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FILE NAME: Metropolitan Life (ML) DATE: 1951 Jan DOC#: ML254 DOCUMENT DESCRIPTION: Experimental Studies of Asbestosis - Saranac Lab Article - Archives of Industrial Hygiene & Occupational Medicine A. M. A > * /> * A R C H I V E S OF and Occupational Medicine ED ITO R IA L BOARD PHILIP DRINKER. Chief Editor 53 Shattuek Street, Boston 15 THEODORE F. HATCH, Pittsburgh FENN E. POOLE, Glendale, Calif. ROBERT A. KEHOE. Cincinnati FRANK PRIN'CI. Cincinnati FRANK A. PATTY, Detroit WILLIAM A. SAWYER, Rochester, N, JAMES 1C STERNER, Rochester. N. Y. RICHARD J. PLUNKETT, M.D., Chicago, Managing E ditor JANUARY 1951 VOLUME 3 NUMBER 1 a PLAINTIFFS ; EXHIBIT Irchhres Volume 3 A- . of Industrial Hygiene end Occupational JANUARY 1951 COPYBIGHT, 195t, BT THB AM EBICA* MEDICAL ASSOCIATION Medicine Number 1 EXPERIMENTAL STUDIES OF ASBESTOSIS ARTHUR J. VORWALD, Ph.D.fPatli.), M.D. THOMAS M. DURKAN AMO PHILIP C. PRATT, M.D. SARANAC LAKE, N. Y. 3BESTO SIS is a form of pneumonoconiosis resulting from protonged inhalation of asbestos dust. The name "asbestos," literally "unburnable," is not that of a specific mineral but is a term applied to a number of different minerals whose characteristic feature is a structure composed of long, parallel, flexible fibers. This structure is unique because the fibers are capable of repeated longitudinal subdivision to units of molecular proportions. In length the fibers vary from a few microns to 6 or more inches (15 or more cm .). Some varieties are 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 to the serpentine and the. amphibole groups. Listed below are the more common*varieties. - . . . . r ; -.^ ' A m phibole g roup: actinolite, am osite, am phibole, anthophyllite. crocidolite and tremolite. .S Serpentine group : chrysotile. . The bulk of the asbestos of commerce is chrysotile, 3M g0.2S i0j.2H.O, which is mined on this continent principally in the Thetford region of the Province of Quebec, Canada, and in Vermont. Crocidolite and amosite also are used commercially 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 blade 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 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE Attention is directed to the mineral brucite, M gO .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'^ 1'^ ; 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, jrw , , ' * - * _ - ^c *r ^ , . 7 . ; A .-- - 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. f Although in man asbestosis is a chronic disease with diffuse pulmo nary fibrosis which requires years to develop, it is possible to reproduce 1. (a) Gardner, L U., and Cummings, D. E .: Studies on Experimental Pneu- mokoniosis: VI. Inhalation of Asbestos Dust; Its Effect upon Prim ary Tuberculous Iaieetion, J. Indust. Hyg. 13 65 and 97, 1931. (6) Gardner, L. U .: Chrysotile Asbestos as an Indicator of Subtile Differences in Animal Tissues, Am. Rev. Tuberc. 45:762, 1942. w r < ; !* * VORWALD ET AL.--STUDIES OF ASBESTOSIS 3 in one or more species of animal characteristic tissue changes which are similar to the lesions of human asbestosis (fig. 1). Since the life span of the experimental animal is relatively short, it is not possible to produce the characteristic lesions in animals under conditions identical with the usual industrial environment. Consequently, to obtain a complete evalu ation of the tissue response to inhaled paniculate 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 diflFerent. the information yielded by animal experiments is invaluable in furnishing a better understanding of the reaction of the human organism to inhaled asbestos dust. E xperimental 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, 8 ft. (2.5 M .) in dimension, in which a cloud of asbestos dust is maintained by 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 a s 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 Jjy 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 they make certain that contact occurs between the dust particles and tissues and because they allow accurate estimation of the dosage and of the potential capacity of that dose to produce reaction. The intratracheal method is particularly valuable when one is dealing with fibrous minerals like asbestos, since it permits observation of the effect of the fibers on pulmonary tissue. T issue S usceptibility 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.1" 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 * 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 *; 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. j .( P ecvlia* C hajuctdustics o r A sbestos Experience has demonstrated that most of the nonfibrous dust particles inhaled into the lungs of man and animal are 10 microns or less T able 1.-- Reaction to Long Fiber Chrysotile in Lungs of Man and Other Species of Animal Spade Men......... .......... Golpei pi*............ B a b b l e . ...... Cat......... ............ White ra t-........... White mouse........ Do*...................... , Mode ot Sxpoaor* Inhalation * Ini)elation end Injection Inhalation and lnjeetkm Inhalation and injection Inhalation and Injection Inhalation Injection FtbroiU * 4+ .. 1+ + + >*. +.. 0 0 Aabaatoila Bodies. Ntumrona Modsately numerous B an and atypical_ B an and a typical Terr ra n B an and atypical Kon' *Tbe symbols 0 to 4 + refer to the decree ol tisane reaction. -- - " , . j 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 rarely 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.234 Their 2. Schuster, N. 6 . : Pulmonary Asbestos in a Dog, J. Path. & Bact. S4 (pt. 2 ): 751, 1931. 3. Vorwald, A. J . : Variations in Individual Susceptibility to Industrial Dusts Inhaled into the Lungs, Am. Rev. Tuberc. 62: (IB ) 13, 1950. 4. Miller, W. S .: The Lung, Springfield, I1L, Charles C Thomas, Publisher, 1937. ........................... VORIVALD ET AL.--STUDIES OF ASBESTOSIS s own essential lining is a low cuboidal type of epithelium but, as their name implies, they actually function in respiration through lateral alveoli distributed along their walls. Either these alveoli or the abrupt change in the character of the lining epithelium, or the small diameter of the respiratory bronchiole, or the combination of all three factors is responsible for retention of the fiber at this site. Only after 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 or T issue Reaction to A sbestos F ibers 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. A sbestosis Bodies The peculiar structure known as the asbestosis body or "curious body" is a specific concomitant of asbestosis.56 : 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.* Gloyne4,1 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; (6) The Asbeitosis Body, Lancet 1:1351, 1932. (c) Gard ner and Cummings.1* 6. Lynch, K. M,, and Smith, W . A .: Asbestosis Bodies in Sputum and Lung, J. A. M. A. 95:659 (Aug. 30) 1930. Simson, F. W., 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.1* Gardner.1*1 Gloyne.5*. *> 6 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE of contact with th?"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' (X 400). 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. VORWALD ET AL.-STUDIES OF ASBESTOSIS 7 ." INHALATION EXPERIMENTS ' ..... '** ' . " . ' ----Four large scale inhalation experiments have been conducted in this laboratory with various forms of asbestos dust. In each of these investi gations, more than 160 animals were used, and the experiments were carried on for periods ranging from two to more than five years. The four kinds of asbestos dust employed are designated as King's floats, short fiber, 100 per cent ball-milled, and long fiber asbestos dust. - K in g ' s F loats Asbestos D u st The first inhalation experiment conducted at the Saranac Laboratory with asbestos dust was begun in 1928. Animals inhaled the dust for T able 2.-- Chemical Analysis of Asbestos Dusting Materials Trpe of Asbestos rtu i tion SIOs FesOs AJtOt CtsO Mao c*o Mro Ne0 KiO co Loi* Total Elat's fio tti.......... . Short fiber............. Loot fiber............. BM 047 ISM 1.74 57.13 9J00 LM 0.14 0.00 046 3&9S 0.14 OJO ose 14.05 100.11 SJSt 0.78 04S OSL BUS 0.0 0j06 047 14.00 55.7 Not determined. Table 3.