table 9). Six animals showed marked local or diffuse extension of the dis ease, and in seven more this spread was of moderate severity. In an additional 9 cases there was slight spread of the process, sc chat a total of 22 animals reacted unfavorably. Cavitation occurred in 2 instances, anpleural extension in 10 of the 30 guinea pigs One animal died from a pneumonic tubercu lous process. 11 Tails 9.--lxf.*nct o) Jnkaltd CommereicJ Hydros Ca/rtww Silitatf D*j< e* Ejptr\r*r*lAl!y Indeed Tubcrcvtonj m Cnix^a pigs Co%rtt oj Predisposition Pht: Aninuis, After Seine Exposed to the Dust for Three Mooths, Were Infected with Tuberde Badlli of the R Straia and Thi Were Immediately Returned to the Dust Room Where Their Dust Exposure Was Continued Until Death GuL04 io: ( 9 10 11 9) K 98 w 96 PC r. \u 106 112 11J 9t lot lli 99 )0t 119 11T 103 1< lit 120 101 102 lie Erporjre to Dui; Atttr IfilKUOO. Dr 0 0 0 0 0 to to to 120 120 UO UO SO IX' UC UO too 100 MS its aea *<x. US 4 US UO UC X Of ex Fttc use g,mi E1D3 FwUaoatrj LmJosi Arr**t*s ToLtrOM, So. TuexrUJOtll / CtrtuUOS i m*C ' DM m*e mu mu JL\14 mu tHU Dt*d m*e md m*o mu ciue mo DkxS DwS m*< mu ms m< m3 IT 9n 16 414 -9 * +-m 11 f 6 +--f -4*-!-9 t 9-9 t Kf -9 4-+ t 4-4- -4 i -4--!4 9-4-- *t--p * -r-r 9 4, 4-9--- 4 -4- s -9 FSrtBi ,, .. -t-9 9-9-9 9-9 9-9 4--9 + -9 -9 4-9. *, V- EHucaXDtUot M1S0647T Lrapo NdOti 2alrf4DU ( TeuulUomr- U*tr -9-9 -4--9-9 ---9 - -- -9- 9- ---9. -- --- -9 9--- -r-r - -9 9-r -- 9 -9 + 9-4f .. .. + 9-9 9- 99- ^ + 99 f -9 9- .. .. .. .. .. .. .. .. .. .. .. .. . .. .. * .. T- Brm&oli; +, iLrbt or loapieat rtAcUoc: 9--9. soOtriu nkc'Jo:; j T. airjpi mtuos; T. QtxrnJotli. i It is of interest to note that the extension tained were so similar to those given in Table of the disease was almost entirely confined 6 that the latter may suffice for the purpose of to the pulmonary tissues, tuberculous foci this paper. being detected in the spleen of one animal only. The reaction in the pulmonary lymph ANALYSIS OF TISSUE OF EXPOSED ANIMALS nodes was not of a significant nature or de- Chemical analysis of lung tissue of unin gret. fected guinea pigs that had inhaled the hy These findings indicate that a tuberculous drous calcium silicate dust for periods up to infection which originates in guinea pigs 36 months yielded the data reported in Table several months after a prolonged exposure 10. It m`!l be noted that as the period of ex of the animals to dust of the hydrous calcium posure became longer the values for the tis silicate product was initiated may be unfa sue ash gradually increased, thus showing vorably influenced by the inhaled dust. A that mineral matter was accumulating in the separate control experiment was set up, us lungs. There was a pronounced increase m. ing 25 guinea pigs, as the predisposition- the silica component up to about 30 months phase experiment was started at a later stage and then a slight decrease. This phenomenon than the preceding studies so that a fresh Rj is illusory, as it is due to the relatively rapid culture had t.o be employed. The results ob rate at which inorganic matte: was deposited 12 rn the Jung at this stage when the tissue reac tion suddenly blossomed forth into the full blown disease process. Comparable results have been obtained in The Saranac Labora tory in inhalation experiments with other dusts. The total amount of silica which accumu lated in the lung was about a third of that which may be demonstrated in the pulmonary- tissue of guinea pigs exposed to quartz dust tor a comparable period. * COWktlS'T The first point which may be considered is that the commercial hydrous calcium sili- As the chrysotile dust is capable of provoicing fibrosis in guinea pigs, it may also in the case of the present study have been the cause of the focal fibrogenic response, so that the hydrous calcium silicate component may have to be exonerated. It seems likely that the giant-cell reaction was largely an effect provoked by the latter component. At tention should be directed to the lack of ob vious necrosis in these cells. In this respect the dust differs quite markedly from quartz dust and silica fume. Indeed, necrosis was seldom a feature in this experimentally in duced disease even in the presence of tuber culosis. It is possible, therefore, that al- Ta>L 10.--Analysis oj the Lungs oj Guinea Pigs Exposed to Co*tttne*eiol Hydrous Calcium Silicate Dus> PtntxJ ot xpour<. Ho. t t 6 5 10 ii r. u r 60 c *6 Jdtfe. Per Out ot Zmsc <.l 4.45 *M t.*e 4.47 4.K 4.K 5.16 l_*S 4.9$ 4JT 54* Mr Htoert) CoBpooat ol D>mt4 Lubc Ptt Oil ouc ouo 0~ 04S 0.4 3 oa: o.r: ose 0.63 0.6* UJ 0.75 0.65 co. Pr C<st 007 0.05 o.w 0.12 0.14 045 0.10 0.17 043 0.15 0,14 ou: 044 PtMr fCOo, t ' 1 .or 0.05 0.95 0.