Document LgnNx2e37LoZ88MzyVEL5DX63

FILE NAME: Metropolitan Life (ML) DATE: 1951 DOC#: ML313 DOCUMENT DESCRIPTION: Suppression of Saranac Cancer Experiment #1 [Part 2 of 2] r Vyp.vgi 'A'i^-fr^ -'S -.. A. M. A R C H I VE S OF Industrial Hygiene and Medicine . EDITO RIAL BOARD PHILIP DRINKER, Chief E ditor . 53 Shattuek Street, Boston 15 THEODORE F. HATCH, Pittsburgh . FENN E. POOLE. Glendale, Calif. ROBERT A. KEHOE. Cincinnati FRANK PR1NCI, Cincinnati FRANK A. PATTY, Detroit WILLIAM A. SAWYER, Rochester, N JAMES IC STERNER, Rochester, N. Y. RICHARD J. PLUNKETT, M.D.. Chicogo, Managing E ditor JANUARY 1951 VOLUME 3 NUMBER 1 -j PLAINTIFF'S EXHIBIT 15 " ^ - A . M A l . . Irchlws of Industrial Hygiene end Occupational Medicine Volume 3 JANUARY 1951 COPYBIGHT, 1 9 5 1 , B T THB A M EBICA* MEDICAL ASSOCIATION N umber 1 EXPERIMENTAL STUDIES OF ASBESTOSIS ARTHUR J. VORWALD, Ph.D.(Path.>, M.D. THOMAS M. DURKAN ANO PHILIP C. PRATT, M.D. SARANAC LAKE, N. Y. 5BESTO SIS is a form of pneumonoconiosis resulting from prolonged 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 (IS 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. - ; .. - . ^ / - V' .. A m phibole group: actinolite, am osite, am phibole, anthophyllite. crocidolite and tremolile. .S ` ' ' ' Serpentine group: chrysotile. . The bulk of the asbestos of commerce is chrysotile, 3M g0.2Si0j.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 f;'l ; \ V f - p - - - ' For many years studies1 have been carried on at the Saranac Laboratory in an investigation of the cause, nature and development of asbestosis. The present paper is devoted to experimental asbestosis, Fig. 1.--Human asbestosis (P-36-144). The photomicrograph reveals a bronchi ole (right center) with a smooth muscle bundle at its inferior margin and with an extensive zone of collagen deposition largely obliterating the surrounding alveolar structure. The black foci are macrophages containing incidental pigment. Asbestosis bodies are present but are not apparent at this magnification ( x 200). and in it are described the experiments made on animals with various kinds of asbestos dust. Another report, to be prepared and issued at a future date, will be concerned with human asbestosis and will cover the health aspects of workers who have been exposed to asbestos dust in an industrial environment. Although in man asbestosis is a chronic disease with diffuse pulmo nary fibrosis which requires years to develop, it is possible to reproduce 1. (a ) Gardner, L U., and Cummings, D. E .: Studies on Experimental Pneu- mokoniosis: V I. Inhalation of Asbestos Dust; Its Effect upon Primary 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. r Jr '. ; t` 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 particulate and fibrous material, it is necessary to accelerate the reaction by employing higher concentra tions of dust than would ordinarily be encountered in industry. While conditions of exposure are thus different, the information yielded by animal experiments is invaluable in furnishing a better understanding of the reaction of the human organism to inhaled asbestos dust. E xperimental M ethods 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* A t intervals during the experiment a few animals are killed and the tissues examined to determine the nature and the extent of the dust reaction. Some animals are exposed for periods up to three years. The injection experiments are used to deter mine in_as short a time as-possible whether or not a particular dust has a potential capacity to produce inflammatory reaction when in direct contact with tissues of the body. The method involves injecting the dust, either dry or suspended in 'fluid, into the animal by the intravenous, the intraperitoneal, the intratracheal or another route. . \ Long term inhalation experiments furnish information on which great reliance is placed when estimating the degree to which a dust might constitute a respiratory hazard to industrial workers. Even