--Ptrographie Analysis of Asbestos Dusting Materials Dor** floete *: Tbe approximate composition. bated on pertidee (except ehrreotlle) smaller tbin to micron* u d reported te percenter obtained from particle eounte, wee ebrreotUe it. serpentine to, marnetlte lx, cerbonate* U, tale it, other mineral* 4. Por ehtreotUe, fiber* np to too micron* lone were Included. _ . , ... _/ Short fiber t: Tbe materiaL before beior bell m lW , contained a prtpondcraneo of fibrous ehrreotlle-and p la tr (nonfibrou*) terpentine. The approximate composition, b r percentace, wee cbrreotD* IT, cerpentln* 55, marnetlte io, Quarts t, brodte i , other mineral*, Indndlnc dolomite. ectlnoUU and tnm olita, U. ... . .. Lon* fiber t: Tbe material emulated prindpafir of tbe fibron* ubeeto* mineral ebm otfie. Shred* of nonieparated fiber* 5 to IS micron* In diameter and np to 50 micron* in lenftb wen present. Tbe approximate composition, b r peneataf*, oaa ebrysotlle 75, terpentine 15, m ar netlte 5, brodte x, other minerals, amour whfcfi were ealeite and ehloritle and micaceous minerals, 3. Onlr a trace of Quarts we* observed. The analrils of tb* O ne's floats asbestos, mad* b r Dr. C. S. Hurlbut I t., of Harvard Colversltr. has been reported elsewhere (Hnrlbot, C. 8., Jr., and William*. C. R.: Tbe Mineralorr of Asbestos Doit, J. Indust. Hr*. A ToxieoL 17:9!. 1535). 1 Eor tbe abort fiber asbestos and tbe loo* fiber asbestos the petrorraphlc analrils was supplemented with x-rar 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 Y\ 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 sixe. It was obtained from the Thetford, Quebec, plant of the Asbestos Corporation of America, and analyses (tables 2 and 3) reveal that the am ount of fibrous chrysotile was only 14 per cent, a rather low value. 8 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE Impinger samples taken sewn after the experiment was started indicated that the dost 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 537 million 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 studies* 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. . T able 4.-- Summary oj Inhalation Experiment with King's Floats Asbestos Dust N atan ot Experiment -l ; . Animals Dust expora* continuous thronst, - S4 guinea pigs out Ills - 9 rabbits IS rats - Dust espoaon followed by prolonced residence In normal air ' - `VU V ts guinea pigs SS guinea pigs 1 rabbit 1 rabbit Tuboeulous Infection a t sta rt o t ' 40 guinea pigs dust expocun Controls to tnlectton: no dust ex 29 guinea plgi posure - ....... Tuberculous Infection * after B mo. of dust expocun, then recideste In normal air Controls to tnlectton: no dust ex posure 12 guinea pigs 12 guinea pigs Maximum Maxi- Survival mum After Dust Ex-. Dust Expotar. pofur*,' Mo. Mo. . ii ^L.C . ... Basalts " --- SS IP 0 Typical-peribronchiolar fibrosis after l< month 0 Foreign body bronchitis 0 little or no reaction 9 SS NooproneetlTt fibrosis 9 St Konptogrseslei fibrosis 9 19 sMo .1I Absorption of lo rd so body reaction - 39 0 Temporary progression of*lafeetlon, followed by bcallnc with fibrosis: 0 SS 1 Beallnc by resolution (one exception) 39 14 No appreciable increese In susceptibility to tuberculous infection: healing with fibrosis 0 t t f Healing by resolution j*1 * The guinea pigs wen Infected with low virulence Ki strain of tubercle badDug. 1 This means the 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 Normoi Guinea Pigs.--Guinea pigs inhaling this dust for periods up to 33 months had a characteristic fibrosis occurring in conicat 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 w ere seldom detected in the lymph nodes. ; After exposure of approximately a year a small amount of cellular reaction had been produced about many respiratory bronchioles (fig. 3 A ) . As more dust was inhaled, it continued to accumulate in the same location, and later stages of the disease (fig. 3 B ) consisted of extensions of the original lesions. Apparently, (the inhaled fibers were caught in the pocket-like alveoli that are given off from the lateral walls of the respiratory bronchioles. There they 7. Fulton, W. B.; Houtz, R. L .; Dooley, A., and Mathews, J. L .: Asbestosis: L The Collection and Counting of Asbestos Dust Encountered in Asbestos Fabri cating Plants, Special Bulletin 37, Pennsylvania Department of Labor and Industry, Harrisburg, 1934. VORWALD ET AL.--STUDIES OF ASBESTOSIS 9 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. I t includes a respirator/ bronchiole, at the left, branching and becoming an alveolar duct, a t the right. f'iNote 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. .T h e field includes a bronchiole, at the center, with peribronchial fibrosis extending; into the walls of adjacent alveoli. Note the cuboidat 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 cuboidat cells. The result was an adenoma-like appearance which frequently accompanies VORWALD ET AL.--STUDIES OF ASBESTOSIS 21 In view of tbe 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 While 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 of inhaled particles by widely scattered dust cells which remained free in air spaces or were transported to the tracheobronchial lymph nodes. No asbestosis bodies were found in the rats, but in the mice there were a very few small, nonhaustrated forms within phagocytes. T able 10.--Analyses of Lungs of Guinea Pigs Exposed to Dust in Inhalation Experiment with 100 per Cent Ball-Milled Asbestos Dust Exposure to Dust, Mo. 1 t 3 s 8 IS 20 24 Period In form al Air, Mo. Amt. of Asb. % of Dried Lune Total SIOj, % of Dried Lune Total SlOt, % of Asb Dust Exposure Continuous Durloc Life 4.55 . 0.21 4.25 0 4.30 0.30 7.05 4.35 . 0.24 5.00 4.86 0.33 4JO 4.00 0.34 T. .0S 0.l 1101 z 5.80 0.70 1147 0 S.0I 0.32 5.38 5.74 0 M 0.77 5.10 0JS 7.51 0 5.05 OM 7.47 , 5.0 . 0J9 7.71 0 txs 0J2 U.88 0 s.ss 6.2 L2S .1 1.45 132 5.40 IjU ' ` 18.B6 5.01 l.U SLB5 A 1.26 22.00 ' . 3-2D LSS 24.il ;9 0.30 ' L55 5.5 1JQ ' 20.06 21.70 Dust Exposure Followed by Frolonced Bealdenee In Normal Air i 7.25 : .7 1 sxs SJ* ISt . 21,63 2J 25.24 0.55 - 112 0.87 14.55 5.38 .I* ) 5.17 0J4 110 044 12.41 Ttsiue Reaction 0 0 0 0 0 . 0 . 2+ The symbols Trselos tbe tissue reaction lo eseb croup represent merely the relative decree of reaction, rsoctnc from 0 to * (questionable) to S+ (tbe maximum observed la this experiment). Tbe relationships apply only within this table and eannot be compared with symbols In other tables. . , - . . ^ : - . v , . ". - ' I : Summary and Interpretation of Inhalation Experiment with 100 per Cent Ball-Milled Asbestos Dust.--The tissue reaction observed in this experiment was not as intense as that in the previous investigation with short fiber asbestos. The reaction 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 fibers does not increase the biologic activity of asbestos inhaled into the lung. 22 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE The finding of long asbestosis bodies in animal* 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, tlie contraction of th fibrous tissue obscures any progression that inay have occurred ; in this experiment, however, since the reaction observed was less mature, its subsequent progress was more readily apparent^*-* \ - . *4- 'V; t .- r .' v I Lone F ibe* 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 rather low content of fibrous chrysotile and contained considerable serpentine and other impurities. Therefore, it was decided to conduct a new inhalation experiment with a purer form of chrysotile which would be richer in long fibers. " < Composition and 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 dost 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 totaling 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 tong fiber dust was chrysotile 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 chrysotile 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 a ir : 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 d u st . . . ______ Guinea pigs, cats, rats and mice were employed m this inhalation experiment The results, summarized in table 12, are described in greater detail below.: VORWALD ET A L --STUDIES OF ASBESTOSIS 23 Reaction m 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 mooth (fig. 7 B ) definite fibrosis was present in these areas as well as around the bronchioles. The fibrous lesion could be seen raacroscopically 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.--Sist-Frequency of Atmospheric Long Fiber and 100 per Cent BallMilled Asbestos Dust Collected Inside Cages Type ol Asbestos Loot fiber.............. BaU-mllled ................ Orslns, % Fibers, % ,------------*------------ ./--------*--------> < 3 3-10 > 10 <10 > 10 Clumps, Microns Microns Microns Microns Microns % . 1.1 0.0 SJ 0.T 1.0 SO.O SB 0.0 0B 04 11 Total 100 100 T able 12.--Summary of Inhalation Experiment with Long Fiber Asbestos Dust j nature of Experiment Dust exporare eon* tlnnous thfootbout Ilfs '-- Dust exposure tollowed by prolooted residence In oonnel elr Anima la lit tuluee pits Scute . S rtU SOmice IS (ulnee p in */ * -. tobies p in lest* .......... -- ' . . . . Maxi mum Duet Expo sure, Mo. . 3 SI 13 . 13 10 IT IS Maximum Survival . After Duet XXpo' eure, Mo. 0 0 0 0 IS IS Results Definite fibrosis In It mo. Slowly deeetoplnt fibrosis first teen a t is mo. Marked peribronchiolar fibroots first seen at IS mo. Limited reaction: no fibrosis Clearint of Inflammatory reaction and definite eootraetloa of fibrous tissue C'earlnt of tnfiammatory reaction and aUtbt contraction of fibrous tissue Similar to continuous exposure troop; tuttestiou of procreation In one of tbe 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 a t 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 dear. 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 lust 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. S B ) . 24 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE In the group exposed lor 27 months and then transferred to a normal atmosphere the response was quite similar to that in the 20 month exposure animats 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. Fig. 7.--Long fiber asbestos inhalation experiment: A , lung of a guinea pig with eight months' dust exposure. The bronchiole at the center already shows an accumulation of phagocytic cells, and there is a slight deposition of collagen. Com pare with figure 6 A , showing the reaction to ball-milled asbestos after 24 months ( x 200). ' B, lung of a guinea pig with 16 months' dust exposure. Again note a bronchiole with its surrounding reaction, consisting of fibrosis and adenomatoid change. Col lagen deposition is now seen in the walls of adjacent alveoli, at the right ( x 200). In the tracheobronchial lymph nodes reaction was first visible at the third, month of exposure. By the eighth month patches of cellular connective tissue- V Q R W A L D E T AL.--ST U D IE S OF A S B E S T O S IS 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 Tower center; the large area above it represents the involvement of alveolar walls. .Compare with figure 7 B and note the increased extent of reaction (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 cells were yellowish from fine pigment granules that stained for iron. No 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 fire months more were visible, chiefly coiled inside giant cells, and at eight months many bodies T able 13.--Analyses of Lungs of Guinea Pigs Exposed to Dust in Inhalation Experiment tvith Long Fiber Asbestos Dust Exposure to Don, Mo. Period In Normal Air, Mo. A nt oi Ash, % o t Dried hung " Total ' 8IOs, %o! Total 610s, Dried Lung -. % ot Aah . Tissue Beactlon * ' Dost Exposure Continuous During Die \ ' 4.55 0.04 L10 1 0 4 AS 0.00 2.11 -- * 447 0.04 * 043 0 s 4.45 - -.1 1 -, 045 V * . ' A '-' 4.48 - XT*- 046-* UJ U8 - i.. ' . - .' ' . r . * - .*?. 4JI : 4 M . . . 0.06 ' 0.06 * * vrv ' - IJO . - 1 . 0 . ` a 448 4-51 046 L it 0.06 v< 1.48 - - dc- ' 4.77 0.06 1.76 1 0 4.08 0.12 2.67 , - - 6.00 0.00 1.77 <jr s 0 It s ' M ' " * 14 V 0 * * 'o * 0 fj : .t 0 10 0 - . -- 4.71 4.9t 4.84 . 447- ' " 641 846 248 243 3.58 344 3.80 348 3.42 ' 3.S2 3.40 3.74 343 3.08 348 0.10 0.00 - * 0.07 048.' 040 * 041 . / 048 . 0.38 044.. 0.43 0.40 042 048 0.20 0.80 0.40 047 0.31 044 241 .. . 140 146 + 540 4.00 840 12.70 12.28 1041 _ 2+ 1242 13.63 1440 + 10.18 840 3+ 1141 13.16 3+ 1046 1142 6.10 ; ' 4+ . I 5.86 040. 840 - U \ 0 ( 8.70 044 _ 12.47 ' . + / n 4.10 0.37 o.ii'. / M 0 t 2.74 ' 046 V 1240 ^ 4 + Dust Exporare Follosrcd by Prolonged Residence In Normal Atr 3.S4 0.41 1242' 0 3.80 0.40 13.83 348 .. 042 1440 . 2+ 20 4 2.02 241 0.21 0.27 743 ' 0.80 2+ 10 10 4.18 4.30 0.24 042 5.78 5.07 2+ 20 14 5.01 5.04 0.21 0.18 4.10 348 + tl 0 3.40 3.74 0.30 1141 0.40 13.18 8+ n s 3.88 244 041 0.13 . 8.80 844 2+ 17 7 3.10 348 0.28 048 7.00 8.72 2+ V ? ! 23..7H8 0.20 043 8.08 841 3+ * Tbe symbol averaging the tissue reaction In eseb group represent merely tbe relstlee decree ot reaction, ranging from 0 to i (Questionable) to 4-*- (the maximum lor tbls experi ment). Tbe relatfoneblps apply only within tbls table and cannot be compared with symbols In otber tables. " were free in connective tissue. They became fairly abundant as exposure con tinued, although in some later animals the asbestosis bodies were only moderately numerous. It is important to note from analyses of the lungs (table 13) that even though the tissue response at any given period of time was much greater in the guinea VORWALD ET AL.--STUDIES OF ASBESTOSIS 27 pigs of this experiment then id those exposed to either short fiber or ball-milled asbestos, the amount of mineral m atter in the lung ash was much less. ' Reaction in Cats.--Four cats inhaled the tong 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. A t that time there were no typical asbestosis bodies, but smooth, pointed, yellow fibers were seen very rarely. W ith 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 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). ... -- * * i 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. F ire 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 experim ent 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'th'e 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, by the use'-o/ 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 n 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. E x pe r im e n t s U sin o I ntra tra ch ea e T e c h n ic As the asbestos minerals do not cause typical advanced fibrosis in extrapulmonary tissue, the intratracheal technic is the preferred way of introducing fibrous dust into the experimental animal. In this method the dust suspension is injected by means of a special needle or catheter deep into the trachea, from which it flows into the lungs. Comparison 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. Tabce 14.-- Comparison of Rtactions to Chrysotils and Serpentins Injected , __ Intratraeheaiy Dosigv: Each animal > u given an Intratracheal Injection ot 0.5 ee. o( a J per cent nupenelon ot tha duat. Two weeks later another similar Injection was (Iren. Total amount ot dost Injected waa <0 me- animate used: Six (roups ot tulnea piss each (one croup tor each type ot dust). Periods a t which animals wars killed: One or two animals In each croup a t 1, X 8, CM and 12 months attar last Injection. Preparation ot dost: ChryeotOe (ball milled) nnheated: Ban milled tor 1,178 hr., dried and reftound In scats mortar. Chrysolite (ball milled) Icnlted: Ball mined chrysolite heated tor t hr. at about TOOC,, then cround In scale m ortar S or 3 min. Chrysolite (tlbraua) nnheated: Ground In scale m ortar to pats too mesb. ChryeotUa (fibrous) Icnlted: SOO-meth material heated for 2 hr. a t about TOO C. Ho further grinding. Serpentine (ball mined) unheated: Ball milled for l.u s hr., dried and recround In scale mortar. Serpentine (ball milled) Icnlted: Ball milled terpentine heated for 2 hr. a t about TOO C., then cround In acate m ortar 2 or 3 min. Mineral Cbrytotlle (ball milled) unbeated CbrysotUe (ball milled) Icnlted Cbytodle (flbrons) unbeated Chrysotils (fibrous) Icnlted Serpentine (ball mined) unheated Serpentine (ball milled) Icnlted Site of Dust Particles 1 microns and lest S microns and less Hesults Grlndlnc destroyed capacity to cause fibrosis. At t mo. considerable Infiammatory edema and cellular prolifera tion and localization of dust particles about bronchi oles: a t 2 mo., only a very alight prollferatlre reaction; at 6, Mi and 12 mo., widely scattered small mononuclear phagocytes. At 12 mo., a tew mleroseopie patches of thin alveolar wall thlekenlnc with some adenomatoid change In portion ot air spaces abutting on thickened bronchi. Ho aabeetoele bodies seen. Beaetlon limited to large foreign body giant calls without production o t fibrous tissue. 30-60 microns approx. 