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Reprinted from the A. M. A. Archives of Industrial Health September 1955, Vol. 12, pp. 348-360 Copyright 1955, by American Medical Association Zffect ofJnkaled Commercia/l ~Jiudrou3 Calcium Silicate 2)uaI on Slnimal SidAueA An Experimental Study G. W. H. SCHERERS, M.D., D.Sc. T. M. DURHAM, M.E. and A. I. DELAHANT, Saranac Lake, N. Y. JI(HU!RUHIUUIIHIIIHtinUtllHUUIIIKIUUUIUtUiUUUUUUUIUIUUHSiniUHnilWUIIUHIUUUIUIUltUlltlinntUUIItHHnHUUUr 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 in 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 car ried 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 IS, 1955. Director, (Dr. Schepers), Associate Director (Mr. Durkan), and Research Associate (Mr. Delahant), The Saranac Laboratory. daily hazardous raw materials used to make a product might not be entirely converted to a nonhazardous 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 30% 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 less than 0.1%. The hydrous calcium silicate product used in The Saranac Laboratory studies was made 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 approximately 80% of the raw mixture had been converted to hydrous calcium silicate and that about 15% was magnesium silicate (Table 1). About 1 % quartz persisted in the final prod uct. Probably the greater part of the mag nesium silicate was present as the fibrous mineral chrysotile, although a portion may have been in the form of serpentine, a min eral similar to chrysotile in chemical compo sition but one which is nonfibrous. EXPERIMENTAL METHOD To determine the effect of inhaled dust of the product on pulmonary tissue, an inhalation experi ment was conducted. In that experiment groups of guinea pigs, rats, and hamsters were exposed in a cubical dust room, 8 ft. in dimension, in which PLAINTIFFS EXHIBIT 01 022 0610 PLAINTIFF'S EXHIBIT V\A/~0QDPSk> 2 Table 1.--Composition of Commercial Hydrous Calcium Silicate Component Analysis Per Cent 8IOa ................................... .8 PaOa, AlaOa, TiOa............................ 3.8 CaO .......................................................... 25-0 MgO ..................................................... 21 NaaO, KaO ........................................ 0.8 Ignition loss ...................................... 19.7 Total .......................... '........... 99.7 A an atmospheric suspension of the hydrous calcium silicate product was created by the action of a paddle which rotated 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 week and for four hours on Saturdays. Some of the animals were exposed to the dust for periods as long as three years. At regular intervals during the experiment a few animal^ were killed, and the organs examined grossly,) and micro scopically to determine the nature and the extent 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 R1 low-virulence strain of the tubercle bacilli was introduced intratracheally by means of the insufflation technique. Dust counts of atmospheric samples collected in the dust room were made regularly by means of the midget impinger. 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. Compound Analysis Per Cent (Approx.) Hydrous calcium silicate.................... 80 Calcium carbonate ............................ 5 Magnesium silicate ............................ 15 Total............................................ 100 EXPOSURE OF UNINFECTED ANIMALS Three species--guinea pig, rat, and ham ster--were employed in this phase of the investigation. Summaries of the findings are 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 intercurrent 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 formation. 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 at 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 to each other in this instance. Table 2.--Biological Action of Commercial Hydrous Calcium Silicate Dust Guinea Pigs, Hamsters, and Rats Were Exposed by Inhalation to an Aerosol of Commercial Hydrous Calcium Silicate Dust Until Death Record of Animals, Exposed to the Dust Alone, Which Died Spontaneously Cause of Death Species Guinea Pig....................... Hamster............................ Rat..................................... Pneumonia Per No. Cent 51* 40.9 10+ 50.0 20i 50.0 Pericarditis Per No. Cent 7 5.6 Peritonitis Per No. Cent 12 9.6 Cervical Adenitis Per ' No. Cent 3 2.4 Total Per No. Cent 73 58.5 m 60.0 20 50.0 *46.5% died within first year of experiment, t100.0% died within first year of experiment. J 0 0% died within first year of experiment. 8 Two deaths from other causes. 01 022 0611 w Symbols: C. cystic change; -f, slight or Incipient reaction; -f-f-, moderate reaction; m 4 Z rj M i tv n if 5 i? 3 -- io r-d 33 3 I - `I w 8 aZ :; `--3 i~* 0-0I *o *** *C--tL-a 2a 2S -a -pf os 0z3 U IS U 8SSSS^52J23t3o*ss ox Pulmonary Reaction to Inhaled Commercial Hydrous Calcium Silicate Dust + : ++' ft :++' + + T + 4- 33 a> ! t+: r<t ^^"t n> wX "r24\*. o!=-i ++, 4+4--44+-- la + +4- ++ a g + + ^3* 5>*. .2 ^ ZT. o I i. 2*. Q 3* *''* o^ S a. 7 a o5 AO ag 53- Z& S?o -U+: _3 > -? 9- H g5a>. 5pri flj c* 1 x "3 o a> o* n o 3 O !J> *< + + + + + T3?1 --33r5n3*2n3 +: : : + +' + +: 1? t, + + -J,bS 3 ZgSS > ?S3 : +: : : : ++: + ++ ++ +, ++ ++ 01 022 0612 marked reaction. 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 36 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 two 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 anirjials. 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 macroscopically 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 hyperplasia 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 start. After the animals had been in the dust atmosphere for 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 zones 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 lesion 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 first year of experiment, the inflammatory 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 year the crenated outlines of the bronchiolar lumina were thrown into sharp relief by a 01 022 0613 0 marked tendency to peribronchiolar atelec tasis, with epithelialization 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 cases 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 iijf the case of the hamsters. The phagocytes 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 quartz 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 plasmacytes. 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 the 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 18-month periods. No acute inflammatory process was demonstrable to account for these results. Asbestos bodies of an elongated, slender, tapering, minute variety were demonstable 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 clubbed 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 18 months of 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 two 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. 01 022 0614 6 Unlike in many other dust experiments, there was no marked tendency toward subpleural congregation of the hydrous calcium silicate dust. Consequently the reaction at this site was minimal and delayed. The most conspicuous changes occurred in the rats where there were occasional subpleural cellu lar condensations and a recurrent tendency to the formation of interlobular adhesions. In some of the guinea pigs which outlasted three years of exposure to the dust, foci of subpleural cellular proliferation attended by giant-cell accumulation were demonstrable. The relatively marked macrophage infil tration of the pulmonary lymph nodes con trasted sharply with the paucity of giant cells 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 of fibrous strands around the lymph nodes followed successive phases of afferent lym 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 which ultimately ensue. One is led to the conclusion, therefore, that the hydrous calcium silicate dust alone, except for its chrysotile component, is nonfibrogenic though it provokes a cellular reaction. EXPOSURE OF