though an atmospheric dust may be potentially dangerous, as indicated b y 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 issu e S usceptibility ' Lnlike 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.lb Fine quartz introduced into various organs f INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE of vanous 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 m other organs of the guinea pig, the rabbit, the cat and the white r a t n 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.r , P eculiar C haracteristics of 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 Table 1.--Reaction to Long Fiber Chrysotile in Lungs of Mon and Other ' _____ Species of Animal . . Sped* ***n...................... Golosa pi*........... _ Rabbit-- ...'.......; c , t ........................ Whit* r a t - ........... Whit* mouse......... *........................ , Mod o t Exporarc Inhalation ' . . Inhalation and Injection Inhalation and Injection Inhalation and injection Inhalation and Injection Inhalation Injection ptbroala* . 4+ . *+ + +. 0 Albettoli Bodies. Xtuserotu . Modaately ouoeroua Bar* and atypical-. Bar* and atypical Very rare Bare and atypical Kona . The annbola 0 to <+ reler to the decree of llaaoc reaction. . j m maximum dimension. Larger particles apparently do not'gain access to the lungs, because, first, large particles settle in air so rapidly that few remain susperided 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.4 Their 2. Schuster. N. 6 . : Pulmonary Asbestos in a Dog, J. Path & B ad 34 (pt: 2 ): 751, 1931. " 3. Vorwald, A . J .: V ariations in Individual Susceptibility to Industrial Dusts Inhaled into the Lungs, Am. Rev. Tuberc. 62: (IB ) 13, 1950. .. . io i 74' M',ler' W ` S " Th* Lun' S Prin*fieW- IU- Charles C Thomas, Publisher, 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. ' R ate o r T issu e 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 .i: ' v 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 haustra'ted 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 ing's F loats Asbestos D ust 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 Type of Asbestos SIOs FesOs AltOt CrjOi MnO CsO Bop's Boats............ 30JX* 8 A4 047 Short fiber............... 37.17 9JJ0 1*40 0.14 0.09 0B6 Loop fiber............... 9.40 SB! 0.78 * ox 0.31 MpO 33 BO 3SB0 4041 JfaeO 0.14 0.0 KtO COs ` 040 0M OB OSI Ipnltion Loss Total li.74 97.13 14.09 100.11 14.00 99.7 .... - Not determined. ' 1' f . .:: . . - 1 (< -* . . ' , . T able 3.--Petrographic Analysis of Asbestos Dusting M aterials BDr*i Hosts : Tbe spproxlmste eompoettloe, based on particle* (except ehrreotlle) smaller tbip l* micron* end reported te percental* obtained Iron partlel# eount*, was ebrysotUe 14. serpentine 40, mapnetlte 1L carbonate* IS, tale It, o4b*r mineral* 4. for ehrreotlle, Bbert np to too micron* lone era Included. . ... Short fiber t: Tim material, bsfort beta ball mfital. contained a preponderance of fibrous ehrreotlle-and p la tr (nonflbroo*) serpentine. The approxlmat* eompoeltlon, by percent**, wa* ehrreotil* IT, eerpentln* M, macnetlte 10, nuarts t, brndto , other mineral*. Inclndlnc dolomite, eetlnollte and tremollta, U. .. . .. ' ' Lone fiber t: The material consisted prlndpaly of the fibrous asbestos mineral ehm otfie. Shreds ol oonseparated fiber* * to B mlaon* In diameter " d np to J?1*" "* *?. **n**h present. Tbe approxlmata eompoeltlon, b r peneatapa, * ehryeoUle if, eerpentln* is, mapnetlte S, brudt* t, other minerals, amont whleh were ealelt* and ehlorltle and micaceous minerals, 3. Only * trace ot Quarts we* observed.......................... The analysis of the Bor's floats asbestos, mod* by Dr. C. 8. Hurlbut Ir ot Hsrrsrd CnlTerelty, has been reported timbers (Hurlbut, C. 8., Jr., and William*. C. R.. The Minsralory of Asbestos Dost, J. Induct. Hyt. A ToxicoL 17:9!. 