2040 microns approx. J microns and 3 microns and less A distinct fibrosis. Beaetlon localised to coonretire tissue about terminal bronchioles: little within those tubes. Contraction caused adenomatoid appearance of air spaces siren off directly from terminal bronchioles. Beaetlon area became smaller with progress ot Urns; no new regions involved. Ho chronic pleurisy even at points abutting Intrapulmonary change- At 1 mo. considerable ... inflammatory edema and foci ot cellular ptoUicratlon; a t 2 mo. well marked cellular proliferation and flbroels occurring locally about respiratory bronchioles. This reaction developed before agbeetosls bodies had formed and was as advanced as th a t produced by 2 yr. Inhala tion of asbestos dust. At 8 mo., reaction less extensive than a t t mo., apparently doe to contraction ot flbrons tissue; asbestosis bodies were abundant. At Mi mo., - reaction etm lees extensive, confined to the Immediate vldaity o t the small terminal bronchioles, where tbs sear tissue was quite dense and was becoming hyaline In char acter. Sometimes It even obliterated tha bronchiole. Asbeatosis bodies bad become scarce. At 12 mo., the well developed peribrooehlal and Intrabronchlal adeno' matold areas of flbroels had produced considerable dis tortion. More peripherally were patches of pneumonitis with eosinophilic Infiltration, some of wbieb was being transformed Into fibrous tissue. These seemed to be pre' cursors ot the localized, diffusa patches o t thin alveolar wall fibrosis seen elsewhere. Beaetlon limited to large foreign body giant eells wlthoot proliferation. Beating 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 8 mo., no ebange except possibly lymphoid cell Infiltration; a t Mi m o, a slight chronic pneumonitis: a t 12 mo., only a little pneumonitis without suggestion of flbroels. Duet relatively Inactive. Reaction essentially the same as for unbeated serpentine. With Ignited, terpentine, lest tendency tor duet to be carried to bronchial nodes. 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 rig h t 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.--STUDIES OF ASBESTOSIS 31 . r >** Kl ... -- The tests were made with long fiber chrysotile, unheate< 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- Fig. 11.--Serpentine injection experiment: Lung of a Guinea pig that had received an intratracheal injection of this dust four months before. A bronchiole 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. V: : ' ,r ' 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 anthophyOite, 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 IS.--Comparison of Reactions to Various Long Fiber Dusts Injected . I ntrairocheelly Do*: Two Injection* ol OlS ee. of I per cent suspension riven two weeks apart. TotaJ do* 11 to m e.. Animals used: From to 9 rulnea pl(> (or *ab dut. Partoda a t which animal* were killed: Usually a t 1, , 8 and 12 month* a (ter laat Injection. SUe ot dart particle: Separated ao th a t moat 8ben were Irom 10 to 80 micron lone. Mineral Cbrreotfle (Lbettord) ChryiotQs (Arizona: low Iron content; 0.2% FesOs) Amotlte Croddoilte ; .. (Bolivia) .. CroeldoUte (S. Atrlea) AntbopbrUt* Tronollte Brnclte Glaaa wool Results Distinct llbroalt. Additional Information siren oppoalte ehrytotlla (fibrous) unbeated. In table 14. __ Reaction virtually Ideotleal with th a t to n e tl o r d ebrysotQe. Both fibrosis and aabeatosla bodies produced wltb an asbestos containing re rr little Iron. Fibrosis occurred as p la n within terminal broaebloles and as finer deposits a t periphery. Fibrosis derdoped before asbestosIs bodies were seen and was in cellular state well formed a t on* month. With ace. fibrous tissue contracted sod occupied smaller atea but was more dens*. Adenomatoid chance* similar to those with n e t l o r d ehrytotlle. Pleurisy limited to Immediate ridnlty ot early reaction about area* oi massive localisation. Asbestosls bodies formed but were lew. At 1 mo. alter Injection, minute lod ol mononuclear proliferation about bronchioles and Ip areas ol atelectasis; a t 1H mo., beery peribronchiolar patebes ol fibrosis ottea with papillary projectIona partially eloslnc lumen ol * bronchiole; adenomatoid appearance marked; eonnaetiee tiers* reaction showed heavy collacen but no byallnltatlon: a t 2 mo., minute lod ot well matured fibrosis about bronchioles; a t I mo., m ature asbestosls fibrosis with evidence o l contraction; considerable chronie pneumonitis with Infiltration ol lymphocytas and eosinophils. A t mo., small intrabrooebloiar fibrous plots with lod ot more delieste fibrosis a t periphery. Typical fibrous eodobroncblolltl* sod peribronchlolltu with formation ol atypical asbcstosU bodies. Banstratlon ot bodies becan below 4th mo. alter Injection, well developed by 8tb mo. Bodies persUt alter 12th mo. , Reaction a t l mo. heavy endobroneblolltis and peribronchiolitis already sbowlny fibrous chant*: atelectasis and fibrosis with some necrosis a t site ot massive localisation ol dust. At 4 mo., heavy, widely scattered endobroncblollU* and peribronchiolitis, now fibrous, with marked delor- mlty ot bronchioles and with an adenomatoid appearaooa. At 8 and 104 mo., reaction In lunc essentially the same a* a t 4 n o . At 12 mo., lod ol fibrous endobroneblolltis and peribronchiolitis still larce, with more dens* tear tissue and more deformity ol bronchial tubes but no extension Into, or atelectasis o t, peripheral parenchyma. _ ^ Advanced fibrous endobroncbloUti* and peribronebloUtla. Beaded asbestosis bodies noted a t 8 a o . At 1 mo., tally fibrous eodobronchloutls and peribronchiolitis: many (la s t a lls and tom* lymphocytic reaction. At 4 mo., small areas ol eadobroncbtoUtta scattered throucbout the lunc; cellular fibrosis. At 8 and U mo., areas ot bronchiolitis smaller because ol contraction ol den* sear tissue; a t It mo., marked lymphocytic infil tration and adenomatoid appearance. Typical advanced fibrous sndobronehlolltis and peribronchiolitis produced by 0.8% suspension (l ee. total dose); most animals would not tolerate usual 8% suspension. Fibrosis well developed before asbestosls bodies teen. At 4 mo., well developed fibrous bronchiolitis with lymphocyte* and ria n t cells and adenomatoid chance. At 8 mo., typical bronchiolitis not quite a t extensive or as heavily fibrous as with a S% suspension, other wise tbe tame. Many deeply stained fibers with r rood proportion ot haustrated asbestos!* bodies. At 12 mo., heavy fibrous bronchiolitis, more peribronchiolitis and endobroneblolltis, with lymphocytes and rian t cells; very marked adenomatoid appearance. Lymphocytic Infiltration and rian t cells but no fibrosis. A very lew atypical asbestosls bodies. At 1 mo., many scattered lod o f lotrabronchlolsr dust without massive localization; lymphocytic Infiltration of walls and a lew ria n t cells. At 8 and 13 mo., little evidence ot dust: a lew bronchioles and bronchi with ria n t cells In adjacent alveoli and with lymphocytic Infiltration ol walls. Fibrosis about bronchioles. At 1 mo., areas ol dust localization wltb col lapse ol alveoli and Infiltration wltb acute Inflsmmstory cells, macrophate* find rian t eella. Within the area were a lew foci ot fibrous tissue and numerous areas ol hypertrophy of alveolar epithelium. Many bron chioles packed with fibers. At 4 mo., renrrsl appearance of lesion unchanred; pleura sllrhtly thiekened over heavy localizations o l dust. An occasional sermented asbestosls body seen. At 8 mo., many tod ol fibers In bronchioles and alveolar duets with eefiulsr reaction a t before: also, tome to d showed distinct eoUateo deposition. At 12 and IS mo., reaction as before with fibrosis about bronchioles more apparent because of con traction and decrease ol Inflammation. Giant cells prominent. Pleura markedly Involved. Typical fibrous endobronehloUtls and peribronchiolitis like reaction to asbestos minerals. At 1 mo., extensive endobroneblolltis and peribron chiolitis with slant cells: dense fibrous loops within bronchioles and cetlnlar fibrosis about tbem: adenomatoid chance present. At 2 mo., heavy IntTsbronchlolar and peribronchiolar fibrosis produdne marked deformity with distortion of tubes sod obliteration ol surroundlnc air spaces: fibrosis pale without byallnlzatlon but with lew nuclei: no necrosis. Typi cal asbestosls bodies seen. At 4 and 8 n o . little chance: fibrous tissue cootrsetlnr. At 104 mo., dense fibrous brooeblolltls with asbestosls bodies. So pleurisy. Ko extension to surroundlnc lunc- No fibrosis within a year. At 1 mo., no reaction Inside bronchioles; in peripheral sir spaces clump* of c<*nt eells packed wltb fine spicules ol slaaa wltb lymphocytic Infiltration ol adjacent wans: no asbestosls bodies. At 2 mo., reaction lees intense than a t 1 mo.; lalr-ttzed clumps ol donrated ctant pbacorytes eontalnloc splenic* and particles of rises: no endohronchltls. At 4 and 8 mo., reaction stll) dlmlnlehlnr. A t-12 mo., local areas of nneumonltle with no fibrosis or endobroochltls: moderate number of smooth Iron-stalnlnr fibers. : VORWALD ET AL__STUDIES OF ASBESTOSIS 33 that produced by the asbestos minerals was obtained (fig. 13 A ) . Since the brucite used contained only 0.90 per cent silica as an impurity, it is obvious that a siliceous component is not an essential factor in the development of asbestosis. Fig. 12.