Document 4ak35Z8pRxYL7qXk3dqegqObx

/ ^ A. M. A. Archives of Industrial Hygiene and Occupational Medicine Volume 3 JANUARY 1951 Copyright, 1951, by the American Medical Association Number 1 79 EXPERIMENTAL STUDIES OF ASBESTOSI?0^ 83 ARTHUR J. VORWALD, Ph.D.fPath.), M.O. 587 THOMAS M. DURKAN I Tj ,.?** to, AND -j-y 399 PHILIP C. PRATT, M.D. C^J SARANAC LAKE, N. Y. A303 SBESTOSIS is a form of pneumonoconiosis resulting from pro> longed inhalation of asbestos dust. The name "asbestos." literally "unburnable," is not that of a specific mineral but is a term applied to a 517 number of different minerals whose characteristic feature is a structure 622 composed of long, parallel, flexible fibers. This structure is unique because the fibers are capable of repeated longitudinal subdivision to 625 units of molecular proportions. In length the fibers varv from a few microns to 6 or more inches (15 or more cm.). Some varieties are 629 stiffer than others, but many are sufficiently flexible to be spun into yarn and woven on modified textile machinery. The asbestos minerals are silicates of variable composition and belong 631 to the serpentine and the amphibole groups. Listed below are the more 647 common varieties. 648 Amphibole group: actinolite, amosite, amphibole, anthophyllite. crocidolite and tremolite. Serpentine group: chrysotile. The bulk of the asbestos of commerce is chrysotile, 3Mg0.2Si0.,.2H20, which is mined on this continent principally in the Thettord region of the Province of Quebec, Canada, and in Vermont. Crocidolite and amosite also are used commercially but in much smaller amounts. Chrysotile occurs as veins in serpentine, a mineral of similar chemical composition, which exists in massive form and is made up of microscopic fibers without the parallel orientation characteristic of chrysotile. The massive, bluish black serpentine, which is smooth and soapy to the touch, is traversed by veins of fibrous chrysotile varying in width from a barely perceptible line to 6 (15 cm.) or more inches. The fibers run across the vein and not lengthwise with the formation. From the Saranac Laboratory of the Edward L. Trudeau Foundation. This series of studies of asbestosis, initiated at the Saranac Laboratory more than twenty years ago by the late Dr. Leroy U. Gardner, director of the laboratory, was nearly completed at the time of his death in October 1946. Although partial reports and informal reviews of some of the experiments had been given from time to time by Dr. Gardner, this paper presents for the first time a complete survey of the entire experimental investigation. 1 2 1XDUSTRIAL HYGIEXE AXD OCCCPATIOXAL MEDICINE Attention is directed to the mineral brucite. Mg0.H,0, which is often found in the same formations with serpentine and chrysotile and may be fibrous in structure. Except for the manufacture of magnesium, brucite has no commercial value at present because its fibers are not sufficiently flexible to be used in textiles, but they are capable of repeated longitudinal subdivision. Unlike other asbestiform minerals, brucite is not a silicate, and for this reason it has been a valuable tool in an experimental evaluation of the action of fibrous minerals on lung tissue. EXPERIMENTAL ASBESTOSIS For many years studies1 have been carried on at the Saranac Laboratory in an investigation of the cause, nature and development of asbestosis. The present paper is devoted to experimental asbestosis, Fig. 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 zone 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. (n) 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 Tissues, Am. Rev. Tuberc. 45:762, 1942. UORH'ALD ET AL.--STUDIES OP 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 usual 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 bv animal experiments is invaluable in furnishing a better understanding of the reaction of the human organism to inhaled asbestos dust. ExrF.HIMF.XTAI. 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. cS ft. (2.5 M.) in dimension, in which a cloud of asbestos dust is maintained bv a rotating paddle in a dust hopper.1" 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 experiments 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 by the intravenous, the intraperitoneal, the intratracheal or another route. Long term inhalation experiments furnish information on which great reliance is placed when estimating the degree to which a dust might constitute a respiratory hazard to industrial workers. Even though an atmospheric dust may be potentially dangerous, as indicated by injection experiments, only inhalation procedures will reveal whether the dust can be inhaled, pass the natural defense barriers of the body and reach the pulmonary tissue in quantities sufficient to cause damage. Injection methods are useful, however, because thev make certain that contact occurs between the dust particles and tissues and because they allow accurate estimation of the dosage and of the potential capacity of that dose to produce reaction. The intratracheal method is particularly valuable when one is dealing with fibrous minerals like asbestos, since it permits observation of the effect of the fibers on pulmonary tissue. Tissue Susceptibility Unlike free silica, asbestos does not produce specific effects in all organs of all species of animals. The comparative data presented in table 1 are based on completed observations and therefore differ slightly from a preliminary report.11' Fine quartz introduced into various organs 4 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE of various animals (guinea pig, rabbit, rat, mouse, cat, dog, chicken and even tadpole) eventually will produce silicotic nodules but at different rates. Similar introduction of long fiber asbestos has resulted in a fibrous reaction in the lung and, to a lesser extent, in the peritoneum but not in other organs of the guinea pig, the rabbit, the cat and the white rat. In our experience the lungs of the dog and the white mouse failed to respond with fibrosis, although Schuster 2 has reported such changes in a dog that lived in an asbestos-fabricating plant. This variation in species and in organ susceptibility is yet to be accounted for 3; it is presumed that in the susceptible animals the greater reaction of the lung to asbestos, far exceeding the reaction of other organ tissues, is due principally to the greater mobility of the lung. Peculiar Characteristics of Asbestos Experience has demonstrated that most of the nonfibrous dust particles inhaled into the lungs of man and animal are 10 microns or less Table 1.--Reaction to Long Fiber Chrysotile in Lungs of Han and Other Species of Animal Species Man........................... Guinea pig.............. Rabbit...................... Cat........................... White rat................ White mouse.......... Dog........................... Mode of Exposure Inhalation Inhalation and injection Inhalation and injection Inhalation and injection Inhalation and injection Inhalation Injection Fibrosis * 4+ 2+ + + + 0 0 Asbestosis Bodies Numerous Moderately numerous Rare and atypical Rare and atypical Very rare Rare and atypical None * The symbols f) to 4+ r^for to the degree of tissue reaction. in maximum dimension. Larger particles apparently do not gain access to the lungs, because, first, large particles settle in air so rapidly that few remain suspended in the atmosphere breathed and. second, large particles are more effectively removed by the protective mechanisms of the upper respiratory tract. In the case of fibrous materials these factors have less influence and fibers 100 and even 200 microns in length have been found in the terminal air spaces of human lungs. In small labora tory animals exposed to asbestos dust the maximum length of fiber found in the lung rarelv exceeds 60 microns. A large proportion of nonfibrous particulate dust inhaled into the lung is found in the terminal air spaces (alveolar ducts, atriums, alveoli) in all parts of the organ; in contrast, inhaled asbestos fibers are first discovered in the respiratory bronchioles. These small passages are immediately distal to bronchioles lined by ciliated epithelium.4 Their 2. Schuster, X. H.: Pulmonary Asbestosis in a Dog, J. Path. S: Bact. 34 (pt. 2): 751, 1931. 3. Vorwald. A. J.: Variations in Individual Susceptibility to Industrial Dusts [nbaled into the Lungs, Am. Rev. Tuberc. 62: (IB) 13, 1950. 4. Miller. W. S.: The Lung, Springfield, 111., Charles C Thomas, Publisher, 1937. VORWALD ET AL--STUDIES OF ASBESTOSIS 5 own essential lining is a low cuboidal type of epithelium but, as their name implies, the}' actually function in respiration through lateral alveoli distributed along their walls. Either these alveoli or the abrupt change in the character of the lining epithelium, or the small diameter of the respiratory' bronchiole, or the combination of all three factors is responsible for retention of the fiber at this site. Only after asbestosis is well established are appreciable numbers of fibers seen in the more peripheral air spaces. Further explanation is required to clarify this observation. Rate of Tissue Reaction to Asbestos Fibers The affected tissues react much more rapidly to asbestos than to quartz dust. For example, in rats receiving asbestos 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 of 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 asbestosis increases for only a short time. Subsequently, the asbestotic fibrous tissue contracts ; this process often distorts the adjacent pulmonary tissue and may. as a result, progressively interfere with cardiorespiratory function. Asbestosis Bodies The peculiar structure known as the asbestosis body' or "curious body" is a specific concomitant of asbestosis.r' The typical body is a golden yellow, beaded or haustrated rod, which may be either straight or curved (fig. 2). Often one or both ends are bulbous like a dumbbell. The bodies vary considerably in length, and dimensions up to 250 microns have been recorded. It is believed that asbestosis bodies are inhaled fibers on which pro tein and iron pigment of tissue origin have been deposited.56 * *G*lo*yne 011 observed reproduction of these bodies in guinea pigs nine months after subcutaneous injection of fibers rendered free of iron. The bodies are abundant in man and in the guinea pig (table 1) but are much larger in the former, probably because the larger-sized air passages admit fibers of greater dimension. In guinea pigs they form after about 70 days 5. Gloyne. S. R.: (a) The Formation of the Asbestosis Body in the Lung, Tubercle 12:398, 1931; (b) The Asbestosis Body, Lancet 1:1351, 1932. (c) Gard ner and Cummings.la 6. Lynch, K. XL, and Smith, W. A.: Asbestosis Bodies in Sputum and Lung, J. A. M. A. 95:659 (Aug. 30) 1930. Simson, F. \V., and Strachan, A. S.: Asbestosis Bodies in the Sputum: A Study of Specimens from 50 Workers in an Asbestos Mill, J. Path. & Bact. 34:1, 1931. Gardner and Cummings.,a Gardner.,b Gloyne. 