1936). t For the short fiber asbestos and tbe loo fiber asbestos the petropraphle analysis was supplement! with x-ray diffraction examination. periods up to 33 months. Some guinea pigs with six and nine months' exposure lived for an additional three years after cessation of their _ exposure. A preliminary report" presented observations after 29 months of exposure. At that time observations covered a period of only 2XA 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 amount of fibrous chrysotile was only 14 per cent, a rather low value. 8 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE Impinger samples taken soon 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 routing 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 man 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 menu. j ' , * * ; . ,,- Ta b u 4.--Su m m ary'oj Inhalation Experim ent w ith ' K in g's' Floatt Asbestos Dusi ,, .. N a ta n ot Experiment ui-. v . . Dust exposure continuous through- 54 oat lit* S IS Dust ezposon follow) by pro- ts loused ra td o e e to normal air IS `1*1 tili;.- 1 1 Tuberculous Infect!on a t sta rt o t dust exposure . .. ' 40 Controls to Infection: no dust ex posure . ...... 23 . . Animals :. guinea pigf rabbits rats guinea pigs guinea pigs rabbit rabbit guinea pigs guinea pigs - Maximum Maxi- 8urrtTal n u a After Dust Ex*Dust Expom . potur** . Mo. Mo. -- SS . 0 IP o * e 0 9 35 9 S7 9 30 | 19 34 .1 39 . 0 0 35 f ' v i i - . :: >-.C . SiTM.--. Basalts ' Trpteal-perlbroaehloler flbroele after IS month* Foreign body bronchitis Little or no reaction Kooprofreeatvc fibrosis Konprogrssslii flbroele Absorption of forclsa body reaction - Thmporary progression orinfection, followed by healing with fibrosis: Healing by resolution (one exception) Tuberculous Infection * after mo. of dust exposure, then residence . In normal air . . Controls to Infection: no dost ex posure - 12 guinea pigs 12 guinea pigs 14 0 ' ! No appreciable Increase In ruiceptlblllty to tuberculous Infection: healing with flbroele Healing by resolution j ' * TJ* m ines pigs were Infected with low virulence Be strain of toberele badDna. f 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 N orm al Guinea P ig s.--Guinea pigs inhaling this dust for periods up to 33 months had a characteristic fibrosis occurring in conical patches about the respiratory bronchioles. During this exposure the peripheral alveoli were not involved. The particulate elements of the dust were transported through the lymphatic system to the bronchial nodes, causing no significant reaction in either site ; the fibrous elements remained fixed at the points of original localization and 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. It includes a respirator/ bronchiole, at the left, branching and becoming an alveolar duct, at the right. ^iNote the accumulation o f 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 IVflite R ais and M ice.-- 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 P igs Exposed to Dust in inhalation Experiment w ith 100 per Cent Ball-Milled Asbestos Dust Exposure to Dust, Mo. 1 t 3 s 8 IS 20 24 23 Period In Normal Air, Mo. Amt. of Asb. % of Dried Ltior T o til SIOj, % of Dried Luos Totsl SlOt. % ol Asb Dust Exposure Continuous Durlos life 4.*$ . 0.21 . 4.28 0 4. 0.30 7.0$ 4-35 . 0J4 5.80 4.86 0.23 4.80 . 0 4.00 0.34 T. .0S o.m 1101 /- ' . 0 5.80 $.01 5.T4 0.70 1147 0J t 8.38 0J8 o.n 5.10 0JS TJ1 0 5.08 0.38 7.47 , 5.0 . 0J0 7.71 0 4.35 OJt U.58 5.55 US .18 ` .0 5.24 . - 1.45 23ES 0 5.40 1At ' ` IMS . 5.01 l.U . 2U5 . 5.65 US .. 22.80 3-2D US 24.81 5.30 ' U6 . ' .06 * 5.95 na ' 21.70 Dust Exposure Followed by Prolonfed Besldence la Normal Air . . TJS ist . 21,53 . * 8.57 2J El 5-23 0.88 . - 1 5 SM 0.87 14.55 8.38 0.54 1105 5.17 0.64 12.11 Reaction 0 0 0 0 0 . 0 . 2+ * The symbols Teresios tbe tissue reaction- la easb sroup represent merely the relative decree of reaction, ranclas from 0 to * (questionable) to 2+ (the maximum observed la this experiment). Tbe relaclootblpe apply only trltbln this table and eannot be compared 1th symbols la other tables. .- . . . . ... - ' f , .... ... . 