TUBERCULOUS ANIMALS 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. In the simultaneous phase, nor mal animals are infected with attenuated tubercle bacilli of the Ri strain by an inhala tion method and then are immediately transferred to the dust room. Thus the tuber culous disease and the tissue reaction to the inhaled dust will develop simultaneously. In the reactivation-phase experiment, the animals, after being infected with the tubercle bacilli, are allowed to live in a normal at mosphere for several months before being exposed to dust. During this period the tuberculous lesions generally regress, and in some instances complete healing by reso lution may take place. Following this inter val, the exposure of the animals to some dusts has no significant effect on the usual course of the tuberculous process, and the lesions continue to regress, while exposure to other dusts may cause the tuberculous process to become active and to spread. A 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 vious dust exposure on the early course of a tuberculous process can be studied. Simultaneous Phase: Reaction in Guinea Pigs Infected with Tubercle Bacilli at Onset of Dust Inhalation In the first experiment in which animals were infected with tubercle bacilli at the time that their exposure to the dust was started, 25 guinea pigs were used, but 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 from progressive tubercu losis, and two others died from undetermined causes but not from tuberculosis. In the re maining 12 animals the course of the tubercu lous disease was followed by killing 1 or 2 animals at 1, 6, 9, 12, 15, 18, and 24 months after infection. In 9 of the 25 animals the le sions were multiple, isolated, and healed, often with central calcification. In six of the remaining animals the lesions were still circumscribed but showed neither a tendency to heal nor to spread even at the end of 18 months. In six other animals there was a moderate tendency to local spread of the tuberculous process, while in the remaining 01 022 0615 7 Table 5.--Causes of Death in Guinea Pigs Exposed by Inhalation to Commercial Hydrous Calcium Silicate Dust and Also Infected ivith Tubercle Bacilli of the Ri Strain Cause of Death Type of Experiment Simultaneous phase........... Reactivation phase............ Predisposition phase ......... Control: Group A ............ Group B ............ Pneumonia Pericarditis Peritonitis Other Causes Total Exposed ,---------- A---------- n ,----------A----------,--------------A---------- n ,----------*---------- \ ,--------- ^\ Animals, Deaths, Per Deaths, Per Deaths, Per Deaths, Per Deaths, Per No. No. Cent No. Cent No. Cent No. Cent No. Cent 30 8 26.6 3 10.0 1 3.3 2 6.6 14 46.6 2 8.0 1 4.0 1 4.0 4 16.0 30 8 26.6 8 20.6 34 4 11.7 4 11.7 3 12.0 1 4.0 2 8.0 6 24.0 animal, which was killed at 15 months, the lesions were widespread throughout the lung 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 inhaled dust upon tuberculosis, the experiment was repeated. Table 6.--Course of Tuberculosis Induced in Guinea Pigs by Infection with Tubercle Bacilli of the Ri Strain Control Study: Infection Only; No Dust Exposure Guinea Pig, No. 62......... 3J1 ... 08 ] 59) eo r -- 63 ) 64 j---- 73......... 87......... 66 j 88......... } 74 J---- 83......... 89 ) 90 j 67 ) 68 j ' 69 j 70 1 71 1 72) 75) 76 j 77 78 79 80 81 82 84 85 86 After Infection, Days GO 120 180 190 200 240 285 300 315 365 Fate Died Killed Killed Killed Killed Killed Killed Died Died Killed Killed Died Killed Killed Died Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Killed Reaction in Pulmonary Pulmonary Lesions Lymph Nodes ------------- -i Arrested Spreading Tubercle Dissemination Tubercles, Tubercu- Casea Calcifi Enlarge Forma- ? No. losis tion cation Pleuritls ment tion Liver 8pleen 3+ 6+ 15 + ++ + T+ T+ ++ + T+ T+ 7 ++ 4 +++ ++ T+ 3 ++ + 5 + ++ H-- ++ ++ ++ T-H-+ T+++ 2 ++ + 4 + ++ + 11 + ++ 2 ++ 0 ++ 4 ++ 1 ++ + 7 ++ 18 + ++ + + ++ -H- ++ ++ + + ++ ++ ++ ++ ++ ++ ++ ++ ++ Symbols: +, slight reaction; ++, moderate reaction; +++. advanced reaction; T, tubercle formation. 