5a> b 6 IXDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE of contact with the tissue. In cats, rabbits and mice a few of the fibers show an atypical coating after much longer residence in the lungs. In rats the bodies are rarely seen, and in dogs none could be found. Although the evidence is incomplete, it appears that the formation of the asbestosis body prevents the fiber from damaging the tissue. Many of the points mentioned above will be elaborated on in subsequent para- 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 tx 4001. 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. f 'ORIVALD ET AL.--STUDIES OP ASBE.STOSIS 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 Asbestos SiO:1 FejOn Al-O.-t CrjOa MtiO (.'ad Mg<> Xa-U KjU (JOl- Ignl- cion Loss Total King's floats............... 39.32 S.S4 ' ` im.7 3.'. .Vi _ 12.74 `*7.13 Short fiber................... 37.17 9.09 1.40 0.14 0.09 U.S5 U.14 u.2o u.9* 14.09 100.11 Long fiber................... 3S.40 5.32 0.7$ * 0.0$ 0.31 40.1$ O.UG 0.CK5 0.57 14.00 99.7G Not determined. Table 3.--Petrographic Analysis of Asbestos Dusting Materials King's floats*: The approximate composition, based on particle? (except ehrysotib1) smaller than 10 microns and reported as percentages obtained from particle counts, ua< chrysotile 14, serpentine 40, magnetite 12, carbonates 1?, talc 12, other minerals 4. For chrysotile, fibers up to 200 microns long were included. Short fiber t: The material, before being hall milled, contained a preponderance of fibrous chrysotile and platy (nonfibrousi serpentine. The approximate composition, by percentage, was chrysotile 17, serpentine 55, magnetite 10, quartz 2, brucite 5, other minerals, including dolomite, actinolite and treinolite, 11. Long fiber f: The material consisted principally of the fibrous asbestos mineral chrysotile. Shreds of nonseparated fibers 5 to 15 microns in diameter and up to 50 micron? in length were present. The approximate composition, by percentage, was chrysotile 75. serpentine 15, mag netite 5, brucite 2, other minerals, among which were ca'cite and ehioritie and micaceous minerals, 3. Only a trace of quartz was observed. * The analysis of the King's flouts asbestos, made by Dr. N. Huribut .lr . of Harvard University, has been reported elsewhere (Huribut, c. S., .Tr., and Williams, c. K : The Min eralogy of Asbestos Dust, .7. Indust. Hyg. & Toxicol. 17: 292, 1935). + For the short fiber asbestos and the long fiber asbestos the petrographic analysis was supplemented with x-ray diffraction examination. periods up to 33 months. Some guinea pigs with six and nine months' exposure lived for an additional three years after cessation of their exposure. A preliminary report presented observations after 29 months of exposure. At that time observations covered a period of only 2,14 years and the conclusions as to the ultimate effects of inhaled asbestos dust were provisional. Those conclusions are substantiated by results of the completed study, which is reported as follows. Composition and Atmospheric Concentration of the Dust.--The dusting material, a commercial variety of asbestos known as King's floats, was composed of short fibers, ranging in length from 1 mm. to 1 micron or less, and of particles which also varied in size. It was obtained from the Thettord. Quebec, plant of the Asbestos Corporation of America, and analyses (tables 2 and 3) reveal that the amount of fibrous chrysotile was only 14 per cent, a rather low value. S INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE Impinger samples taken soon after the experiment was started indicated that the dust concentration was at first quite low, the average dust count being only 6.0 million particles per cubic foot of air by the standard light field technic and 0.8 million for particles and fibers greater than 10 microns. After the inhalation experiment had been under way for about two years, the speed of the rotating paddle in the dusting machine was increased and for the remaining 10 months of the experi ment considerably more dust was dispersed into the atmosphere. The average dust count of impinger samples collected after this change was 53.7 miltion by the usual light field method and 1.6 million for particles and fibers larger than 10 microns. It is probable, however, that the true values of the dust concentration were higher than the counts given in this paragraph. The impinger samples for the King's floats experiment were collected in water, but later studies7 have shown that counts of impinger samples of asbestos dust taken in water are not reliable. Ethyl alcohol instead of water was used as the collecting fluid in all subsequent experi ments. Table 4.--Summary of Inhalation Experiment with King's Floats Asbestos Dust Nature of Experiment Dust exposure continuous through* out life Dust exposure followed by prolonged residence in normal air Tuberculous infection * at start of dust exposure Control.'! to infection: no dust expusure Tuberculous infection * after 23 mo. of dust exposure, then resilience in normal air Control5 to infection: nu dust exposure Animals 54 guinea pigs 9 rabbits 18 rats 25 guinea pigs 25 guinea pigs 1 rabbit l rabbit 40 guinea pigs 25 guinea pigs 12 guinea pigs 12 guinea pigs Maximum Maxi* Survival mum After Dust Ex Dust Exposure. posure, MO. Mo. 33 0 19 0 60 6 35 9 37 6 30 l 19 24 J 35 0 0 35 t Results Typical peribronchiolar fibrosis after 16 months Foreign body bronchitis l ittle or no reaction Nonprogressive fibrosis N'onprogressive fibrosis Absorption of foreign body reaction Temporary progression of infection, followed by healing with fibrosis Healing by resolution (one exception) 26 14 N'o appreciable increase in susceptibility to tuberculous infection; healing with flbrosi5 0 19 t Healing by resolution The guinea pig- were infected with low virulence Iti strain of tubercle bacillus, f This means th- survival period following infection. Results of the investigation, briefly summarized in table 4, show that inhalation of King's floats asbestos dust produced a typical peribronchiolar fibrosis in guinea pigs but not in rabbits or rats. Reaction in Normal Guinea Pigs.--Guinea pigs inhaling this dust for periods up to 35 months had a characteristic fibrosis occurring in conical patches about the respiratory bronchioles. During this exposure the peripheral alveoli were not involved. The particulate elements of the dust were transported through the lymphatic system to the bronchial nodes, causing no significant reaction.in either site; the fibrous elements remained fixed at the points of original localization and were seldom detected in the lymph nodes. After exposure of approximately a year a small amount of cellular reaction had been produced about many respiratory bronchioles (fig. 3.4). 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 they 7. Fulton, \V. B.; Houtz, R. L.; Dooley, A., and Mathews. T. 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.--STL'DIES Of ASBESTOSIS 9 were phagocytosed, and many of them were carried into the wall by migratory cells. Mononuclear leukocytes attracted to the area caused an appreciable thicken ing of the bronchiolar wall. After 16 months a delicate fibrosis made its appearance. The process evolved gradually, and the number of fine intercellular collagenous fibers steadily increased. As this fibrous deposit contracted, it partially closed and Fig. 3.--King's floats inhalation experiment: A, lung of a guinea pig with 12 months' exposure. It includes a respiratory bronchiole, at the left, branching and becoming an alveolar duct, at the right. Note the accumulation of cells in the wall of the bronchiole and in adjacent alveoli (X 130). B, lung of a guinea pig with 28 months' exposure. The field includes a bronchiole, at the center, with peribronchial fibrosis extending into the walls of adjacent alveoli. Note the cuboidal epithelium lining these alveoli. This is the so-called "adenomatoid'' appearance (x 200). distorted the alveoli, and with this change the alveoli became lined with cuboidal cells. The result was an adenoma-like appearance which frequently accompanies 10 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE chronic pulmonary inflammation resulting front many causes. Willis 8 described a similar structure in the lungs of guinea pigs inhaling silicon carbide. The longer asbestos exposures resulted only in more thickening of the walls of the air spaces, largely due to an increase in the amount of fibrosis. The fibrous tissue always remained cellular and failed to show the hyalinization characteristic of silicosis. 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 1x 200). 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. VORU'ALD ET AL.--STUDIES OE ASBESTOSIS 11 Asbestosis bodies (fig. 2B), first seen in the lungs of the guinea pigs that had inhaled dust for about two months, became more numerous and more distinctly segmented with increasing exposure. The reaction produced in guinea pigs exposed for six and nine months did not progress significantly during a subsequent period of 35 and 37 months when the animals lived in a normal atmosphere (fig. 4). Between eight and 11 months after exposure ceased, the cellular reaction in the lung had been completely replaced by thin strands of fibrous tissue. At later periods the scar tissue was less in amount, but in the last animal killed, 37 months after discontinuing dust exposure, some fibrosis was still visible. Reaction in Guinea Pigs Infected unth Tubercle Bacilli at the Onset of Dust Inhalation.--Of the group of 40 guinea pigs infected with attenuated tubercle bacilli, Ri strain,9 *a*t the time that dust exposure was begun. 31 died or were killed before the completion of two years of the exposure and were reported in the paper by Gardner and Cummings.12 Seventeen of these died from intercurrent pneumonia. Briefly, the results were as follows: Ten revealed some evidence of spread of the tuberculous process (fig. 5.4); 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 were more 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. 5Z?). In the remaining five the tuberculous foci showed evidence of having previously spread locally ; in four of them, by the time of autopsy, the foci were healed, with excessive fibrosis: in the fifth animal there was a generalized chronic tuberculous pneumonia in one lobe, and in the other lobes there were isolated primary tubercles, which were still active but had not spread. Reaction in Guinea Pigs Infected with Tubercle Bacilli After Establishment of 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 months after infection. The subpleural tubercles were no more numerous in the dusted animals than in the nondusted controls, but a considerable number were found in the depths of the lung about foci of asbestosis. The tuberculous component of the combined reaction showed only slight local extension about lesions in the lungs and tracheobronchial lymph nodes. Caseation was found in tubercles 1 x/2 months old, but by 5^2 months it had completely disappeared, leaving only scar tissue. Foci of fibrosis still persisted in the last animal, which was killed 14 months after infection. Reaction in Rabbits.