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 tio n 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.-^:** ^ \ M V; t.- r .'v a r ' 'Vr .4 Lon-e F ibe*A sbestos__Duvs.t" ; - - S f r i f s .- . Since inhalation of short fiber and of 100 per cent ball-milled asbestos dust did not result in acceleration of the tissue reaction in comparison with that produced by King's floats, the hypothesis that short fibers of asbestos were of minor importance in the etiology of asbestosis was given added support, and attention was directed to the view that the long fibers were of primary significance in that etiology. - The King's floats asbestos used in the first inhalation experiment had a rather low content of fibrous 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 Dost.--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 dust . . . _______ . Guinea pigs, cats, rats and mice were employed m this inhalation experiment T he 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. T o 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 T able 11.--Sist-Frequency of Atm ospheric Long Fiber and 100 per Cent BallMilled Asbestos Dust Collected Inside Cages Type ol Asbestos Orslns, % Fibers, % ,------------*------------ ./--------*--------> < 3 3-10 > 10 <10 > 10 Clumps, Microns Microns Microns Microns Microns % Loot fiber.............. . 1.1 0.0 SJ 0.T 1.0 BaU-mllled ................ . SO.O SB 0.0 0B 04 11 Total 100 100 T able 12.--Summary of Inhalation Experim ent with Long Fiber Asbestos Dust j nature of Experiment Anima la Maximum Maxi Survival mum . After Duet Duet Expo XXpo- sure, 'sure, Mo. Mo. Results Dust exporare eon* l it tuluee pits . 3 tlnnous thfootb- out Ilfs '-- * Scute . . , SI S rtU 13 SOmice . 13 0 Definite fibrosis In It mo. 0 Slowly deeetoplnt fibrosis first teen . a t is mo. 0 Marked peribronchiolar fibroots first seen at IS mo. . 0 Limited reaction: no fibrosis - Dust exposure tollowed by prolooted residence In oonnel elr IS (ulnee p in 10 */ * . -. tobies p in IT lest* . IS .......... -- ' . . . . IS Clearint of Inflammatory reaction and definite eootraetloa of fibrous tissue -. C'earlnt of tnfiammatory reaction and aUtbt contraction of fibrous tissue - IS -' 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 at any point Some thicken ing of the larger bronchi with a chronic inflammatory infiltration was revealed, but it was considered no more than would be produced by a similar period of inhalation of any dust In guinea pigs exposed to the dust for 20 months and then removed to normal air, there was a marked tendency for cellular inflammatory reaction to 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 e response was quite similar to that in the 20 month exposure animals mentioned above. Small foci were always visible on gross inspection of sections of all guinea pigs of the 27 month series, but in no instance was there evidence of the reaction. . . if 5 'J ! r `Lo?ff *5er inhalation experiment: A , lung of a guinea p ic du<t tpo?!,r*- J b bronchiole at the center already shows an f Pi a? ocr iC ce,ls' and ,here " sl,ght deposition of collagen. Com- (X 2 0 0 * * * 6 A ' S^W,ng the react,on to baU-",iHd asbestos after 24 months J ? oi * ??ine* piR.with 16 !nonths' dust exposure. Again note a bronchiole iTJiL j 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- VQRWALD ET AL.--STUDIES OF ASBESTOSIS 25 began to appear in the medulla, and by the fourteenth month most of the node had been replaced by cellular connective tissue. This picture, which resembled that in early silicosis, persisted to the end of the experiment Some animals showed, as a variant, heavy sheets of diffusely distributed monocytes and large active giant cells, but there was never any necrosis or hyaline formation. The spindle-shaped Fig. 8.--Long fiber asbestos inhalation ecperiment: 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). * - .5 , lung of a guinea pig with 20 months* dust exposure and then 14 months' jiving 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 . . 