--Amosite and croddotite mjeetipn experiments: A , lung of a guinea pig four months after an intratracheal injection of amosite. The inflammatory reaction exhibits pronounced acctsndbisoa of cells and collagen deposition ( x 200). B, lung of a guinea pig. four months after an intratracheal injection of croddolite. As in A, peribronchiolar amrnmlation of cells and deposition of collagen are shown ( x 200). Third, no fibrosis resulted from the injection of glass wool fibers ( fig. 13 B ) , even though glass wool resembles asbestos in many ways. 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 oonsilideous 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). aB, lung of a guinea pig which four months before had received an intratracheal injection of glass w ool 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 2 0 0 ). - ........................... VORWALD ET AL.--STUDIES OF ASBESTOSIS 35 fibrosis. .Experimental studies concerning this observation will be reported in a separate publication. ..t - -- . Table 16.--Comparison of Reactions Produced by Long Fiber and Short Fiber Dusts Injected IntratracheaUy Dosage: Two Injections of OS ee. oi a 6 per cent suspension ( I r a two woeki opart. dote wat JO me. Animala need: Six tro u p i of guinea pica. Perioda a t which animala wen killed: l, 2, 9, SH and U montha attar Injection. Total Mineral ChrriotUa (Thetford) Amoilta CroeldoUta (Boll ria) AntbopbyWt* Tremoli te Brnclta 81 of Duat P art Idei Lone Ober. 20-60 microns Short ber, Smlcrona andlaaa Long Aber, 20-60 microna Short fiber, 2D microna andleaa Lone fiber, 9060 microna Short fiber. 30 microna and le Lone fiber, 9060 microna Short fiber, 3 mlerona and le Lone fiber, 9060 microna Short fiber, 90 microna Lone fiber. 9040 mlerona Short fiber (made by crurblnc lone fiber* with rubber police man) Baaulto A distinct flbroeta. Safer to chrysotlla (fibrona) an tirated In table 14. Vo fibroita. Baler to cbrraotlla (ball mined) nnheated In table II. Typical fibrona eodobronehloutla and peribronchiolitis. Refer to table U. Reaction limited to pbacoertoala with lymphocytic infil tration of adjacent walla. Sbort fibers packed Inside swollen sbacocytea; longer o n tree: some coated to form typical asbatoala bodies. At 1 mo. alter injec tion, alreoll contained good-slxed giant cells; most phagocytes were within air spaces and bad not migrated to walla. At I mo., tret extracellular libera had worked tbemaetve* Into Interstitial tissue, where there was extensive proliferation ol lymphoid cells and monocytes bat no fibrosis. At I mo. foreign body reaction with tome pneumonitis, no bronchiolitis. Typical aebMtoela bodies present. Advanced fibrona endobrooehiolltla and peribronchio litis. Baler to table 16. No fibrosis. At 1 m o, air spaces compressed and largely filled with giant cells packed with duat needles. Walla heavily Infiltrated with monocytes and lymph oid cent-. At 4 .m o, a moderate degree of eellnlar Infiltration of walla: small giant cells packed with dost splenica. At 6 and t t t mo., m ass of giant Us, containing mineral partId , in small bronchi but not . In respiratory broochiolte; smaller o n widely scat tered la terminal air apse. Numerous aabestoela - bodies. No reaction In connective tissue. No endo bronchial proliferation. At It m o , many scattered small monocytes packed with dost. No endohronebltla. No peripheral fibrosis. In lymph node, alight reticulosis; no fibrosis. Lymphocytic infiltration and giant eeOa but no definite flbrofte. B i te to tiM i 10 No fibrosis and practically no asbestos!* bodies. At 1 m o , local collection* of dust-filled monocytes and a few giant cells: a t 4 m o, tome adenomatoid epithelial reaction: a t > m o, simple pneumonitis with phago cytosis of short fibers; a t It m o . Isolated and sharply localised collection* of duet ceils Inside air spaces about terminal artertoln. Beaction in walls limited to lymphoid .cell tafittratlon. No fibrosis. In lymph node, reaction limited to slight prominence of reticu lum. fibrosis about bronehtolca. Refer to table IS. Simple foreign body inaction. No acute Inflammation. No accumulation of dost In or about terminal bron. - ehloles. No endobroorhitis. At 1 m o , a tte n d email giant *** and eonafdmaMe Infiltration of adjacent walls with moooeytn and lymphoid cell*. At 4 mo., little change except m on cellular Infiltration of eonneettve tissue. At I mo., lymphoid Infiltration and " thickening of walls about m e but not all terminal brooehlol. Typical fibrona endobronebkXItls and peribronchiolitis like reaction to asbestos minerals. Refer to table 16. Inert type of reaction. At 1 mo. after Injection, small monoeytee. widely scattered through air space: focus of atelectasis with lymphoid Infiltration of eompreaiad air-spa walls. No endobronchial reaction a t with ebrysotue. At 2 m o, reaction similar to that at 1. mo.: typical asbestoal* bodl seen. At 12 m o, small clumps of Inactive dnst-filled phagocytes; no fibrosis. No reaction in lymph nod. 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. ! ' . E xperiments U sing I ntravenous `T echnic The experiments summarized in table 17, in which the intravenous method of injection was employed, show that the asbestos minerals are for 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 by the results given in the table. The reason for the early deaths in the experiment with chrysotile particles is not clear. E xperiments U sing I ntraperitoneal T echnic 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. W., and Rae. V. M .: Effect of Asbestos, and of Asbestos and Aluminum, on Lungs of Rabbits, Thorax 1:188, 1946; abstracted, IndusL Hyg. Digest, 1947, vol. 11 (Feb.), no. 234. . Tam.e 17.--Summary of Injection Expriment by Intravenous Technic .............' ........ ~7~ ..................... ~ ~ ~ h*: Total sm ouot of d u it vaa 1.0 O n., divided luto 20 equal dowa (each done waa 6 ce. o | a 1 per cent sospciielou) which were glveu twice a week lor 10 wcoka. ; Mineral Chryitolllo (Titel lord) Ainosite (ro c Mollte Anthophylllte Trrinollte (sods Iron) Yrumollt (d a) Size o( Dual Particles 1 microns aol leva (hall milled 192 hr.) / Habblta Uaed a mlerona and leaa t * (ground lo gale mortar) Tmlcrons aod leaa 4 ` (ground lo agate mortar) I mlerona and leaa 4 (hall milled l.iuuhr.) S mlerona and leaa 4 (hall milled HO h r .) 1 mlerona and leaa 4 (ball milled 48 hr.) Maximum Survival Alter l.aet lojectlou. Mo. . 17 tl 1 U iii . Results 1 | The rabblta did uot tolerate lutravenoua Injeetlooe of finely ground chryaotlle and fi ol the 0 died a lte r l to 0 Injection ol even diluted auaiteualoua; the other animal died alter 27 lujcctfooe ol one* q uarter etrenglli auapcnelou (00day alter Aret lujeclloti). Reaction limited to lew large giant pltago* cytea o l Inactive typo In liver, eplecu and luoge. No thrombi o l dual celle aeen lu tmlioouary capil larit. No deflulte explaoatlon lor latalltlea discovered, but material may have been retalued In ; heart, causing local thrombi. Advanced pulmonary Inlcctloo killed 1 animale a t 0, U and 17 mo. alter laat Injection, shortening Intended duration ol experiment aod complicating picture. However, rabbits killed earlier (S aud 0 mo.) showed only loert phagocytosis with no progreselou lu the 6 mo. atilmal. The laat two (11 aod 17 mo.) w en probably ibe same although local necrosis o l tho Uver and amyloid ol the spleen made Interpretation difficult. Reaction waa th a t to no loert substance with no chaoge lo 12 mo. (Other observations a t t, 1 and il mo.) Blniple phagocytosis of particles. No teudeucy to agglomrats and no cliaoge lu adjacent tissue. Grinding the dust to aises of 3 mlerona aud uuder destroyed the fibrous structure of this mineral, aod the Injected material resembled plates rather than filters. Reaction essentially that of an Inert mineral. Observations made a t 3, A, 12 aod 24 mo. Only sugges tion o t Irritating properties manifested lo spleen ami lymph nodes, but n o t liver, of the 24 mo. rabbit. In thla aoltual there had been proliferation of mononuclear and giant cells th at was not present In eittier spleen or lymph node of 12 mo. animal. The absence ol eesodeted fibroblastie reaction In these orgaos and of any chauge In the liver condition Justifies the claeslilcstion of enihopfayUlt s en Inert silicate. No fibers were retalued In lung to demonstrate whether esbeetoels bodies would develop. Reaction essentially th at o t an Inert mineral. I.aet animal killed allowed a little prolifrratl'>n end lymphocytle Infiltration In liver, not seeu earlier (e t 3, d and 12 mo.). No evidence of any activity In lesions In other organa. An Inert foreign body reaction with no change In 24 mo. Olwervatlone made a t 3, 0, 12 and 24 mo. n ; . I 1:vitl.l;f:-ifr C ' t- jprnsCTrrjr* mtrynr: >- T able 18.