--Rabbits exposed to the asbestos dust for periods up to 19 months showed a foreign body type of reaction of low grade, but no fibrosis. Although their lungs contained particulate elements of the dust, fibers were not present, indicating that the upper respiratory mechanism of the rabbit is adequate to exclude fibrous foreign bodies. Two rabbits, after inhaling dust for six and 19 9. Steenken, \\\, Jr., and Gardner, L. U.: Ri Strain of Tubercle Bacillus: Its Dissociation and Virulence of Variants in Normal and Silicotic Guinea Pigs, Am. Rev. Tuberc. 54:51, 1946. 12 INDUSTRIAL HYGIEXE AXD OCCLPATIOXAL MEDICINE months, lived in normal air for more than two years. At autopsy neither animal showed any evidence of cellular reaction or fibrosis in the terminal bronchioles, nor were there any asbestosis bodies. Reaction in ll'hitc Rats.--All the rats had acquired an infection, resulting in the formation of pulmonary abscesses, before they came to autopsy. Apparently, so much heavy mucus obstructed their bronchi that very few fibers could have entered Fig. 5.--King's float inhalation experiment: A. 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. Xote the lack of encapsulation and the peripheral epithelioid cell pneumonia, which illustrate a spreading tuber culous process (x 200). B, lung of a guinea pig infected with Ri tubercle bacilli and then exposed to dust for 35 months. Xote 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). I'ORIVALD ET AL--STUDIES OF ASBESTOSIS 13 their lungs. In a few of the rats, an occasional asbestosis body was discovered, but there was no fibrosis. This phase of the experiment was considered unsuc cessful. Summary and Interpretation oj Inhalation Experiment zvith King'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 of asbestos dust, the infectious process continues to progress and eventually causes the death of the animals. On the other hand, infected animals exposed to a harmless dust like iron oxide do not show any progression of the infection.1" Guinea pigs infected with attenuated tubercle bacilli after the termination of two years' asbestos dust exposure did not show progressive disease. The only modification of the infection was in its localization, a few bacilli being retained in the peribronchiolar fibrous tissue, with tubercles forming there in addition to the usual tubercles beneath the pleura. In view of the variability of the results, the unusual nature of the response and the high proportion 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 tuberculous infection are justified bv this experiment. Short Fiber Asbestos Dost Since hazardous dusts like quartz are most effective' in producing fibrosis when the particles are 3 microns and less in size, an inhalation experiment was performed to determine whether this condition is true for asbestos dust. It was thought that a short fiber asbestos dust consisting almost entirely of fibers and particles smaller than 3 microns would initiate an accelerated tissue response and produce an advanced reaction in a shorter time than did the King's floats dust, which con tained fibers from 1 mm. to 1 micron and less in length as well as much particulate matter. Composition and Atmospheric Concentration of the Dust.--The dusting material tor this experiment was the remains of fibers collected in dust bins of an asbestos fabricating plant after a carding operation and screened to pass 200 mesh. Since 10. Yorwald. A. J.; Pratt, P. C.; Durkan, T. M.: Delahant, A. B.. and Bailey, D. A.: Siderosis : A Benign Pneumoconiosis Due to the Inhalation of Iron Dust, Indust. Med. & Surg. 19:170, 1950. 14 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE the material as received contained many long fibers, it was ground in a steel ball mill to reduce practically all the 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 chopper, and it became necessary to mix one volume of the unground material with three volumes of the ground to generate a satisfactory dust cloud. It is pertinent to mention here that the addition of the small quantity of unground asbestos was unfortunate, because it confused the interpretation of results. The composition of the short fiber asbestos as received is disclosed by the chemical and petrographic analyses given in tables 2 and 3. Samples taken before and after grinding yielded about the same values on analysis, indicating that there was no contamination from the mill or loss of water content. The dust concentration varied during the experiment, the light field counts for atmospheric samples collected inside the animal cages with the impinger apparatus ranging from 83 million to 182 million. The average of counts was 130 million for the first year of the experiment, 134 million for the second year and 140 million for the third year. Size-frequency measurements of air-floated dust from inside the cages at a magnification of 1.300 X revealed a great preponderance of fine particles, nearly Table 5.--Summary of Inhalation Experiment ivith Short Tiber Asbestos Dust Nature of Experiment Dust exposure continuous through out life Animals 46 guinea pigs 73 rats IS cats 7 rabbits Dust exposure followed by prolonged residence in normal air 13 guinea pigs 2 eats 1 rabbit Maximum Maxi- Survival mum After Dust Ex- Dust Exposure, posure. Mo. Mo. 34 0 32 0 54 * 0 47 * 0 20 14 31 24 62 * 6 Results Rate of reaction about the same as in experi ment with King's floats asbestos but extent of involvement very much less t Characteristic patches of peribronchiolar .fibro sis; no asbestosis bodies Subpleural reaction only No fibrosis seen grossly; microscopic evidence of alveolar wall thickening after 40 months' exposure Progression after removal from dust doubt ful--neither clearly established nor definitely excluded Same as for continuous exposure Similar to continuous exposure; evidence of slight regression * After 33 months the animals were exposed to 100 per cent hall-milled asbestos. t The reaction was probably due to long fibers in the onground material which was mixed with the ground asbestos dust to 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 5, are presented in 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 the extent of involvement was very much less. After 16 to 24 months of exposure only a very few small foci of reaction, which generally required microscopic examination for detection, had been produced in the guinea pigs. * : i } !: s VORWALD ET AL.--STUDIES OF ASBESTOSES 15 Only after exposures had continued for approximately one year was there an appreciable tendency for dust-containing phagocytes to gather into clumps. By 16 months phagocytes had collected about the walls of a few of the 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'' 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 hyalinized. Diffuse chronic pleurisy was present in a few animals without evidence of pul- Table 6.--.4ua/_v.$Y\r of Lungs of Guinea Pigs After Prolonged Inhalation of Short Fiber Asbestos Dust Exposure to Dust, Mo. Period in Normal Air, Mo. Amount of Ash, % of Dried Lung Total SiO=, % of Dried Lung Total SiOa, % of Ash Tissue Reaction * Dust Exposure Continuous During Life 1 5.02 0.51 10.23 12 0 ] 4.5S 0.46 10.08 [ 5.16 0.54 10.54 { 5.00 0.49 9.96 15 0 4.76 0.43 9.00 [ 4.95 0.53 10.60 20 n ( 5.SO ( 6.43 0.85 0.90 14.46 14.07 2+ 024 ( 5.42 0.78 14.48 } 5.50 0.7S 14.20 3+ no 0 \ 5.35 ( G.55 0.96 1.27 17.S9 19.46 4+ 34 S 606 0.75 12.37 l 6.35 0.96 15.11 4+ Dust Exposure Followed by Prolonged Residence in Normal Air 20 4 J 5.10 j 5.11 0.4S 0.30 9.30 7.16 2+ s20 10 6.11 l 3.98 0.02 0.34 10.21 S.51 3+ ( 4.77 20 14 \ 5.IS l 4.77 0.25 0.26 0.22 5.31 5.00 4.60 2 + * The symbols averaging the tissue reaction in each group of guinea pigs represent merely the relative degree of reaction, ranging from - (questionable) to 4+ (the maximum for this experiment). The relationships apply only within this table and cannot he compared with symbols in other tables. monary infection. This suggests that pleurisy may be a specific concomitant of asbestosis, but the evidence is not adequate to establish this point. The reaction of the tracheobronchial lymph nodes was more pronounced than in the previous experiment with King'? floats asbestos, probably because more fine particles had been transported to the nodes in animals inhaling short fiber asbestos. The nodal reaction was essentially an increase in reticulum, rather than a fibrosis, with the original cells being preserved between the thickened reticular fibers. In the group removed to normal air after 20 months' inhalation of dust, progres sion of disease was not definitely demonstrated, but neither could it be absolutely disproved, owing to the variability of the response in different animals. The reactions, from mild to severe, occurred sporadically and bore 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 in lungs with widely different amounts of tissue change. For example, the ash 16 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE and silica values were quite similar for three animals living in dust 20 months and then in normal air for 14 months, yet the tissue reaction was severe in one animal, mild in another and only doubtful in the third. The formation of asbestosis bodies was at first extremely limited in both groups. After five months' exposure only a very rare short body could be found, usually inside a cell. Some of the finest intracellular particles were surrounded by yellow deposits having the same color as the asbestosis body. One year's exposure had per mitted an accumulation of many longer fibers, a number of which were coated and seen as typical asbestosis bodies. Most of these were still short enough to be partially or entirely within phagocytic cells. By the twentieth month and thereafter they Table 7.--Analyses of Lungs of White Rats That Had Inhaled Short Fiber Asbestos Dust Duration of Expo sure, Mo. n* Amt. of Ash, % of Dried Lung ,3.9 4.3 2.9 .3.6 .3.3 .3.9 3.4 f 3.."> J n.e 1 2.9 U.2 3.3 3.6 l 3.4 Total SiOs, % of Dried Lung 0.00 0.00 0.00 0.00 0.00 0.00 0.08 0.1.3 0.09 it.03 0.08 o.n 0.07 Total S1O2, % of Ash 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.1 3.5 3.2 1.5 2.3 3.0 2.2 Duration of Expo sure, Mo. Amt. of Ash, So of Dried Lung 3.3 3.4 3.7 3.9 3.6 8 3.5 4.4 . 3.7 (4.9 10 4.6 1 5.3 14.7 Total SiOs, Sc of Dried Lung 0.07 0.05 0.04 o.os 0.1s 0.15 0.17 0.16 0.1$ 0.15 0.15 0.13 Total S1O2, % of Ash 2.1 1.5 1.1 2.2 5.5 3.4 4.0 4.2 3.S 3.3 2.8 2.S * Normal control.* Coo dust exposure). Table 8.--Average Values of Ash and Total Silica for Lungs of White Rats Inhaling Various Dusts for Various Periods (Lungs Only, Without Included Lymph Nodes) Amt. of Ash, c/c of Dried Lung Dura Total SiOs, % of Dried Lung Total SiOs, Sc of Ash tion Short Gypsum- Short Gypsum- Short Gypsum- of Ex Fiber Ferru Quartz Fiber Ferru Quartz Fiber Ferru Quartz posure, Asbes ginous Mix Asbes ginous Mix Asbes ginous Mix Mo. tos Quartz Chert ture tos Quartz Chert ture tos Quartz Chert ture 2 .3.3 4.3 5.9 2.9 0.00 0.51 0.25 O.OS 2.6 11.7 3.9 2.i! 4 3.4 4.5 5.9 3.S 0.09 0.51 0.32 0.07 2 5 11.4 3.6 2.0 6 3.5 7.1 9.9 3.4 0.05 2.94 3.45 0.11 1.6 41.5 34.4 3.4 S 3.S 4.6 9.0 3.6 0.15 1.44 2.40 0.32 5.9 29.4 26.5 9.1 10 4.9 7.S 14.1 4.1 0.15 4.40 6.09 0.2S 3.2 56.6 43.2 6.7 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 tor periods up to 32 months. During the first 10 months animals were killed bimonthly and tor the remainder of the experiment at less frequent intervals. Up to eight months the dust cells were widely scattered and existed in loci only sporadically. Reaction was limited to occasional slight thicken ing of the septums about small accumulations of dust cells. 