01 022 0616 8 Table 7.--Influence of Inhaled Commercial Hydrous Calcium Silicate Dust on the Course of Experimentally Induced Tuberculosis in Guinea Pigs Simultaneous Phase: Animals Received Their Tuberculous Infection and Started Their Period of Dust Exposure Simultaneously Exposure Guinea to Dust, Pig, No. Pays 4 40 i 60 o 60 6 111 3 120 5 120 24 135 25 160 10 178 7 180 8 180 30 185 14 204 22 230 9 240 13 240 28 255 11 365 12 365 17 415 20 420 15 480 16 450 27 517 26 580 18 680 21 690 19 730 23 730 29 730 Fate Died Killed Pied Died Killed Killed Died Died Died Killed Killed Died Died Died Killed Killed Died Killed Killed Died Died Killed Killed Died Died Died Died Killed Killed Killed Dissemination Pulmonary Lesions Arrested Spreading Tubercles, Tuber No. culosis Caicitation 2 /6 30 2 6 + +++ k* 0 +++ ++ ++ 3 7 1 2 7+ 5+ 1 +++ +++ 9 3 +++ 5 ++ ++ 2 2 5 -H- " Pleuritis + ++ +++ ++ + +++ *f++ ++ +++ +++ -H-+ ++ +++ ++ ++ Pulmonary 3 Enlarge Tuber ment culous ++ + + + + + ++ + ++ ++ ++ + + + ++ + + ++ ++ ++ + + +++ ++ + + + + ++ + + + ++ + ++ ++ ++ ++ -i+ + + Liver N+ N+ T++ T+ 8pleen N+++ F+++ F+-H- Symbols: +, slight reaction; ++, moderate reaction +*+*+> marked reaction; F, fibrosis; N, necrosis; T, tuberculosis. 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 a 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% as against the high rate of 46.6% found in the animals which were caused to inhale dust. As these experiments were conducted simultaneously and in the same laboratory, except that the animals receiving dust ex posure were even better protected from out side contacts, the different death rates may have some significance after all. 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 was a marked spread which caused the death of the guinea 01 022 0017 H 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 groins of four healthy guinea pigs. These animals developed local abortive lesions char acteristic of the Rx bacillus, and no systemic tuberculosis ensued. By this study it was fairly satisfactorily demonstrated that Guinea Pig 73 was unduly susceptible to tuberculbsis. In 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^ve 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 Rx tubercle infection. Reactivation Phase: Course of Tuberculosis in Guinea Pigs Which Were Exposed to Hydrous Calcium Silicate Dust Several Months After Infection with R, Tubercle Bacilli Infected guinea pigs were placed in the dust chambers at intervals of two months, i. e., 10 at two months, 8 at four months, and 8 more at six months after infection. In 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 killed 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 minimal 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 Rx 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 normal pattern of tissue reaction to attenuated tubercle bacilli, whether the infection is combined with dust exposure or not, such animals must be ex cluded in assaying the effect of an inhaled dust upon a tuberculous infection. It is be lieved that "sports" are animals whose na tive resistance to the attenuated Rx organ ism is unusually low. Support for this beliet is given by experience with quartz dust, a definitely hazardous material which reacti vates an inhalation infection produced by attenuated Rx bacilli but ordinarily is not as sociated with tuberculous extension to organs other than the lungs and pulmonary lymph nodes. 01 022 0618 10 Table 8.--Influence of Inhaled Commercial Hydrous Calcium Silicate Dust on the Course of Experimentally Induced Tuberculosis in Guinea Pigs Reactivation Phase: Animals, After Receiving Their Tuberculous Infection Were Allowed to Live in a Normal Environment for a Period of Two to Six Months Before They Were Exposed to Dust Pulmonary Lesions Exposure Guinea to Dust, Pig, No. Days Fate SpreadArrested ing Tubercles, Tuber- No. culosis Caseation Fibrosis PleurltJs Exposure to Dust Was Started Two Months After Infection 47 9 Died 51 11 Died 0 + *. + +.. +.. 