--Sum m ary of Injection Experiments by Intraperitoneal Technic Do*: Each Mineral Chryaotlle (Thetford) animal * received a Ingle Intraperitoneal Injection Size of Duat Particle* Guinea Pigs Uaed Maximum Survival After Injecth Mo. 1 micron* and lea* 15 SO (ball milled t i t hr.) Chryaotlle L oot fiber 4 8 (Thetford) (through lOOvneah) AmoiiU 8 micron* and lea* 0 It (ground In agate m o rtar) Oroeldolita S microns and Icaa; T It alao aotna ions plcutes (ground In a u to mortar) . Anthophylllte 1 mlerona aod leaa 0 tt (ball milled 1,400 lr.) Anthophyllll* Moatly 8 mlerona ft It ! (originally and leaa; acme . labeled tale) fiber* 80 micron* or more ton* (ground In agata . mortar) Itcm ollto 8 mlerona and lea* 6 u i (eoda-lron) (ball milled hr.) Tremollta 8 mlerona and leaa 8 18 (coda) (ball milled t 48 hr.) 1 Anthophylllte Pyrophytltte (fibromi) Pyrophylllte 1 (crystalline) 100 micron* and leva 100 micron* and Irfla 100 mlerona and lea* 6 It 6 It 6 It ot 1 cc. of a 10 per cent duet euapenalon. Total amount of duet Injected wa< o.l Om. i | Reeulta i Mo flbroal* or aebeetoela bodlea. Duet particle Ingmted b f phagocyte*, chiefly multlnucleated variety. Six mo. aft* Injection ftbrou* elementa ol dust appear to hare dtaaolved leaving only the Inaolubla magnetlU, a eontamlnanL No reaction In anrrouodlng fa t or areolar tlaaue. Mo tranaportlng of duat to mglone! lymph node*. Obaervatlooa mada a t Intervale from 1 to M mo. a tta r Injection. Deflnlta llbroua reaction produced, delicate and nonhyaline. Atypical aabeatoala bodle* developed, but all were unueually amall. Mo evidence of extra long flbera aean. Obaervatlona only a t g mo. Infection Interfered with Interpretation. Duat reaction appeared to be of Inart type and limited to pbagoeytoala with a moderate tendency to lymphocytic Infiltration. Obaervatlona a t I. t . t and It mo. Duat tod eoneleted only of large mononuclear and giant phagocyte* aurrounded by a minimum amount o t cellular connective tlaeue. The Injected duat contained not only On* material th at In grinding bad bean maabed Into Irregular plataa b ut alao many long apleulae 10 mlerona o r more In length. Mo aabeatoala bodlea aacn. although the longer apleulaa appeared lightly awollea and greenlah. Obaervatlona a t 1, t , ana I t mo. EaaentlaDy Inert foreign body reaction. In early animal* (I, I aod I mo.) tocua of monocyte* and amall giant ceil* and a nttle central oecroela. In the g mo. animal there waa alao alight peripheral ftbroala. A t I t and t t mo., nonprograaatva maaa of monocyte* and giant call*; no Abroala. Raaetlon, which eonalated of vary large giant call* aurrounded by a variable number of lympho- . eytea, waa much heavier to the* unintentionally long flbera than to the fine duat In the experiment above. Thera waa more or lea* proliferation of flbroblaata producing cellular connective tlaaue vlalbl* la area* where the quantity of foreign partial** waa not ao g n a t th a t It obaeured the raaetlon. Obaervatlona a t I, I, S and I t mo. 1 i i Inert type of reaponce never progreaalng beyond tbe atage of vary alight lymphocytic nactlon about maaaea of duat-IIlied phagocyte*. - Mo flbroal*. Obaervatlona a t 1, I, 8 and I t mo. Inert nonprofttealv* foreign body type ol raactlpn. Mo flbroal*. Obaervatlona a t I, I, 8, I t and I t mo. Dlatloet early flbroal* produced by aotbophylllt* and flbrou* pyrophyUlte with aubaequent regrea- - eloo; cryatalllne pyrophylllte Inert throughout. At l mo., giant cella about long thick apllntera; a t I mo., definite flbroal* replacing giant cell* of anthophylllte and llbroua pyrophylllte reaction; a t 8 mo., flbroal* which atarted a t 4 mo. bad decreaaed, eapedally with flbrou pyrophylllte. At 12 mo., reaction to all three duata eonalated of foreign body giant cell* with lymphocyte but without neeroala or flbroal*. No aabeatoala bodlea. ' * Each animal recatvlog long fiber ehryaotlle waa given an Injection of t ee. of a OA par cant duat euapenalon. VORWALD ET AL--STUDIES OF ASBESTOSIS 39 . "PioraatTE Aenow or Aluuinuv Compoonos " W hen colloidal aluminum hydroxide had been added to a suspension of long fiber chrysotile prior to injecting this suspension intratracheally into rats, the aluminum compound did not prevent the irritation of tissue due to chrysotile. If anything, the acute inflammatory response evoked by the injected fibrous mineral was accelerated. One month after the last injection of the dust suspension the bronchiolitis was becoming fibrous. King and his associates also found that aluminum failed to protect pulmonary tissue from the irritation caused by asbestos fibers u ; in their experiments metallic aluminum was used instead of the hydroxide. FoutATioir or A sbestosis B odies 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 chrysotile were injected subcutaneously into the groin of a guinea pig--one kind containing 2 per cent and the other 0.2 per cent ferric oxide--the asbestosis bodies were equally numerous at both sites of injection and showed no difference in their reaction to prussian blue, the reagent which stains iron. This finding is in agreement with that of Giroux.1* / T issu e R eaction to A sbestosis Bodies Asbestosis bodies recovered from human lung tissue and injected intratracheally into guinea pigs failed to produce a fibrous reaction. The material forjnjection 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 rather resistant coating which is not destroyed by moderate hypochlorite treatment, which may be maintained in vivo for a year or longer and which renders the fiber incapable of producing fibrosis. It thus appears that the coating is a protective mechanism. This thought was expressed by Beintker as early as 1934." THEORY o r 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 12. Giroux, M.: Amiantose exprimentale: valeur pathognomonique du "corps d'amiante," Laval md. 8 239, 1943. 13. Beintker, E. : ber die Asbestosiskrperchen : Bemerkungen zu der Arbeit von Beger, Virchows Arch. i. path. A nat 293:527, 1934. ................. 40 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE content of only 0.90 per cent, caused typical fibrosis like that produced by 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 Jung-- ar.d that a fibrous reaction can be produced by injecting r * *. T able 19.-- Analysts of Fibrous Minerals ~ __ nbroa* liberal AfflOdte..................... Amphibol.................. AntbopbrHlt_.......... B m dte-..................... OhnriotOt.................. CroeMoUte................ T re o o U te ................... 8IO % 46JB 66.04 SOJO 0J0 68.66 64.6 66JO TtiOt % 1.06 6.06 OAT 6.71 166 16JT 7St y0 % SSJS ..I.. #J6 4J> ........... Alto C0 %%_ U ro % 1MJ.m ' U S 1.60 1U6 BM OJt 0.44SU7 . OlBS 0.46 0.04 66J 0.64 0.06 88J6 101 0J0 1166 0A6 \ 4.44 60JS N |0. % 0JS 0.11 0M 0.60 6Jt 6.16 Irnltlao Lom KlO < I96 0 . >106 a Tow % % % % 0J0 .0.66 . 6JS 61 0.11 -V. 0J6 6JO 66 0.16 0JJ . 6.06 WJt 0.1 J 0A> U M M.3 046 ' 4J0 U M 98Ji 0JT 0.01 166 96J) 0J0 . 