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 clump 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 ot 150 diameters or more, consisted of patches along rORU'ALD ET AL.--STUDIES Of ASBESTOSIS 17 alveolar ducts in which the walls of the associated air spaces were very thick, owing to swollen collagen framework. Connective tissue and Foot-Bielschowsky silver preparations revealed complete loss of capillary bed locally. Outside the collagen was a thin layer of epithelial cells. This did not resemble the "adenomatoid" change characteristic of guinea pig asbestosis. Near the lesions the air spaces were filled with phagocytes containing gray to yellow particulate dust and a rare, long, naked asbestos fiber. Careful search failed to reveal even a suggestion of an asbestosis body. Pleurisy was absent. The tracheobronchial nodes showed com pact focal collections of monocytic cells at 12 months and. at 20 months, some diffuse thickening of the reticulum. In a few rats there was definite fibrosis along the margins of the node, extending into the mediastinal areolar tissue. Results of chemical analyses made on the 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 for 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 one-half that of the gypsum-quartz mixture and was one-fifth that of the ferruginous chert. Since the values for asbestos are low. it might be 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 from 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 continuously 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 exposure of 31 months: one of these was killed five months, and the other 24 months, later. In general, the tissue response was confined to microscopic foci of fibrosis, which were in the walls of groups of subpleural alveoli rather than in the peribronchiolar areas. In one animal the change was extensive enough to be visualized on gross inspection of the section. Only in the animal with the longest exposure---54 months--did the roentgenogram reveal definitely abnormal shadows. A roentgenogram made after 30 months revealed no abnormality ; after 45 months, a faint mottling could be detected throughout both lungs. At autopsy, nine months later, there was only microscopic fibrosis in the subpleural zone plus heavy lymphocytic infiltration about small bronchioles. Asbestosis bodies were rare. On prolonged search a fewyellow atypical bodies, smooth and without haustrations, were found in two animals exposed for more than a year. Reaction in Rabbits.--Eight rabbits were exposed to dust for periods extending from one to more than five years; the last animal was removed from the dust room and left in normal air six months before being killed. There was never enough pulmonary fibrosis to be detected grossly, and there was no chronic adhesive pleurisy. Microscopic evidence of alveolar wall thickening was first detected in one animal after about three years of exposure and was seen in all five animals examined thereafter, including the one removed to normal air. One animal that died of paralysis after nearly four years of exposure exhibited a reaction visible on gross inspection of tissue sections. The possibility of pulmonary infection in this animal could not be excluded. In another animal dying two years later the focal fibrosis was not nearly as obvious or as advanced. Areas of involvement, which were largely visualized because of phagocytic reaction within the air spaces, tended microscopically to become more fibrous with the passage of time, but 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 that died early in the experiment but were seen in all animals that had been exposed to the dust for more than three years. 18 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE Summary and Interpretation of Inhalation Experiment with Short Fiber Asbestos Dust.--The original purpose of the experiment was to evaluate the role of short asbestos fibers in the genesis of asbestosis. It was felt also that if the tissues reacted more rapidly and more extensively to short fiber asbestos than to -thing's floats there would be a basis for believing that the action of asbestos is in part, at least, a chemical one as postulated for quartz. This experiment, in which the tissue reaction was slower and less extensive than that in the previous experiment with King's floats dust, indicates that the capacity of inhaled asbestos fibers to produce fibrosis is determined primarily by factors not chemical in nature. Of the four species exposed in this experiment, only the guinea pig and to a lesser extent the white rat responded with characteristic peri bronchiolar fibrosis. The cat reacted with atypical subpleural fibrosis and the rabbit with only slight parenchymal fibrosis. Ball-Milled Asbestos Dust In the inhalation experiment with short fiber asbestos dust a small quantity of unground short fiber asbestos was mixed with the ballmilled product in order to generate a suitable dust cloud. When that experiment failed to produce an accelerated tissue reaction, in com parison with the response initiated by King's floats, it became apparent that the biologic activity of asbestos is not increased by a reduction of fiber size. Thus the possibility arose that the tissue reaction observed was due solely to the relatively few long fibers of the unground asbestos and that the short fibers of asbestos had no more than a very insignificant role in the production of asbestosis. a concept not in accord with previous experiments concerning pneumonoconiosis. Consequently another inhalation experiment was started in which only ball-milled asbestos was used. Composition and Atmospheric Concentration of the Dust.--The dusting material was the ball-milled, short fiber asbestos used in the previous inhalation experiment, but unground material was not mixed with it. Owing to the tendency of the material to form small spherules which prevented much of the fibrous portion from floating out of the dusting machine, the dispersal of the dust was not entirely satisfactory. Therefore, after an initial seven months of operation, steel wire brushes were attached to the inside surface of the hopper and to the rotating paddle to disintegrate the spherules and release the fibers. This arrangement gave satis factory results and was used for the remaining 21 months of the experiment. The composition of the raw asbestos used is shown in tables 2 and 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 the atmospheric dust was somewhat lower than 15 per cent, but reliable values were not obtained. The dust concentration during the first seven months of the experiment was about 100 million particles per cubic foot of air. After the wire brushes 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 150 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 be classified as clumps or particles; only about 1 to 1.5 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 Guitica 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 of inflammatory cells Table 9.--Summary of Inhalation Experiment with 100 per Cent Ball-Milled Asbestos Dust Nature of Experiment Dust exposure continuous throughout life Dust exposure fol lowed by pro longed residence in normal air Animals 84 guinea pigs 40 rats 24 mice 10 guinea pigs Maximum Maxi- Survival mum After Dust Dust Expo Expo sure, sure, Mo. Mo. 24 0 20 0 12 0 23 12 Results No appreciable pulmonarv reaction No suggestion of asbestosis No suggestion of asbestosis Fibrosis typical of asbestosis was present 12 mo. after exposure ceased in an amount'sufficient to be visible grossly; smaller foci could be seen microscopically at 2 mo. and S mo. after termina tion of exposure could be seen. At 24 months (fig. 6.4) there was still no change large enough to be seen with a hand lens, although microscopic examination revealed cellular accumulations about terminal bronchioles and many more asbestosis bodies, chiefly within cells. The lungs of animals exposed for the full dusting period of 28 months and afterward living in normal air for two months revealed the changes described above and also very slight peribronchiolar fibrosis. For exposed animals living eight months in normal air the findings were similar, but at 12 months three of four animals showed grossly visible characteristic peribronchiolar fibrosis with adenomatoid change (fig. 6 B). The tracheobronchial nodes were essentially normal until exposure had been continued for more than a year and a half. Animals killed at 12 months and at 16 months revealed a few minute collections of phagocytes containing particles but practically no fibers large enough to be recognized as such. After 20 months of exposure many monocytes filled with yellow granules were present. At 30 months there had been a slight increase in reticulum but no fibrosis. No further changes occurred in the nodes. Asbestosis bodies were not seen in the nodes of any of the guinea pigs. Minute asbestosis bodies were observed in the lungs as early as three months after exposure began, but they did not become numerous until 16 months had elapsed. The bodies were short and practically all were intracellular, although at 20 months some were long enough to project beyond the cell borders. It is 20 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE important to note that in the later months of exposure there was a distinct increase in the number of long fibers, up to 70 microns in length, in the lungs with the formation of characteristic long asbestosis bodies. Chemical analyses (table 10) of the lungs revealed that considerable dust had been retained in the lungs. After 24 months of continuous exposure the average Fig. 6.-- Ball-milled asbestos inhalation experiment: A, lung of a guinea pig with 24 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 28 months' dust exposure and then 12 months' inhalation of normal air. The reaction is much like that shown in A, but there is 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 animals exposed 24 months to the short fiber asbestos dust. VORIVALD ET AL.--STUDIES Of ASBESTOSIS 21 In view of the high values for silica obtained with the animals exposed to 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 fiber 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 fibers. Reaction in JVhite 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 to phagocytosis ol inhaled particles by widely scattered dust cells which remained free in air spaces or were transported to the tracheobronchial lymph nodes. Xo asbestosis bodies were found in the rats, but in the mice there were a very lew small, nonliaustrated forms within phagocytes. Table 10.