9 Although asbejtosis 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 Tabi 13. Analyses of Lungs of Guinea P igs Exposed to D ust in Inhalation _________________ Experiment w ith Long Fiber Asbestos Dust Expoeanto Dost, Mo. Period in D o ra ti Air, Mo. A m t Ot . Total Ash, % ot 8IOs, % of Dried Lung . Dried Lung - f Total 610s, % otAih . Tliioc Beaetloa* Dost Exposure Coatlaooos During U te - N- 1 . o _ * %* s . 0 ... . 4-. . .. .* l.i s .. 0 !, 1 0 4.36 ' 0.04 4a s 0.00 <7 , ' 0.04 4.46 sa4AS : ", j . . 0.08 4.88 ' 046 448 046 4At - 0.08 4. n 0.06 4.88 0.12 6.08 . 0.00 - ti " * ' L10 2.11 048 IP c. u s . ut . 140. LU : 1.48 1.76 2.87 , .. 1.77 0 ` \ - - 4.72 0.10 241 ... a 0 . 4.0S ' 0.00 - 140 4.S4 . ..' 0.07 , 146 + It . . 0 M * . . . 0 ` *' * . - . * ,, *. * . ? rV; ' * 0 ` * a v 0 4SI. 60S . 5J 2.08 243 3.18 344 3.80 ' 348 14 3.82 048 ; 040 . 041 . 048 . 0.86 044 :. 0.43 0.40 042 046 , 0.20 540 4.00 64 12.70 * - 12.26 -- 1041 . 2+ . 1242 13.83 144 2+ 10.18 ~~- 84 3+ fj : 0 to. 0 *a * A V |5 0 3.40 3.74 343 3.08 348 | 5.86 ( 8.70 4.10 2.74 0.80 0.40 047 0.31 044 040. . 044 _ . 0.87 046 -. 1141 13.16 1046 1142 8.10 . 840 12.47 ' ' . ' 0.11'. ' 1240 ^ . 8+ .- 4+ . 4+/ / 4+ Dust Exporar* Followed by Prolonged Bealdenee la Normal Air JO 0 3.84 0.43 3.80 0.40 48 .. : 042 1242' 13.83 144 . 2+ 20 4 2.02 0.21 748 ` 241 0.27 0.60 2+ 20 10 4.18 4.30 0.24 042 5.73 5.07 2+ 20 14 5.01 0.21 4.1 5.04 0.18 348 + fl 0 3.40 0.30 1141 3.74 0.40 13.15 8+ n s 3.68 041 8.50 244 0.18 . 844 2+ tt ' 7 3.10 0.28 7.00 348 048 6.72 2+ *7 0 3.21 0.2 8.08 2.76 048 841 2+ 'rviboli averaging tbs tim e reectloa la eeeb troop reprereat merely the reletlre o em ot reietloa. r e a tla r from 0 to * (questionable) to 4-- (the maximum lor this experl- nieot). Toe relationships apply only within this table aad eaaaot be compared with symbols id otoer tablet. f 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 o f mineral matter in the lung ash was much less. ' ' Reaction in Cate.--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. With continued exposure, up to 42 months, reaction in the locations noted progressed to the formation of cellular con nective tissue which made well defined sheaths about the respiratory bronchioles and arterioles, marked lymphoid hyperplasia and lymphoid infiltration of bronchiolar walls (fig. 9 ). Typical asbestosis bodies were not formed, although there was Fig. 9.--Long fiber asbestos inhalation experiment: Lung of a cat with 42 months' dust exposure. Tw o bronchioles are shown with adjacent cellular reaction and collagen deposition ( X 200). . . ... -- ~ * J 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 Rate.--Although 20 rats were placed in the dust room, many died from pneumonia and were not suitable for study. Fire 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. A ll 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 M ite.--Out of 20 white mice used in this experiment, 11 lived a year or more in dust and died or were killed without showing an appreciable degree of pulmonary infection. The reaction to the inhaled dust was limited to phago cytosis by mononuclear cells. Usually these were widely scattered through the air spaces; a limited number were grouped about the terminal bronchioles, producing some thickening of their walls. There was no suggestion of fibrosis. Numerous asbestosis bodies were observed in animals killed late in the experi m ent Thus these animals exhibited asbestosis bodies without fibrosis. ...... Summary and Interpretation of Inhalation Experiment with Long . Asbestos Dust.-- The purpose of this experiment was to evaluate me 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 m this experiment than in previous experiments in spite of a smaller concentration of atmospheric dust and a lower mineral content of the ungs. . Furthermore, typical peribronchiolar fibrosis' was produced in cats, although in a previous experiment with short fiber dust peribron chiolar fibrosis did not develop in this species. ; The cause of The cellular fibrosis in the lymph nodes of the guinea- pigs is not clear. It did not occur in other inhalation experiments with asbestos.