0M US MS asbestos fibers into the peritoneum, where there is also a degree of mobility, but not by injecting them into other extrapulmonary organs such as the liver; the spleen and subcutaneous tissue. COMPLICATIONS The experimental investigations with asbestos minerals were con cerned primarily with the effect of the dust on normal tissue, but some attention was given to other phases, such as susceptibility to infection. The only experiment in which the effect of 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. SuscEmsnjTY to T ubebcUlol's I nfection i 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 A L --STUDIES OF ASBESTOSIS 41 dust.like quartz but above an inert dust such as iron oxide. In an im als 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. SusatPTTsn.iTY to Nontubmculous iN n c n o x 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. ' *v -- *' - COMMENT AND SU'MMARY 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 asbestos after being exposed by inhalation or intratracheal injection to long chrysotile asbestos fibers, 1 ' i - 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 arc 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. r-; <-; - j- V C The mode of action of the long asbestos fiber in the production of -vv 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) . r rr D. Typical experimental asbestosis was produced by the inhalation ofan atmospheric suspension containing an average of 138 million V asbestos particles per cubic foot of air by light field count, of which. less than J 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. i / 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 panicles per cubic foot of air, and size-frequency determinations disclosed that 6.7 per cent of the air-suspended material consisted of fibers longer than 10 microns (table 11). Thus, by calculation, it is estimated that the con centration of the longer fibers was 2.7 million (6.7 per cent X 40 mil lion). The lungs of animals exposed to the long fiber asbestos dust revealed that the pulmonary reaction developed in approximately onehalf the exposure time required for its development in animals inhaling the ball-milled product, for which the concentration of the longer fibers ~ .was only 0.8 million (0.6 per cent X 138 million). F. Established experimental asbestosis ceases to progress on discon- ,. tinuance of dust exposure. . y >. ' The experimental investigation shows, in fact, thafon discontinuance of exposure there was an appreciable clearing of the mature pulmonary -CSK-v V'HV -> :.t;. VORWALD ET AL.--STUDIES OF ASBESTOSIS 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. > } y ; . .. / " " ................. * - ,T ..............................' " * This investigation was made possible by the generous' financial support of a ' group of companies of the asbestos industry. ... 20 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE important to note that in th 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 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 .celts 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 (X2Q0). 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. , **' 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 m il lio n .____ 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 far the short fiber experiment Guinea pigs, rats and mice were used in the inhalation experiment with the 100 per cent bail-milled asbestos d u st The results are summarized in table 9. Reaction in Guinea Pigs.--The experiment was started with 100 guinea pigs. As the dust exposure proceeded, there were 39 accidental deaths, 32 of these being due to pneumonia in an epidemic. The 61 pigs remaining exposed to the dust were killed at intervals during exposure, except for 16 guinea pigs transferred to normal air after 28 months of dusting. For the first year of exposure practically the only reaction to the dust was the presence of scattered phagocytes and an occasional minute asbestosis body. At 16 and 20 months no gross response was visible on the tissue section, but microscopically peribronchiolar foci of inflammatory cells T able 9.--.Summary of Inhalation Experiment with 100 per Cent Ball-Milled Asbestos Dust .Nature of Experiment Dust exposure eonUauous tbroutbout llte Dust expoeure followed by prolooted residence In normal air - Animals M tulata pits 40rt tlm leo U rdnea rite f - ' Maxi mum Dust Expo sure. Mo. M SO 11 a Maximum Survival Alter Dust Expo sure, Mo. 0 0 0 IS f Beeults No appreciable pnlmooary reaction No sucreetioo ot asbestosis No euctettlon ot ast*stoeia nbroals typical o t asbestosis was present i t mo. after exposure ceased In an amount soffldent to be visible crossly! smaller tod ' eould be seen microscopically a t t mo. and 8 mo. alter termina tion ot exposura could be seen. At 24 months (fig. 6 A ) there was soil no change large enough to be seen with a hand tens, 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 peribroochiolar fibrosis. For exposed animals living eight months in normal air the findings were similar, but at 12 months three ot four animals showed grossly visible characteristic peribronchiolar fibrosis with adenomatoid change (fig. 6 5 ) . , . The tracheobronchial nodes were essentially normal until exposure had been continued for more than a year and a halt. 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 A t 30 months there had bee 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  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 King's floats there would be a basis for believing that the action of asbestos is in part, at least, a chemical one as postulated for quartz. This experiment, in which the tissue reaction was slower and less extensive than that in the previous experiment with King's floats dust, indicates that the capacity of inhaled asbestos fibers to produce fibrosis is determined primarily by factors not chemical in nature. 'c / . . ...i; ' l - ' .--.* . . -.i. * 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. B all-M illed A sbestos D ust . 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 doud._ 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. Competition and Atmospheric Concentration oj the Dust.--The dusting: material was the ball-milled, short fiber asbestos used in the previous inhalation experiment, but unground material was not mixed with i t 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 cen t 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 r. VORWALD 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 ot 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 Cali.--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 fod 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 few yellow 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 <me animal after about three years of exposure and was seen in all five animals examined thereafter, including the ooe 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 anothef- 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 rabb.its that died varlv in the experiment but were seen in all animals that had been exposed to the dust for more than three years. 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 asbestos 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* mined 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 T able 7.-- Analyses of Lungs of While Rats That Had Inhaled Short Fiber Asbestos Dust " ' - ^ 4 . ' - - ' Duration of Expoaure. Ilo. 0* 4 Amt. of Ash, % ol Dried Lune 34 44 !S SB 34 34 34 SB 3B It 4 SB SB 3.4 Total 8IOa, % of Dried Lune 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.08 0.1S 0.00 0.06 0.08 0.U 0B7 Total SiOa. % of Asb 0.0 0.0 0.0 0.0 04 0.0 0.0 SJ 3.5 SB IB SB S.0 SB ' Amt. of - ' Duration--. Asb.% ol Expo- ol Dried sura. Ho. Lune SB 8 S.l S.7 Total 810a. % . of Dried lun . 0.07 0.05 . 0.04 - Total SlOa. % o lA ab _ 11 . .. IB 1.1 SB SB 8/ SB 4.4 S.7 048 ' 14 0.18 64 0.16 3. 0.17 4.0 0.18 4.1 f 4.0 ^ 0.18 34 10 1 4.8 1 SB 0.13 0.18 34 SB 1.4.7 0.1S ^ . ! B . * Nora*) controls (no d n it expocurc). T able 8.--Average Values of Ash and Total Silica for Lungs of W hite Rats Inhaling Various Dusts for Various Periods (Lungs Only, Without Included Lymph :Nodes) Amt. ol Aah, % of Dried Lun* Dora- ,------------------- * ,. tlOD Short , Gypaum- of Ex- Fiber Femi- Quarts t>orarc( Aabes- elnous . Mix Mo. toa Quarts C b m ture 2 SB 4.3 SB 2 B 4 S.l IB SB 3.8 6 SB 7.1 9B 3.4 8 3B 4.8 9.0 3.8 10 4B 7.8 14B IB Tota) 610, % ot Dried Lons Short Fiber Asbes tos _ 0.0 ; 0.0 - 0.08 0.15 0.15 Quarts 0B1 0B1 2.94 1.44 4.40 Ferroelnous Cbert OBS 0B2 3.45 2.40 8.0 GrpenmQuarti Mix ture 0.08 0.07 0.11 0.32 ' 0BS i Total 6IOt. % of Asb . Short Fiber Asbes tos Quartz GypsumFemi- Quarti elnous Mix Cben ture 2.8 a .: 3B 2.8 2.5 11.4 3.8 2.0 L8 41.5 34.4 3.1 3. 