--Analyses of Lungs of Guinea Pigs Exposed to Dust in Inhalation Experiment zvith 100 per Cent Ball-Milled Asbestos Dust Exposure to Dust, .Mo. Period iu Normal Air, Mo. Amt. of Ash, 9c of Dried Lung Total SiOs, 9c of Dried Lung Totul SiOg, 9c of Ash Tissue Reuction Dus t Exposure Continuous During Life ( 4.55 0.21 4.2$ 1 0 { 4..50 0.30 i .05 [ 4.35 0.24 5.60 0 | 4.66 0.23 4.90 2 0 4 4.60 0.34 7.40 l 5.05 0.61 12.01 0 | 5.60 0.70 12.47 3 0 i 3.07 0.32 6.3$ 1 5.74 0.56 9.77 0 | 5.10 0.3$ 7.51 5 0 \ 5.0$ 0.3$ 7.47 [ 5.02 0.39 7.72 0 3 0 4.35 0.52 11.$6 0 12 0 \ 5.6.) ( 6.24 1.25 22.16 1.45 23.2$ 0 i16 0 \ 5.40 5.01 1.02 15.96 1.11 21 95 + o20 0 f 5.65 t 5.20 1.25 22.60 1.2$ 24.61 1 6.30 1.S5 29.05 "4 1 5.56 1.21 21.70 Dust Exposure Followed by Prolonged Resilience in Normal Air 2S 1 7.2-5 " l $.67 1.57 21.63 2.19 25.24 + 2S ) .>.25 ) 5.90 0.6$ 12.99 o.ST 14.55 + 2S | 6.3$ " l 5.17 0.64 15.(f$ 0 .61 12.41 2+ * The symbols averaging the tissue reaction in each group represent merely the relative degree of reaction, ranging from o to (questionable) to 2 -- (the maximum observed in thic experiment). The relationships apply only within this table and cannot be compared with symbols in other tables. Summary and Interpretation of Inhalation Experiment ultli W0 per Cent Ball-Milled Asbestos Dust.--The tissue reaction observed in this experiment was not as intense as that in the previous investigation with short fiber asbestos. The reaction was slower in development and less extensive even though more dust accumulated in the lungs. Since there were fewer fibers longer than 3 microns in the material used in this experiment, the results tend to confirm the interpretation made in the summary of the previous short fiber experiment that the reaction is not primarily chemical in nature, and to support the impression that reduc tion in size of asbestos fillers does not increase the biologic activity of asbestos inhaled into the lung. 22 INDUSTRIAL HYGIENE AXD OCCUPATIONAL MEDICINE The finding of long asbestosis bodies in animals that had inhaled the ball-milled material is an example of the difficulty of completely eliminat ing long fibers from a large volume of asbestos as required for an inhalation experiment. In regard to the progression of the tissue reaction after the animals had been removed from the dust, observed in this experiment but not in the others, the following interpretation is offered: When the reaction is well developed at the termination of exposure, the contraction of the fibrous tissue obscures any progression that may have occurred; in this experiment, however, since the reaction observed was less mature, its subsequent progress was more readily apparent. Loxr, Fiber Asbestos Dust Since inhalation of short fiber and of 100 per cent ball-milled asbestos dust did not result in acceleration of the tissue reaction in comparison with that produced by King's floats, the hypothesis that short fibers of asbestos were of minor importance in the etiology of asbestosis was given added support, and attention was directed to the view that the long fibers were of primary significance in that etiology. The King's floats asbestos used in the first inhalation experiment had a rather low content of fibrous chrvsotile and contained considerable serpentine and other impurities. Therefore, it was decided to conduct a new inhalation experiment with a purer form of chrvsotile which would be richer in long fibers. Composition ami Atmospheric Concentration of the Dust.--The dusting material employed in this investigation was obtained from an asbestos fabricating plant. Samples of several varieties of long fiber asbestos dust were first submitted to the Saranac Laboratory for examination, and one of these, which was low in magnetite and chromite and had a fibrous content estimated to be about 75 per cent, was selected as most suitable. Steel wire brushes, fastened to the inside surface of the hopper and to the rotating paddle as in the preceding inhalation experiment, were used to open up the bundles of asbestos and liberate more fibers into the atmosphere. The 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 chrvsotile and about 20 per cent serpentine; as already noted, the composition of a similar air-floated sample of ball-milled, short fiber dust was 15 per cent chrvsotile 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 light field counts was 32 million particles per cubic foot of air; for 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 the electrostatic precipitator. It will be noted that there was far more fibrous material in the long fiber dust. Guinea pigs, cats, rats and mice were employed in this inhalation experiment. The results, summarized in table 12. are described in greater detail below. VORIVALD ET At.--STUDIES OF ASBESTOSIS 23 Reaction in Guinea Pigs.--The experiment was started with 100 guinea pigs. After exposure had been carried on for a year, a severe epidemic of pneumonia arose in the dust 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 macroscopically at 20 months. From this time on the reaction increased in extent and in the amount of collagen, and by the thirty-fourth month, it had fanned out Table 11.--Size-Frequency of Atmospheric Long Fiber and 100 per Cent BallMilled Asbestos Dust Collected Inside Cages Type of Asbestos Long fiber................ Ball-milled ............... Grains, % Fibers, % <3 Microns 90.6 3-20 Microns 4.S >10 Microns 0.0 0.0 <30 Microns 25.S 0.S >30 Microns 6.7 0.6 Clumps, % 1.0 3.2 Total 100 100 Table 12.--Nummary of Inhalation Experiment with Long Fiber Asbestos Dust Nature of Experiment Dust exposure con* tinuous throughout life Dust exposure followed by pro longed residence in normal air Animals 117 guinea pigs 4 cats 20 rats 20 mice 12 guinea pigs 9 guinea pigs 2 cats Maximum Maxi Survival mum After Dust Dust Expo Expo sure, sure, Mo. Mo. 3<> 0 42 0 25 0 25 0 20 14 o- 9 13 24 Results Definite fibrosis iu 16 mo. Slowly developing fibrosis first seen at 24 mo. Marked peribronchiolar fibrosis first seen at 24 nio. Limited reaction: no fibrosis Clearing of inflammatory reaction aod definite contraction of fibrous tissue C'caring of inflammatory reaction and slight contraction of fthrous tissue Similar to continuous exposure group: suggestion of progression in one of the two animals considerably into the parenchyma (fig. 8 A). The lesions were rather sharply localized and the extensions from different bronchioles showed no tendency to fuse, even in animals exposed for the maximum period of three years. Although the intrapulmonary reaction sometimes reached the pleura, there was no involve ment of that membrane. Emphysema was not detected at any point. Some thicken ing of the larger bronchi with a chronic inflammatory infiltration was revealed, but it was considered no more than would be produced by a similar period of inhalation of any dust. In guinea pigs exposed to the dust for 20 months and then removed to normal air, there was a marked tendency for cellular inflammatory reaction to clear. This effect, accompanied by contraction of the fibrous tissue, resulted in a diminishing size of the focal lesions. None of these animals, killed at various periods up to 14 months after exposure, revealed lesions as large as those in the group killed at the end of the 20 month exposure period or those in animals which remained in the dust room for more than 20 months. Fourteen months after dust exposure ceased, the foci in four of the six remaining guinea pigs were so small that they were visible only with a hand lens (fig. SB). 24 ISDUSTRIAL HYGIESE AND OCCUPATIONAL MEDICINE In the group exposed for 27 months and then transferred to a normal atmosphere the response was quite similar to that in the 20 month exposure animals "mentioned above. Small foci were always visible on gross inspection of sections of all guinea pigs of the 27 month series, but in no instance was there evidence of the reaction. Mg. 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). /->, lung of a guinea pig with 16 months' dust exposure. Again note a bronchiole uith 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 VORWALD ET AL.--STVDIES OE ASBESTOSIS 25 began to appear in the medulla, and by the fourteenth month most of the 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 Fig. 8.--Long fiber asbestos inhalation experiment: A. lung of a guinea pig with 34 months' dust exposure. A bronchiole is seen at the lower center; the large area above it represents the involvement of alveolar walls. Compare with figure 7 B and note the increased extent of reaction f X 200). B, lung of a guinea pig with 20 months' dust exposure and then 14 months' living in normal air. The reaction is essentially like that shown in figure 7 B : The bronchiole at the right center is surrounded by fibrous tissue with adenomatoid 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 ( X 200). new ceils were yellowish from fine pigment granules that stained for iron. Xo fibers or asbestosis bodies were seen. 26 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE Although asbestosis bodies were found in the lung as early as one month after exposure began, they were rare and hard to find. At five months more were visible, chiefly coiled inside giant cells, and at eight months many bodies Table 13.--Analyses of Lungs of Guinea Pigs Exposed to Dust in Inhalation Experiment with Long Fiber Asbestos Dust Exposure to Dust, Mo. Period in Normal Air, Mo. Amt, of Ash, %of Dried Lung Total SiOs, % of Dried Lung Total S1C>2, % of Ash Tissue Reaction * Dust Exposure Continuous During Life I 4.35 0.04 1.10 1 0 { 4.33 0.00 2.11 l 4.37 0.04 0.93 0 f 4.45 0.05 1.23 2 0 4.4S 0.06 1.18 1 4.35 0.06 1.46 + [ 4.33 0.05 1.20 3 0 \ 4.38 0.05 1.13 [ 4.51 0.06 1.48 j 4.77 0.08 1.75 5 0 { 4.03 0.12 2.67 [ 5.08 0.09 1.77 f 4.72 0.10 2.21 3 0 j 4.32 0.09 1.90 [ 4.34 0.07 1.58 *- 1 4.S7 0.25 5.20 12 0 i 5.02 0.20 4.09 1 5.16 0.31 5.99 + 1 2.9s 0.38 12.70 16 0 { 2.83 0.35 12.25 [ 3.16 0.34 10.91 2+ f 3.54 0.43 12.22 20 0 i 3.60 1 3.5S 0.49 13.63 0.52 14.59 2+ 1 3.42 0.35 10.18 24 t 3.52 0.29 S.29 3+ \ 3.40 0.39 11.51 27 ( 3.74 0.49 13.15 3+ I 3.53 0.37 10.55 30 u \ 3.03 [ 3.88 0.34 11.22 0.24 a in 4 + i 3.85 0.50 8.60 34 i 0.70 0.S4 12.47 4+ i 4.10 0.37 9.11 36 i 2.74 0.35 12.80 4+ Dust Exposure Followed by Prolonged Residence in Normal Air f 3.54 0.43 12.22 20 0 { 3.60 0.49 13.63 1 3.58 0.52 14.59 2+ 1 2.92 0.21 7.23 20 ) 2.81 0.27 9.50 2-f- 20 10 1 4.IS l 4.30 0.24 0.22 5.75 5.07 2+ 20 14 \ 5.01 i 5.04 0.21 0.18 4.19 3.58 + \ 3.40 f 3.74 \ 3.56 ) 2.54 0.39 11.51 0.49 13.15 3+ 0.31 S.59 0.18 6.94 2 + j 3.19 0.25 7.99 } 3.IS 0.28 8.72 2+ 9 i 3.21 i 2.75 0.29 0.23 8.96 8.31 2+ * The symbol* averaging the tissue reaction in each group represent merely the relative degree of reaction, ranging from o to -z (questionable) to 4-i- (the maximum for this experi ment). The relationships apply only within this table and cannot he compared with symbol? in other tabic?. 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 VORWALD ET AL.