* 0.15 0.15 0J9 040 0.07 0.17 0.06 04? 045 XlMrxi Coapo&tsu o'. Pe8rtCOci,at 4-E 4.65 54* 6*5 trr 1042 J0.4C U.4S li.) 11.74 19.91 14.OS U.4f P*c7*Oot.Ol \M 1.94 144 146 i. i.9i ISO >4 1.75 111 i<: :.X 11". Ac P**7r00*,si ' 1 50 J .64 . 110 543 4- 1.74 1.46 Mi o.r. 341 IS* cate product, studied in the experiments just described, really provoked two underlying pathological processes ascribsble, respec tively, to the calcium silicate and to the chrysotile components. The terminal syn drome of peribronchiolar atelectasis, fibrosis, and adenomatoid change was, in fact, of the same kind as that which has been repeatedly produced in The Saranac Laboratory by means of inhaled asbestos dust. The lesion of experimental asbestosis has somewhat more fibrosis to it and shows less of a cellular reaction and the giant cells seen in the pres ent case are less commonly observed. In character with the asbestos lesion is the ab sence, in the present experiments, of fibrosis of the pulmonary lymph nodes in which no asbestos bodies could be found either. though the hydrous calcium silicate dust stimulated the proliferation or local accumu lation of cells it did not kill these cells. This poinx is well illustrated in the case of the bronchial epithelium, which proliferated a; pan of the process of chronic bronchiolar in flammation but did not read:!)' necrose or be come desquamated as in animals exposed tc quartz dust. In many of the worst seeming adenomatoid or cystic lesions, the bronchia' epithelium even retained ms ciliated epithehasuriace. The evolution of the adenomatoid reaction is clearly revealed to compnse cryptic dis tention of the bronchioles, secondary papiilomatous ingrowths into these da'.zr.dzi lumina, and peripheral epithehabzation of atelectatic alveoli. 13 Why the alveolar ducts and bronchioles should have dilated at an early phase in the xats was not clearly revealed. N*o proximal obstruction could be displayed which could account for the distention on mechanical principles. Possibly the cause may be re lated to the tendency to peribronchiolar atelectasis. Perhaps a neurogenic mechanism was at the root of it all. To be true, the larger cystic distentions were usually found within areas of chronic diffuse consolida tion, and it is possible that is such instances the distention of the trapped air passages resulted mechanically from-cicatricial con traction. The origin of two types of emphysema which were discovered may have similar ex planations. While that which developed in the guinea pigs was definitely compensatory to the foci of atelectasis and fibrosis which it surrounded and the onphysema found in the rats and hamsters was of t&e hyper trophic variety, both may have had a com mon origin in the damage to the bronchioles. The mild to moderate tubercuiogenic ef fect of the hydrous calcium silicate dust mani fested itself in local or even diffuse spreads, delayed healing, and dissemination to other organ*. It is not yet possible at this stage to attribute this phenomenon definitely to dtber the chrysotile or hydrous calcium silicate components. The fact that the tuberculous process did not persist in the pulmonarylymph nodes, whereas it was activated in the lungs, parallels the observation that only in the lungs were asbestos bodies found. It must be presumed that the hydrous calcium silicate dust was transported from the lungs to the lymph nodes, to judge by macrophage accumulation at the latter sites, and it is probable that the dust was more densely con centrated in the lymph nodes than in the pul monary tissues. This suggests that the stimulation to perpetuation and spread of the tuberculous process may have derived from the chrysotile fibers rather than from the hydrous calcium silicate. Such a con clusion naturally requires further confirma tion through experimental investigation. SUMMARY Inhalation studies have been conducted using a commercial product composed of hydrous calcium silicate and chrysotile on normal guinea pigs, rats, and hamsters and on guinea pigs infected with tubercle bacilli of the R: strain. The dust caused marked chronic bronchio litis, with terminal peribronchiolar focal fibrosis, bronchieerasia, and epithelialication of atelectatic alveoli. The final lesions closely resembled those, j/yrd tnjMtpgrimenral asbestosi* and included asbestos bodies. The codrse of experimentally induced tu berculosis was mildly to moderately ad versely affected by the prolonged inhalation of the dust. It seems likely that the chrysotile compo nent of the commercial product, rather than the hydrous calcium silicate, was the cause of the deleterious effects on the tissues of the cx'perimental animals. ' Pnmri **4 > thf [jnitri S'tirt