-*-.- -- ... . /. -v. * - ~ ..`C > . r 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'.of controlled dosages, the" relative capacities of various asbestos minerals to produce reaction could be compared. In these injection.-experiments, guinea pigs, rabbits, rats and dogs were used, and the mineral dust was injected by the intratracheal, the intraperitoneal and the intravenous technic, but not all the technics were used for each species. For the purpose of simplification the findings in each series of tests, except for dogs, have been condensed and reported in tables to which reference will be made later. In the case of dogs, only one test was made, and since the findings were negative, no detailed report is included. E xperiments U sing I ntratracheal T echnic 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. T abu; 14.--Comparison of Reactions to Chrysolite and Serpentine Injected -- . __ _ Intratracheaily Doaasv: Each animal u given an Intratracheal Injection ot OJ ee. ot a 5 per cent suspension ot Um duet. Two weeks later anotber similar Injection w u (Iren. Total amount ot dual Injected was <0 m i. Animals used: Six troupe ot tuloee pits eeeb (one croup tor eaeb type ot dust). - Periods a t which animals wars killed: One or two animals In each croup a t 1, 2, 8, Hi and 12 months attar last Injection. > Preparation ot dost: ChryeotDa (ball milled) nnheated: Ban milled tor 1,17 hr., dried and retround In scats mortar. Cbrysotil* (ball milled) Icnlted: Ball mined cbrrtotUe heated tor t hr. at about TOOC,, then cround In acate m ortar 2 or J min. Chrraotlle (Hbroua) nnheated: Ground In acate m ortar to pats 3 mesb. - ChrreotUs (fibrous) Icnlted: 200-meeb material heated tor 2 hr. a t about TOO C. So further crlndlac. Serpentine (ball milled) unhealed: Ball milled tor l.US hr., dried and recround In acata mortar. Serpentine (ball milled) Icnlted: Ball milled terpentine heated (or 2 br. a t about 7S0 C,, then cround In acate m ortar 2 or t min. Mineral Cbryeotlle (ball milled) nnheated Chrysotils (bell milled) Icnlted Cbyeotlle (flbroua) unbested Cbryeotlle (fibrous) Ignited Serpentine (ball mined) unheated Serpentine (ball milled) Ignited Site of Dust Particles 1 mlcrooe and lest S microns and ; lesa Hesults Grlndlnc destroyed eapaelty to cause flbroili. At t mo. considerable Inflammatory edema and cellular prolifera tion and localization of dust particles about bronchi oles: a t 2 mo., only a very allcbt prollleratlre reaction; at C, and 12 mo., widely scattered small mononuclear phagocytes. At 12 mo., a tew microscopic patches ot thin alveolar wall thlckenlnc with some adenomatoid chance In portion ot air spaces abuttlnc on tblekened bronchi. No aabeetoels bodies seen. Beaetlon limited to larce forclcn body clant cells without production o t flbroua tissue. 30-60 microns . approx. {MO mieroaa approx. v ` 3 microoi tod 1M 3 mlcrooi to d leas A distinct fibrosis. Beaetlon localised to connective tisane about terminal bronchioles; Uttla within those tubes. Contraction ceased adenomatoid appearance ot air spaces riven off directly from terminal bronchioles. Beaetlon a n a became smaller with procrees ot time; no new reclona Involved. No chronic pleurisy even at points abuttlnc Intrapulmonary chance. At 1 mo. considerable ... Inflammatory edema and to d ot cellular proliferation; a t 2 mo. well marked cellular proliferation and fibrosis oecnrrlni totally about respiratory bronchioles. This reaction developed before aabeetoels bodies had formed and was as advanced as th a t produced by 2 yr. tnbaladon ot asbestos dust. At 8 mo., traction less extensive than a t t mo., apparently due to contraction ot fibrous tissue; asbestosla bodies w en abundant. At Mi mo., - reaction atm less extensive, confined