--STUDIES OF ASBESTOSIS 27 pigs 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 asbestosis 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 asbestosis bodies were not formed, although there was .A- ..Y< ft- 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. Roentgenograms of cats made after exposure periods of 25, 33 and 42 months, respectively, 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. Five animals, of which one was exposed for 19 months and four for 25 months, were free from pulmonary infection and offered a basis for tentative conclusions. In the 19 month 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 asbestosis bodies were found in the 19 month animal and none was found in the 25 month animal. Thus these animals exhibited fibrosis without asbestosis bodies or fibrosis accompanied by only a very infrequent asbestosis body. 28 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE Reaction in Mice.--Out of 20 white mice used in this experiment, 11 lived a year or more in dust and died or were killed without showing an appreciable degree of pulmonary infection. The reaction to the inhaled dust was limited to phago cytosis by mononuclear cells. Usually these were widely scattered through the air spaces; a limited number were grouped about the terminal bronchioles, producing some thickening of their walls. There was no suggestion of fibrosis. Numerous asbestosis bodies were observed in animals killed late in the experi ment. Thus these animals exhibited asbestosis bodies without fibrosis. Summary and Interpretation of Inhalation Experiment with Long Fiber Asbestos Dust.--The purpose of this experiment was to evaluate the importance of long fibers in the tissue response to inhaled asbestos. The results, in comparison with those of previous investigations, indicate strongly that long fibers are chiefly responsible for asbestosis. Thus, the reaction in guinea pigs developed earlier and became more extensive in this experiment than in previous experiments in spite of a smaller concentration of atmospheric dust and a lower mineral content of the lungs. Furthermore, typical peribronchiolar fibrosis was produced in cats, although in a previous experiment with short fiber dust peribron chiolar fibrosis did not develop in this species. The cause of the cellular fibrosis in the lymph nodes of the guinea pigs is not clear. It did not occur in other inhalation experiments with asbestos. INJECTION' EXPERIMENTS Since the inhalation experiments reported above strongly suggested that long fibers of asbestos are the significant factor in the causation of asbestosis. a series of injection experiments was inaugurated wherein the dosage and the length of the fibers could be controlled more precisely. Also, bv the use of controlled dosages, the relative capacities of various asbestos minerals to produce reaction could be compared. In these injection experiments, guinea pigs, rabbits, rats and dogs were used, and the mineral dust was injected by the intratracheal, the 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 I.ntratrachf.ai. Technic As the asbestos minerals do not cause typical advanced fibrosis in extrapulmonary tissue, the intratracheal technic is the preferred way of introducing fibrous dust into the experimental animal. In this method the dust suspension is injected bv means of a special needle or catheter deep into the trachea, from which it flows into the lungs. Comparison of Fibrous and Nonfibrous Dusts.--To demonstrate that the ability of asbestos to produce fibrosis resides in its fibrous character, the series of injection experiments reported in table 14 were performed. Table 14.--Comparison of Reactions to Chrysolite and Serpentine Injected Intratracheally Dosage: Each animal was given an intratracheal injection of 0.3 cc. of a 3 per cent suspension of the dust. Two weeks later another similar injection was given. Total amount of dust injected was 50 mg. Animals used: Six groups of 9 guinea pigs each (one group for each type of dust). Periods at which animals were killed: One or two animals in each group at l, 2, 6, months after last injection. and 12 Preparation of dust: Chrysotile (ball milled) unheated: Ball milled for l,176 hr., dried and reground in agate mortar. Chrysotile (ball milled) ignited: Ball milled chrysotile heated for 2 hr. at about 700 C., then ground in agate mortar 2 or 3 min. Chrysotile (fibrous) unheated: Ground in agate mortar to pass 200 mesh. Chrysotile (fibrous) ignited: 200-mesh material heated for 2 hr. at about 700 C. No further grinding. Serpentine (ball milled) unheated: Ball milled for 1.4S8 hr., dried and reground in agate mortar. Serpentine (ball milled) ignited: Ball milled serpentine heated for 2 hr. at about 700 c\, then ground in agate mortar 2 or 3 min. Mineral Chrysotile (ball milled) unheated Chrysotile (ball milled) ignited Chysotile (fibrous) unheated Chrysotile (fibrous) ignited Serpentine (ball milled) unheated Serpentine (ball milled) ignited Size of Dust Particles 3 microns and less 3 microns and less Results Grinding destroyed capacity to cause fibrosis. At 1 mo. considerable inflammatory edema and cellular prolifera tion and localization of dust particles about bronchi oles; at 2 mo., only a very slight proliferative reaction; at G, S% and 12 mo., widely scattered small mononuclear phagocytes. At 12 mo., n few microscopic patches of thin alveolar wall thickening with sonic adenomatoid change in portion of air spaces abutting on thickened bronchi. No asbestosis bodies seen. Reaction limited to large foreigu body giant cells without production of fibrous tissue. 20-30 microns approx. *20-50 microns approx. 3 microns and less 3 microns and less A distinct fibrosis. Reaction localized to connective tissue about terminal bronchioles: little within those tubes. Contraction caused adenomatoid appearance of air spaces given off directly from terminal bronchioles. Reaction area became smaller with progress of time: no new regions involved. No chronic pleurisy even at points abutting intrapulmonary change. At l mo. considerable inflammatory edema and foci of cellular proliferation; at 2 mo. well marked cellular proliferation and fibrosis occurring focally about respiratory bronchioles. This reaction developed before asbestosis bodies had formed and was as udvaneed as that produced by 2 yr. inhala tion of asbestos dust. At G mo., reaction less extensive than at 2 mo., apparently due to contraction of fibrous tissue; asbestosis bodies were abundant. At 5% mo., reaction still less extensive, confined to the immediate vicinity of the small terminal bronchioles, where the scar tissue was quite dense and was becoming hyaline in char acter. Sometimes it even obliterated the bronchiole. Asbestosis bodies had become scarce. At 12 mo., the well developed peribronchial and intrabronchial adeno matoid areas of fibrosis had produced considerable dis tortion. More peripherally were patches of pneumonitis with eosinophilic infiltration, some of which was being transformed into fibrous tissue. These seemed to be pre cursors of the localized, diffuse patches of thin alveolar wall fibrosis seen elsewhere. Reaction limited to large foreign body giant cells without proliferation. Heating the fibers, which made them brittle, destroyed their capacity to produce significant reaction. Dust relatively inactive. At 1 and 2 mo., simple phago cytosis without proliferation: at 6 mo., no change except possibly lymphoid cell infiltration; at SVs mo., a slight chronic pneumonitis: at 12 mo., only a little pneumonitis without suggestion of fibrosis. Dust relatively inactive. Reaction essentially the same as for unheated serpentine. With ignited serpentine, less tendency for dust to be carried to bronchial nodes. 29 T 30 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE 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 (x 200). VORWALD ET AL.--STCDIES 0I: ASBESTOSIS 31 The tests were made with long fiber chrysotile, unheated, and with chrysotile that had been ignited to destroy its flexible structure or ball 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 chrysotile but is nonfibrous. A review of the findings reveals that only the unheated, long fiber chrysotile 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 11). 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. Experi- is shown at the left center. The phagocytic cells exhibit little predilection for the bronchiole and collagen deposition is absent (X 200). mental studies concerning this observation will be reported in a separate publication. Comparison of Various Long Fiber Dusts.--Some very interesting findings are disclosed by the results of the experiments recorded in table 15. First, all the long fiber asbestos minerals tested, with the exception of anthophvllite, produced typical fibrosis. The characteristic peri bronchiolar reaction caused by three representative long fiber asbestos minerals--chrysotile, amosite and crocidolite--is shown in figures 10 A and 12. Why anthophyllite behaved differently from the other asbestos minerals is not entirely clear. Second, with the mineral brucite. which is not a silicate but is a fibrous form of magnesium hydroxide, a characteristic fibrosis like Table 15--Comparison of Reactions to Farwus Long Fiber Dusts Injected Intratracheally Dosage: Two injections of o.5 cc. of a 5 per cent suspension given two weeks apart. Total dose was 50 mg, Am'iuals used: From <i to 9 guinea pigs for each dust. Periods at which animals were killed: Usually at 1, 4, 8 uml 12 months after last injection. Size of dust particles: Separated so that most fibers were from 20 to 50 microns long. Mineral Chrysotile (Thetford; Chrysotile (Arizona: low iron content; U.2<Tc FesOa) Amosite Crocidolite (Bolivia; Crocidolite (S. Africa) Anthophvllit*' Tremolite Brucite GIa= wool Results Distinct fibrosis. Additional information given opposite chrysotile (fibrous) unheated, in table 14. Reaction virtually identical with that to Thetford chrysotile. Both fibrosis and asbestosis bodies produced with an asbestos containing very little irou. Fibrosis occurred as plugs within terminal bronchioles and as finer deposits at periphery. Fibrosis developed before asbestosis bodies were seen and was in cellular state well formed at one month. With age, fibrous tissue contracted and occupied smaller area but was more dense. Adenomatoid changes similar to those with Thetford chrysotile. Pleurisy limited to immediate vicinity of early reaction about areas of massive localization. Asbestosis bodies formed hut were few. At 1 mo. after injection, minute foci of mononuclear proliferation about bronchioles and in areus of atelectasis: at 1% nio., heavy peribronchiolar patches of fibrosis often with papillary projections partially closing lumen of bronchiole; adenomatoid appearance marked; connective tissue reaction showed heavy collagen but no hyalinization: at 2 mo., minute foci of well matured fibrosis about bronchioles; at G mo., mature asbestosis fibrosis with evidence of contraction: considerable chronic pneumonitis with infiltration of lymphocytes and eosinophils. At 9 mo., small intrabronchiolar fibrous plugs with foci of more delicate fibrosis at periphery. Typical fibrous endohronchiolitis