29.4 28B 9.1 3B 66.6 43.2 8.7 were relatively numerous although still rare in comparison with the findings in the King's floats experiment Reaction in W hile Rats.--Seventy-three white rats were exposed to atmospheric short fiber asbestos dust for periods up to 32 months. During the first 10 months animals were killed bimonthly and for the remainder of the experiment at less frequent intervals. Up to eight months the dust cells were widely scattered and existed in foci only sporadically. Reaction was limited to occasional slight thicken ing of the septums about small accumulations of dust 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 of 150 diameters or more, consisted of patches along VORIVALD ET AL.--STUDIES OF ASBESTOSIS 15 Only aiter exposures had continued for approximately one year was there an appreciable tendency for dust-containing phagocytes to gather into dum ps. 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 multinudeated cells, but they were of the inert, foreign body type. At 20 to 24 months the cellular dumps were sometimes quite prominent, and sometimes changes in the epithelium resulted in the adenoma-like or "adenomatoid" appearance (fig. Z B ) previously described in the section review ing the experiment with the King's floats dust. In most of the subsequent members oi 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 byalinized. Diffuse chronic pleurisy was present in a few animals without evidence of pul- T able 6.--Analyses of Lungs of Guinea Pigs A fter Prolonged Inhalation of Short Fiber Asbestos Dust Exposure to Dust. Mo. 12 13 to U JO 34 *r 20 20 Period in Normal Air. Mo. Amount ot Asb, % of Dried Lung Total 3IOt, % ot Dried Lunt Total SIO;, % ot Ash Dust Exposure Continuous During Lite. 5. 0.51 10.23 0 4.58 0.4 10.0 s.ts 0-54 10.54 5.00 0.40 0.00 0 4.7 0.4S 0.00 4.05 0.53 10.00 0 SM 6.4S 0.85 0.00 14.4 14.07 Au 5.41 5. 0.78 0.78 14.48 14.20 A 5J5 0.06 1130 V -55 1.27 19.4 A 0 0.75 11.37 V JS 0.0 15.11 Dust Exposure Followed by Protested Residence In Normal Air < . { a?* 0.43 0JO 0 . 7J J . i tii 0.62 10.21 0.34 8J1 (4.77 0.1$ 5.31 14 1 5.1S 0.26 5.00 [ 4.77 0JS 4 AO Tissue Reaction' s+ 3+ 4+ 4+ 2+ 3+ 2 + * Tbe erm bola sre rsfln c tbe tissue reaction to each croup ot rulnea pics represent merely the relative decree of reaction, ranctbc from 2: (questionable) to 4+ (the maximum for this experiment). Tbe relationships apply only within this table and cannot be 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's floats asbestos, probably because more fine particles had been transported to the nodes in animals inhaling short fiber asbestos. The nodal reaction was eissentially 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 'i lungs with widely different amounts of tissue change. For example, the ash / 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 hopper, 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 resu ltar 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 V"t"** . r-";; * The dust concentration varied during.the experiment, the light field counts for atmospheric samples collected inside the ninni cages with the impinger apparatus' ranging from 83 million to 182 million. The average f 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 ___1 t ""-T'- ': : " '1 ' " V V ' -- ' T able S.-- Summary of Inhalation Experiment with Short Fiber Asbestos Dust Nitore of Experiment Dust exposure eonttaoous throogb- out Ills Dust exposure followed by prolonged residence In norm el sir Animile ^ 4S guinea pigs 3 rete IS eats 1 rabbits IS guinea pigi t cete 1 rabbit Maxi- Survival SQUID After Dust x- Dust Exposare. potare. Ho... H o.,.' 34 0 33- 0 54 * p 47 * * : U 31 34 -<* ' Besultt'r-yv Bate ot reaction about tbe tame aa In exiwit meat with King's floats asbestos but exteet of Involvement very mueb lest t Characteristic patches ot peribronchiolar Abie ala: no asbestoit bodies Subpleura] reaction only > Ho fibrosis teen grossly; microscopie evideaai of alveolar wall thickening after SOmontw exposure . - ./ Procreation after removal from dust doubt fnl--neither dearly established nor defloitdr excluded Same aa tor continuous exposure Similar to continuous exposure; evidence ef slight regression*10 After S3 months the animals were exposed to 100 per cent ball-milled asbestos. t Tbe resetlon was probably due to long libera In tbe unground material wbleb was mixed with the ground asbestot to produce a aaUslactory 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 tra c t 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. ' -'VV-'V.?'-'.*' r" : VORWALD 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. 'T ' t r r y^--- .y - - .......... .................................................. Summary and Interpretation of Inhalation Experiment with 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-dst 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 oh the course of tuberculous infection are justified by this experiment. : = ` ? .*/ S hoct F ra u A sbestos D ust 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 for 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. Since10 10. Vorwald, 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.  12 INDUSTRIAL HYGIENE AND OCCUPATIONAL 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 W hite Rate.--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.--Kind'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 clT'infiltration of the wall. Xote the lack of encapsulation and the peripheral epithelioid cell pneumonia, which illustrate a spreading tuber culous process (X 2 0 0 ). B, lung of -a guinea ,pig infected with Ri tubercle bacilli and then exposed to dust for 35 months. Note the subpleural distinctly encapsulated caseous focus, the calcification at the right border of the lesion and the absence of cells in adjacent alveoli, all of which illustrate a healing tuberculous process ( X 200). VORW ALD E T AL.--STU D IES OF ASBESTO SIS 11 Asbestos bodies (fig. 2 5 ) , 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. A t 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 with Tubercle Bacilli at the Onset of Dust inhalation.--Of the group of 40 guinea pigs infected with attenuated tubercle bacilli, Ri strain,' at the time that dust exposure was begun, 31 died or were killed before the completion of two years of the exposure and were reported in the paper by Gardner and Cummings.11 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 A ) : in 6 of these it was confined to the lungs, and in the other 4 the abdominal viscera also were involved. Extension of the infection was first seen after seven months of dust inhalation; during the next 20 months more than half of the animals showed actively spreading tuber culosis, and in 3 of them small cavities had developed. During the last eight months no animals exhibited any evidence of active infection although in half oi 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. 5 5 ) . In the remaining five-the tuberculous foci showed evidence of having previously spread locally; in four of them, by fhe 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. . V * 5' ' Reaction in Guinea Pigs Infected with Tubercle Bacilli A fter 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 1H months old, but by 555 months it had completely disappeared, leaving only scar tissue. Foci of fibrosis still persisted in the last animal, which was (filled 14 months after infection. ,. ___ ____ Reaction in Rabbits.--Rabbits exposed to the asbestos dust lor 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 199* 9. Steenken. W., Jr., and Gardner, L. U .: R Strain of Tubercle Bacillus: Its Dissociation and Virulence of Variants in Normal gnd Silicotic Guinea Pigs, Am. Rev. Tuberc. 54:51, 1946. f 10 l SDL'STRIAL HYGIENE AND OCCUPATIONAL MEDICINE chronic pulmonary inflammation resulting from many causes. Willis * 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. . \ j Fig. 4.--King's floats inhalation experiment: A , lung of a guinea pig with six months' dust exposure followed by 35 months' inhalation of normal air. The reaction is rather slight, but distinct fibrosis is present (X 2 0 0 ). 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 8. Willis, H. S., and Brutsaert, P .: Tumor-like Structures in the Lungs of Guinea Pigs Artificially Exposed to Silica D ust Am. Rev. Tuberc. 17:268, 1928.