34 40 Died 2 31 60 Killed 8 32 60 Killed 8 + .. 35 120 Killed 3 36 120 Killed 37 240 Killed 4 7 + 38 240 Killed 2 33 365 Killed 8 39 365 Killed 19 ++ + 40 365 Killed 9 ++ ++ Exposure to Dust Was Started Four Months After Infection 45 120 Killed 2 43 120 Killed 41 240 Killed 4* 42 240 Killed 3 +++ +4- 43 365 Killed 2 44 365 Killed 2 Exposure to Dust Was Started Six Months After Infection 49 117 Died 5 50 120 Killed 3+ 52 120 Killed 53 240 Killed 7+ 1 54 240 Killed 9 + + ++ ++ +++ 55 365 Killed 56 365 Killed 5 + + ++ Dissemination Pulmonary Lympb Nodes Enlarge- Tuber- ment culosis Liver + -4- T+ + ++ ++ + + ++ 4++ ~i--h ++ ++ ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++ + + + + + + + + + + + + + + + Symbols: +, slight; ++, moderate; +++, advanced; T, tuberculosis. Spleen T+ T-H-+ Predisposition Phase : Reaction in Guinea Pigs Infected with Tubercle Bacilli Three Months After Their Exposure to Dust Was Started An experiment of this type is a severe test, 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 carried 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 of the eight animals that died from pneumonia 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 in these animals prior to their terminal fatal illness. The prevalence of such spreads in this series of guinea pigs does indeed appear to 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, so that a total of 22 animals reacted unfavorably. Cavitation occurred in 2 instances, and pleural extension in 10 of the 30 guinea pigs. One animal died from a pneumonic tubercu lous process. 01 022 0619 11 Table 9.--Influence of Inhaled Commercial Hydrous Calcium Silicate Dust on the Course of Experimentally Induced Tuberculosis in Guinea Pigs Predisposition Phase: Animals, After Being Exposed to the Dust for Three Months, Were Infected with Tubercle Bacilli of the Ri Strain and Then Were Immediately Returned to the Dust Room Where Their Dust Exposure Was Continued Until Death Exposure After Guinea Infection, Pig, No. Days 107 0 80 90 10 0 11 0 91 30 92 60 93 60 94 120 95 120 96 180 97 180 114 210 105 230 112 240 113 240 98 300 106 300 115 345 99 365 100 365 119 405 117 435 103 450 104 465 118 480 120 480 101 630 102 630 116 630 Fate Killed Killed Killed Killed Killed Died Killed Killed Killed Killed Killed Killed Died Died Killed Killed Killed Killed Died Killed Killed Died Died Died Died Killed Killed Killed Killed Killed Pulmonary Lesions 1 . . . Spreading Tubercles, Tuber Cavita No. culosis tion / Pleuritis Dissemination Pulmonary . Enlarge Tuber ment culosis Liver -- > Spleen i 17 21 16 14 7 11 6 3 6 30 # 2.1 3 2 4 4 5 3 + + + + ++ + +++ ++ + ++ ++ +++ ++ +++ +++ ++ +++ +++ ++ + + + +++ ++ ++ +++ ++ ++ ++ + + + ++ ++ ++ ++ ++ ++ ++ ++ + + + ++ ++ ++ + +++ +++ + ++ + ++ + -h + ++ + + + + + + + + + + + + + + + + + T+ Symbols: +, slight or incipient reaction; ++, moderate reaction; +++, marked reaction; T, tuberculosis. 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 gree. 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 will 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 in 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 to be employed. The results ob rate at which inorganic matter was deposited 01 022 0620 1 12 in the lung 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. * COMMENT The first point which may be considered is that the commercial hydrous calcium sili- As the chrysotile dust is capable of pro voking 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- Table 10.