and peribronchiolitis with formation of atypical asbestosis bodies. Haustration of bodies began before 4th mo. after injection, well developed by Sth mo. Bodies persist after 12th mo. Reaction at l mo. heavy endohronchiolitis and peribronchiolitis already showing fibrous changes: ateleetnsis and fibrosis with some necrosis at site of massive localization of dust. At 4 mo., heavy, widely scattered endohronchiolitis and peribronchiolitis, now fibrous, with marked defor mity of bronchioles and with an adenomatoid appearance. At 8 and 10% mo., reaction in lung essentially the same ns at 4 mo. At 12 mo., foci of fibrous endohronchiolitis and peribronchiolitis still large, with more dense scar tissue ami more deformity of bronchial tubes but no extension into, or atelectasis of, peripheral parenchyma. Advanced fibrous endohronchiolitis and peribronchiolitis. Beaded asbes tosis bodies noted at 8 mo. At 1 mo., early fibrous endohronchiolitis and peribronchiolitis: many giant colls and some lymphocytic reaction. At 4 mo., small areas of endohronchiolitis scattered throughout the lung: cellular fibrosis. At S and 12 mo., areas of bronchiolitis smaller because of contraction of dense scar tissue; at 12 mo., marked lymphocytic infil tration and adenomatoid appearanee. Typical advanced fibrous endohronchiolitis and peribronchiolitis produced by 0.5^ suspension (l cc. total dose): most animals would not tolerate usual 5% suspension. Fibrosis well developed before asbestosis bodies ceen. At 4 mo., well developed fibrous bronchiolitis with lymphocytes and giant cells and adenomatoid change. At 8 mo., typical bronchiolitis not quite as extensive or as heavily fibrous as with a 5% suspension, other wise the same. Many deeply stained fibers with a good proportion of haustrated asbestosis bodies. At 12 mo., heavy fibrous bronchiolitis, more peribronchiolitis and endohronchiolitis, with lymphocytes and giant cells: very marked adenomatoid appearance. Lymphocytic infiltration and giant cells but no fibrosis. A very few atypical asbestosis hodies. At 1 inn., many scattered foci of intrabronchiolar dust without massive localization: lymphocytic infiltration of walls and a few giant cells. At ? and 12 mo., little evidence of dust: a few bronchioles and bronchi with giant colls in adjacent alveoli and with lymphocytic infiltration of walls. Fibrosis about bronchioles. At 1 mo., area5 of dust localization with col lapse of alveoli and infiltration with acute inflammatory cells, macro phages and giant cells. Within the area were a few foci of fibrous tissue and numerous areas of hypertrophy of alveolar epithelium. Many bron chioles parked with fibers. At 4 mo., general appearance of lesion unchanged: pleura slightly thickened over heavy localizations of dust. An occasional segmented asbestosis body seen. At < mo., many foci of fibers in bronchioles and alveolar ducts with cellular reaction as before: also, some foci showed distinct collagen deposition. At 12 and 18 mo., reaction as before with fibrosis about bronchioles mure apparent because of con traction and decrease of inflammation. Giant cells prominent. Pleura markedly involved. Typical flhrou- endohronchiolitis and peribronchiolitis like reaction to ashesto- minerals. At 1 mo., extensive endohronchiolitis and peribron chiolitis with giant cells: dense fibrous loops within bronchioles and cellu lar flhro=i= about them: adenomatoid change present. At 2 mo., heavy intrabronchiolar and peribronchiolar fihrosis producing marked deformity with distortion of tubes and obliteration of surrounding air spaces: fibrosis pale without hyalinization hut with few nuclei: no necrosis. Typi cal a=be<tosis bodies seen. At 4 and s mo.. little change: fibrous tissue contracting. At 10% mo., dense fibrous bronchiolitis with asbestosis bodies. Xo pleurisy. No extension to surrounding lung. Xo fibrosis within a year. At l mo., no reaction inside bronchioles: in peripheral air spaces clump5 of giant cells packed with fine spicules of glass with lymphocytic infiltration of adjacent walls; no asbestosis bodies. At 2 mo., reaction lo;s intense than at 1 mo.: fair-sized clumps of elon gated giant phagocytes containing spicules and particles of glass: no endohronchitis. At 4 and s mo., reaction still diminishing. At 12 mo., focal area< of pneumonitis with no fihrosi* or endohronchitis; moderate number of smooth iron-staining fibers. UORIVALD ET AL--STUDIES OF ASBESTOSIS 33 ! that produced by the asbestos minerals was obtained (fig. 13 A). Since f 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. si Fig. 12.--Amosite and crocidolite injection experiments: lung of a guinea pig four months after an intratracheal injection of amosite. The inflammatory reaction exhibits pronounced accumulation of cells and collagen deposition { X 200). B, lung of a guinea pig four months after an intratracheal injection of crocidnlite. As in A. peribronchiolar accumulation of cells and deposition of collagen are shown (X 200). Third, no fibrosis resulted from the injection of glass wool fibers (fig. 13/?), even though glass wool resembles asbestos in many wavs. However, there are fundamental differences. A glass wool fiber 3 microns 34 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE in diameter is a solid rod which in short lengths is fairly rigid, 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 capacity of a mineral to produce peribronchiolar Fig. 13.--Brucite and glass wool injection experiments: A, lung of a guinea pig which four months before had received an intratracheal injection of brucite. Even with this nonsiliceous 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 (x 200). VORWALD 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 Intratraeheally Dosage: Two injections of 0.5 cc. of a 5 per cent suspension given two weeks apart. dose was 50 mg. Animals used: Six groups of guinea pigs. Periods at which animals were killed: 1, 2, 6, and 12 months after injection. Total Mineral Ohrysotile (Thetford) Amosite Crocldolite (Bolivia) AnthophyUite Tremolite Brucite Size of Dust Particles Long fiber, 20-50 microns Short fiber, 3 microns and less Long fiber, 20-50 microns Short fiber, 20 microns and less Long fiber, 20-50 microns Short fiber, 20 microns and less Long fiber, 20-50 microns Short fiber, 3 microns and less Long fiber, 20-50 microns Short fiber, 20 microns and less Long fiber, 20-50 microns Short fiber (made by crushing long fibers with rubber police man) Results A distinct fibrosis. Refer to chrysotile (fibrous) un heated in table 14. No fibrosis. Refer to chrysotile (ball milled) unheated in table H. Typical fibrous cndobronchiolitis and peribronchiolitis. Refer to table 15. Reaction limited to phagocytosis with lymphocytic infil tration of adjacent walls. Short fibers packed insi<je swollen phagocytes; longer ones free; some coated to form typical asbestosis bodies. At 1 mo. after injec tion, alveoli contained good-sized giant cells; most phagocytes were within air spaces and had not migrated to walls. At 4 mo., free extracellular fibers had worked themselves into interstitial tissue, where there was extensive proliferation of lymphoid cells and monocytes but no fibrosis. At S mo. foreign body reaction with some pneumonitis, no bronchiolitis. Typical asbestosis bodies present. Advanced fibrous endobronchiolitis and peribronchio litis. Refer to table 15. No fibrosis. At 1 mo., air spaces compressed and largely filled with giant cells packed with dust needles. Walls heavily infiltrated with monocytes and lymph oid cells. At 4 mo., a moderate degree of cellular infiltration of walls; small giant cells packed with dust spicules. At 6 and mo., masses of giant cells, containing mineral particles, in small bronchi but not in respiratory bronchioles; 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 scattered small monocytes packed with dust. No endohronchitis. No peripheral fibrosis. In lymph node, slight reticulosis; no fibrosis. Lymphocytic infiltration and giant cells but no definite fibrosis. Refer to table 15. No fibrosis and practically no asbestosis bodies. At 1 mo., focal collections of dust-filled monocytes and a few giant cells; at 4 ino., some adenomatoid epithelial reaction; at S mo., simple pneumonitis with phago cytosis of short fibers; at 12 mo., isolated and sharply localized collections of dust cells inside air spaces about terminal arterioles. Reaction in walls limited to lymphoid cell infiltration. No fibrosis. In lymph node, reaction limited to slight prominence of reticu lum. Tibrosis about bronchioles. Refer to table 15. Simple foreign body reaction. No acute inflammation. No accumulation of dust in or about terminal bron chioles. No endobronchitis. At 1 mo., scattered small giant cells and considerable infiltration of adjacent walls with monocytes and lymphoid cells. At 4 mo., little change except more cellular infiltration of con nective tissue. At 3 mo., lymphoid infiltration and thickening of wall? about some but not all terminal bronchioles. Typical fibrous endobronchiolitis and peribronchiolitis like reaction to asbestos minerals. Refer to table 15. Inert type of reaction. At 1 mo. after injection, small monocytes widely scattered through air spaces; focus of atelectasis with lymphoid infiltration of compressed air-space walls. No endobronchial reaction as with chrysotile. At 2 mo., reaction similar to that at l mo.; typical asbestosis bodies seen. At 12 mo., small clumps of inactive dust-filled phagocytes; no fibrosis. No reaction in lymph nodes. Comparison of Long Fiber and Short Fiber Dusts.--With quartz dust it has been demonstrated that the smaller the particles the more 36 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE intense is the tissue reaction and that particles larger than 3 microns in diameter cause little reaction. In the case of asbestos, however, the reverse is true and apparently only long fibers have any specific effect, as was suggested by the inhalation experiments. This is confirmed by the data of table 16, in which a series of tests with fibrous minerals is reported. When the injected dust consisted of fibers 20 to 50 microns long, all the fibrous minerals tested except anthophyllite, as noted in the preceding section, produced fibrosis; when the material was prepared by first grinding the fibrous dust until the length of fibers was reduced to 20 microns and less or. in some cases, to 3 microns and less, none of the injected dusts caused fibrosis. These results differ from those of King, Clegg and Rae,11 who reported the production of reticulosis comparable to the experimental silicotic nodule in rabbits receiving monthly intratracheal injections of 100 mg. of Rhodesian asbestos fibers, 15 microns long, and the produc tion of diffuse interstitial fibrosis in rabbits receiving similar injections of short fibers, 2.5 microns in length. We believe this dose, especially in the long term rabbits, is highly excessive. In our experiments the dosage was kept low in order to minimize untoward reactions which might obscure the peribronchiolar type of fibrosis which characterizes early human asbestosis. Experiments Using Intravenous Technic The experiments summarized in table 17, in which the intravenous method of injection was employed, show that the asbestos minerals are far different from quartz in their action on tissue. It has been repeatedly demonstrated that intravenous injection of quartz particles 3 microns and less in diameter will cause a typical tissue reaction with the development of hyalinized fibrotic lesions in extrapulmonary sites, such as the liver and the spleen. Asbestos minerals, however, on intravenous injection generally produce only an inert type of reaction, as is revealed bv 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, vol. 11 (Feb.), no. 234. Dosage: 'I'o tiil a m o u n t o f d u s t was 1.0 G in., divided In to 20 equal doses (each dose was S cc. o f u 1 per cent suspension) which were given tw ice u week fo r 10 weeks. 