to the Immediate vicinity o t the small terminal bronchioles, where the sear tissue was quits dense and was beeomtnc hyaline In char acter. Sometimes It even obliterated tbs bronchiole. Aabeatosis bodies bad become scarce. At 12 mo., the well developed pertbrooehlat and Intrabronchlal sdeno' matold areas of fibrosis had produced conaldsnble dis tortion. More peripherally were patches ot pneumonitis with eosinophilic Infiltration, corns ot wbieb was belnc transformed Into fibrous tissue. These seemed to be pro ' cursors ot the localized, diffuse patches o t thin alveolar wall fibrosis seen elsewhere. Beaetlon limited to larte forelrn body ria n t " without proliferation. H eattnr the fibers, which made them brittle, destroyed their capacity to produce alcnlfleant reaction. Dust relatively 'Inactive. At 1 and 2 mo., simple pbagoeytosis without proliferation; at 8 mo., no ebange exeept possibly lymphoid cell Infiltration; a t Mi m o, a alight chronic pneumonitis: a t 12 mo., only a little pneumonitis without suggestion ot fibrosis. Dust relatively Inactive. Beaetlon essentially the same as for unheated serpentine. With Ignited terpentine, less tendency tor dust to be canted to bronchial nodes. 30 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE _ Fig. 10.-- Comparison of reactions provoked by injected long fiber and ball milled asbestos dusts: A , lung of a guinea pig which four months before had received an intratracheal injection of long fiber asbestos dust N ote 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 righ t In cootrast 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 The tests were made with long fiber chrysotile, unheated; and with chrysotile that had been, ignited to- destroy its flexible structure or ball milled to reduce the length of fiber to 3 microns and less. At the same time control tests were made with serpentine, which has the same chemical composition as chrysotile but is nonfibrous. A review of the findings reveals that only the unheated, long fiber chrysotile produced typical peribronchiolar fibrosis and that ball-milled material containing only fibers less than 3 microns in length failed to cause fibrosis (figs. 10 and 11). Fibers subjected to ignition also had lost their capacity to cause serious tissue damage. Ignition produced important changes in the chrysotile fibers, among them being loss of water, an alteration from a flexible to a brittle structure and possibly other changes. Experi- 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. ' - - .+ - Comparison of Various Long Fiber Dusts.--Some very interesting findings are disclosed, by the results of the experiments recorded in table IS. 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 ll.-Comparison of Rtactions to Various Long Fiber Dusts Injected ________________________ IntrairacheoUy d o w * u M*mlf.)*etl0ni oi M ** * * ver sent auapenalon siren two w k i part. ToUJ AbIidbI a n d : from A to (ulne* p io (or aeb duat. Pwiod. t whleb animal were killed: Oauaflr e t 1. 4, 8 end 12 m onth. .( te r le .t Injection. SI ot dart particle: Separated eo th a t moat ber were Irom 10 to 80 micron lone. Mineral Cbryaotlle (Ihettord) ChmotOe (Arizona: low 0Ir.2o%n cFoenaOteas)t; Amotltc Croddollto i (Bollria) - .. . Croeldollte ' (S. Attica) AntbopbrQlte Reaulta ^ M b e a w d T n 'u b le lii*011*1 ln,onn*tkm **Ten oppoaltaehrytotlU (flbroui) RVoCd 10 'n i,tio rd eteraotOe. Botn flbrotla ,*d arbertorla bodice produced with an arbe*tof eontalnlne yry little dcnoaiu^ jft^n jrin h jf^ SI1LbSr0U*f1** dlPte*dnnblantaolnbraoinbcehaitooleel*a abnoddleaaa wlineerer *' " Uuiar atau well torinad a t one month. With ace. eootraeted and oceoplcd (mailer a n a bnt waa m on deni*. nmiSi. " S "" * boae with Tbetlord ehmotlle. Pleurlay bout anaa ol maaaire r7 " " * tlon-, ~b*atoal bodlea tormed bnt w en few. At 1 mo. attar JT n 2 ' Ul U- >od * m? nf "a*k " proliferation boot broneblolea and * Obmllf*?. .WpUlBt1y,4 paro<jJe-'ctibo*n,T*T pparetriiablrloyncehliooalainrr plautembeena oott ih o ^ d hl'. r.n"*" kL.,.W**1'?nei, " **! eonnaettn tlaan* reaction but no byallnlaatlon: a t 2 m o- minute tod ot flbroda*wlihl ` i 001 broneblol; at mo., m atun aaheetoala -V.Ti!.