--Analysis of the Lungs of Guinea Pigs Exposed to Commercial Hydrous Calcium Silicate Dust Period of Exposure, Mo. 2 4 6 8 10 12 18 21 24 27 30 33 36 Ash, Per Cent of Desiccated Lung 4.63 4.45 4.44 4.46 4.47 4.92 4.86 5.16 4.36 4.95 5.57 5.54 5.47 Mineral Components of Desiccated Lung SiOaJ Per Crat 0.22 0.20 0.22 0.29 0.43 0.52 0.72 0.59 0.63 0.64 1.11 0.78 0.68 CaO, Per Cent 0.07 0.08 0.09 0.12 0.14 0.15 0.10 0.17 0.13 0.13 0.14 0.12 0.14 MgO, Per Cent 0.07 0.08 0.08 0.09 0.18 0.15 0.29 0.10 0.07 0.17 0.05 0.19 0.16 Mineral Components of Lung Asb SiOa, Per Cent 4.SI 4.65 ' 5.59 6.66 9.27 10.52 10.46 11.42 12.61 12.74 19.91 14.05 12.49 CaO, Per Cent 1.54 1.94 2.34 2.59 2.62 2.98 1.95 3.29 2.78 2.49 2.42 2.26 2.62 MgO, Per Cent 1.60 1.94 2.09 2.10 3.66 3.13 5.93 1.74 1.46 3.42 0.92 3.51 2.94 cate product, studied in the experiments just described, really provoked two underlying pathological processes ascribable, 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 point is well illustrated in the case of the bronchial epithelium, which proliferated as part of the process of chronic bronchiolar in flammation but did not readily necrose or be come desquamated as in animals exposed to quartz dust. In many of the worst seeming adenomatoid or cystic lesions, the bronchial epithelium even retained its ciliated epithelial surface. The evolution of the adenomatoid reaction is clearly revealed to comprise cryptic dis tention of the bronchioles, secondary papil lomatous ingrowths into these distended lumina, and peripheral epithelialization of atelectatic alveoli. 01 022 0&21 13 Why the alveolar ducts and bronchioles must be presumed that the hydrous calcium should have dilated at an early phase in the silicate dust was transported from the lungs rats was not clearly revealed. No proximal to the lymph nodes, to judge by macrophage (obstruction could be displayed which could accumulation at the latter sites, and it is account for the distention on mechanical probable that the dust was more densely con principles. Possibly the cause may be re centrated in the lymph nodes than in the pul lated to the tendency to peribronchiolar monary tissues. This suggests that the atelectasis. Perhaps a neurogenic mechanism stimulation to perpetuation and spread of was at the root of it all. To be true, the the tuberculous process may have derived larger cystic distentions were usually found from the chrysotile fibers rather than from within areas of chronic diffuse consolida the hydrous calcium silicate. Such a con tion, and it is possible that in such instances clusion naturally requires further confirma- the distention of the trapped air passages * tion through experimental investigation. resulted mechanically from cicatricial con traction. SUMMARY The origin of two types of emphysema Inhalation studies have been conducted which were discovered may have similar ex using a commercial product composed of planations. While that which developed in hydrous calcium silicate and chrysotile on the guinea pigs was definitely compensatory normal guinea pigs, rats, and hamsters and to the foci of atelectasis and fibrosis which on guinea pigs infected with tubercle bacilli it surrounded and the emphysema found in of the R! strain. the rats and hamsters was of tfie hyper The dust caused marked chronic bronchio trophic variety, both may have had a com litis, with terminal peribronchiolar focal mon origin in the damage to the bronchioles. fibrosis, bronchiectasia, and epithelialization The mild to moderate tuberculogenic ef of atelectatic alveoli. The final lesions closely fect of the hydrous calcium silicate dust mani resembled those found-in experimental as- fested itself in local or even diffuse spreads, bestosis and included asbestos^bodies. delayed healing, and dissemination to other The course of experimentally induced tu organs. It is not yet possible at this stage to berculosis was mildly to moderately ad attribute this phenomenon definitely to either versely- affected by the prolonged inhalation the chrysotile or hydrous calcium silicate of the dust. components. The fact that the tuberculous It seems likely that the chrysotile compo process did not persist in the pulmonary- nent of the commercial product, rather than lymph nodes, whereas it was activated in the the hydrous calcium silicate, was the cause lungs, parallels the observation that only in of the deleterious effects on the tissues of the the lungs were asbestos bodies found. It experimental animals. Printed and Published in the United States of America 01