37 VORWALD ET AL.--STUDIES OF ASBESTOSIS 39 Protective Action of Aluminum Compounds When colloidal aluminum hydroxide had been added to a suspension of long fiber chrvsotile prior to injecting this suspension 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 11; in their experiments metallic aluminum was used instead of the hydroxide. Formation of Asbestosis Bodif.s 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.12 ,:.sf 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 be seen in the guinea pigs for at least a year after injection. This experiment shows that the asbestosis body has a 3 8 rather resistant coating which is not destroyed by moderate hypochlorite treatment, which may be maintained in vivo for a vear 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 1934.13 THEORY OF IRRITANT ACTION Two hypotheses have been proposed to explain the tissue irritation and reaction caused by asbestos fibers: the chemical and the mechanical. In the chemical theory, which is based on experience with quartz, it is assumed that the asbestos minerals dissolve in the body fluids and that in this process their bases are leached away to leave silica in a form capable of irritating tissues. According to this hypothesis asbestosis is merely an indirect silicosis. Several facts make the chemical theory untenable: Intratracheal injection of brucite fibers, which had a silica d'amiante," Laval med. 8:239, 1943. 13. Beintker, E.: fiber die Asbestosiskorperchen: Bemerkungen zu der Arbeit von Beger, Virchows Arch. f. path. Anat. 293:527, 1934. 40 IXni'STRlAL HYGIEXE AXD OCCUPATIOXAL MEDICINE content of only 0.90 per cent, caused typical fibrosis like that produced bv the asbestos minerals; free silica particles increase in potency as the particle size becomes less, but asbestos fibers shorter than about 10 to 20 microns are relatively innocuous; aluminum hydroxide neutralizes the irritating effect of quartz but not of asbestos; serpentine has the same chemical composition as long fiber chrysotile, but it produced only an inert type of tissue reaction; there is a wide range in the chemical composition of the minerals which do cause asbestosis (table 19). In view of this evidence it seems more likely that asbestosis is caused by an unusual mechanical irritation due to long asbestos fibers, this irritation being related to the peculiar filamented structure of the fiber and the associated flexibility, which are possessed by no other foreign body studied. Thus, ignition of chrysotile fibers changed their structure and made them inert, although the same fibers, before being heated, would have produced fibrosis (table 14). Further support for the theory of mechanical irritation is that asbestosis occurs in an organ of high mobility --the lung--and that a fibrous reaction can be produced by injecting < | Table 19.--Analyses of Fibrous Minerals Fibrous Minerals Amosite......................... Amphibole.................... Anthophyllite.............. Brueite........................... Chrysotile.................... Croeidollte................... Tremolite...................... SiOj 46.23 o 3.04 39.SO 0.90 3S.56 34.99 50.20 Ft? 2 O.t 4.06 3.06 0.57 6.78 2.33 16.27 7.21 KeO a 33.53 9.36 4.29 AlsOs 1.09 1.09 0.32 0.46 0.34 1.01 0.56 CaO % 2.01 2.22 0.44 0.04 0.03 0.80 4.44 MrO % 6.28 23.29 33.37 33.99 38.95 12.2> 20.52 N'asO % 0.33 0,43 0.52 0.91 0.29 6.92 6.73 KaO % 0.20 0.12 0.15 0.16 O.OS 0.57 0.99 Ignition Loss < 105 C. > 105 C. %% 0.66 6.28 0.32 3.00 0.27 4.08 0.53 26.66 4.30 14.99 0.02 2.56 0.34 2.78 Total % 99.97 99.77 99.52 99.79 99.89 99.63 99.S2 asbestos fibers into the peritoneum W here there is also a degree of mobility, but not by injecting them into other extrapulmonary organs such as the liver, the spleen and subcutaneous tissue. 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. Susceptibility to Tuberculous Infection 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 OF ASBESTOSIS 41 dust like quartz but above an inert dust such as iron oxide. In animals infected with attenuated tubercle bacilli, quartz causes the infectious process to progress until the animal dies of tuberculosis. Inert dusts have no effect on the infection, and the lesions usually heal and the disease disappears. 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. Susceptibility to Nontuberculous Inff.ctiox There was no specific experiment concerning the effect of inhaled asbestos dust on nontuberculous 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 nontuberculous pulmonary infection. COMMENT AND SUMMARY Owing to the vast amount of data included in this investigation, it seems most convenient to summarize and to state as concisely as possible the various observations which emerged from the experiments and to follow each with a brief resume of the evidence. A. Various species of animals, including the guinea pig. the rat and the rabbit, but not the mouse and the dog, develop peribronchiolar fibrosis of the lung similar to human asbestosis after being exposed bv inhalation or intratracheal injection to long chrvsotile asbestos fibers. Both inhalation and injection experiments provide ample support for this statement. Figure 8 A reveals the cellular fibrosis that occurs in guinea pigs following inhalation of long fiber asbestos; figure 9 shows the fibrosis caused in the cat by inhalation of long fiber asbestos dust. Similar but less extensive fibrosis occurred also in rats and rabbits (table 1). Mice and dogs failed to respond. This variation in response of different species to identical dust exposures is still to be accounted for. B. Long asbestos fibers are essential in the production of the peribron chiolar fibrosis; short fibers are incapable of producing this reaction. 42 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE Inhalation experiments with asbestos dust suggest, and intra tracheal injection experiments confirm, that peribronchiolar fibrosis is produced by asbestos fibers between 20 and 50 microns in length but not by particles shorter than 20 microns (tables 16 and 18). This indicates that the minimum length of fiber possessing the capacity to produce the typical peribronchiolar fibrosis in animals is somewhere between 20 and 50 microns. Pointed studies have not been carried out to determine the upper limit of effective fiber length. It appears, however, that that limit will be determined by the inhalability of the fiber. C. The mode of action of the long asbestos fiber in the production of asbestosis is primarily mechanical rather than chemical in nature. The evidence for this conclusion has been reviewed in a preceding section, page 39. The flexible filamented structure of asbestos fibers plays an essential part in the irritating action, since the solid, inflexible fibers of glass wool do not produce fibrosis (fig. 13 B). D. Typical experimental asbestosis was produced by the inhalation of an atmospheric suspension 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 fibers longer than 10 microns. In the inhalation experiment with 100 per cent ball-milled asbestos dust containing 0.6 per cent of fibers longer than 10 microns (table 11) typical fibrosis was obtained (table 9). The evidence presented shows at least that an atmospheric concentration of asbestos dust containing less than 1 million (0.6 per cent X 138 million) fibers longer than 10 microns per cubic foot of air is capable of producing experimental asbestosis in guinea pigs. The actual lower limit of concentration of long fibers necessary to produce asbestosis in animals cannot be estab lished from these studies. E. The duration of exposure required to develop the pulmonary reaction to inhaled asbestos dust is inversely proportional to the concentration of long fibers in the atmosphere; as the concentration is increased, the reaction develops in shorter time. The basis for this statement appears in the data of the inhalation experiment with long 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, bv 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 VORWALD ET AL.--STUDIES OF ASBESTOSES 43 lesions, due to contraction of the fibrous tissue. In contrast, an imma ture tissue response, evidenced primarily by cells with little or no fibrosis, continued to progress. It is assumed that, following attainment of fibrotic maturity, the same process of contraction would ensue as was noted for the mature lesion. G. The formation of asbestosis bodies represents a coating of the fibers 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 chrysotile asbestos prior to intratracheal injection did not retard or prevent the development of asbestosis in rats. I. Inhalation of asbestos dust did not alter significantly the final outcome of experimental tuberculosis in two series of guinea pigs exposed to the dust. The apparently mild influence of asbestos dust is in distinct contrast to the stimulating effect exerted by inhaled quartz on a tuberculous process in the lung. The interpretation must remain tentative, however, since it is based on an investigation limited to two series of guinea pigs exposed to only one kind of asbestos, namely. King's floats: Table 4 shows that when the infection was coincidental with the onset of dust exposure, there was temporary progression of the infectious process, with subsequent healing; when infection was initiated after 26 months of dust exposure, the course of the tuberculosis was not appreciably altered. The latter finding is quite different from our usual experience with quartz dust or with mixed dusts containing quartz, wherein the adverse influence of quartz on a tuberculous infection is manifested most strikingly when infection is initiated after a period of dust exposure, viz., superimposed on a background of established silicosis. As indicated above, this more sensitive test, when applied to asbestos dust, failed to demonstrate that the latter had an adverse influence on a tuberculous infection. The inability of asbestos dust in that experiment to affect unfavorably the tuberculous process furnishes strong support for the interpretation that inhaled asbestos dust has no more than a mildly unfavorable effect on pulmonary tuberculosis. This investigation was made possible by the generous financial support of a group of companies of the asbestos industry.