*L-b.,_TfcB?* ot, contraction; conaldrrable ehronle pneumonltia ol p 'Pboeyte* and eoalnopblla. A t * mo., email lntra- brooehloiar flbroua pint* with tod ot m o n delleat* Dbroalt a t periphery. TIt!S iil.ibJiL.*n?02!2.D.d,,oUJ l* *Dd Peribronchlolltla with lormatlon ot IIS Banattatlon ot bodlea b e ta s bato n 4tb mo. t 'J f L i ? _ V 0,11' dre*>0ped by 8th mo. Bodice peraiat after 12th mo. ' A* 1 " O' beary endobronehlolltla and peribronchlolltla already f i ? * nd bbroela with tome necroala a t loe>l.lMt> * <*"** At I mo., beary. widely acattcred TMii2bI ? nKh o 2i*. ,n d Peribronchtolltte, now flbroua, with marked delor- ' 2L bronchiole and with an adenomatoid appearance. At 8 and l i mo., reaction In tunc eeeentlally the am* aa a t 4 n o . At 12 mo., tod ot *Bd b*ribronehlolltl. atlD larce, with more deoe* oorr*aaitzaiiIeeMtaa^l.air1o,ft,,1^pLeri2pihie0r,a?l1pJta7re0n,chbyromnac.hial t_ub^e* but no extenavlon Into,* Advanced _flbraa endobroncblolltl and peribronchlolltla. Beaded aabee- ** * '?? A t }. " 0l> bbrou* endobronchloutli and **5* " u* *nd (ome lymphocytic reaction. A t 4 S n ia iD i 5 ? * * * ? l. fal,n^d?5Ilt0"m*ob-l0lalrtela,colft<btrrodncbtihorUotulithaornoatDetrhebelcoanueae; trstion i m sdenomstold sppesrsnes.* 11 " r mlrk<d lymphocytic Infll- T^ l`nl,.^ ^ * * * d i f br?,Uf ndobronehlolltle and peribroneblolltte produced ,0 *g:Kto t*1 do); moat animal would not tolerate Wbroeto wen deraloped b a to n aabeetoete bodice !E?*t S S 1' derrioged flbrone bronehloUtie with lymphocyte* and fja n t cell and adenomatoid ebanin. At 8 m o- typical broncblolIU not S i^th ! dbrota a with * 6 % euepenilon. other. deeply talned flben with r food proportion of m n n ^ Mh. V >!*Lz?i, f* .At 1J " -> beary flbroua brooeblolltli, 2nl? ,n d udobronchlolltl, with lymphocyte* and slant ceuf, Terr marked adenomatoid appearance, . . L^ ^ V * h ! 5 / ! i i , *tl0.D, ,nd r l " t **" bot o dbroale. A rery few atypical l!? h l!l? ia l>0<!tei*i AU 0* nJ1Jr icttU rtd fod o f iotrabronehiolar duat localization; lymphocytic Inflltratlon of walU and a few fn ifw S 'iL i * i d " nj-., Httle erldenee ot dut: a tew bronchiole* totU tntten'ot^w all/ *"* " li> ta *dJ* " nt IreoU and with lymphocytic Tremoli tc Brsclte Glaat wool , #b25?J5l!00*2!0!*i:,. At. 1 t"0,1 *" * ' o t d u ,t localliatlon with col l i ,TJ!`>'*.,n d Inflltratlon with acute Inflammatory cell, macrophase And slant cell*, within the area were a few foci of flbrotu tlaaue !* I hypertrophy of alreolar epItbeUum. Many bronenioiet packed with flhen. At 4 mo., feofral appearance of talon onchaofad: pteura illkhtlr tblekenad over h e arr localiaatiooi of duat An occaiiooal aermented asbeatotia body aaen. At 8 mo,, many tod of fiber* in broochlota and alveolar duct* with cellular reaction aa before: also, aome foci abowed dlatinet eoUaten depoaltion. At and 18 mo., reaetlon aa before with obroala about broochlota more apparent becauae of con* *TtcL0P._#D<* decrease of inflammation. Giant ceila prominent. Pleura markedly invoiced. r ypicai tiSSI nnroua Z'JES?" ?e1*n*dobAr.on1cJhlnooii"tlai i and perfbroocblolltla like reaction to Un,lTe odobroncbloUtl* and perlbron- chiolltla with aiaot celts: dense fibrous loops within bronchioles and cellu lar Abrasi* about them: adenomatoid chance present. At f mo., heavy b r o o c h f l r and peribronchiolar fibrosis producine marked deformity with distortion of tubes sod obliteration of rurrooadlnc sir spacci: ccflaoblnratarasalbseetpslnatolfes. lawiAbthot odui1e0tsHbsyeametnfe.n.l,sAadttelon4naeabnufdltb8rwointtuho Mfbewrloitanteluebcllcoehil:latlnn*goewn: eitfchirborsoaiusb.se*tTitsoysputie bodies. >o pleurisy. No extension to surrounding long. No flhroria within a year. A t 1 mo., no reaction Infide bronchiole*; tn peripheral elr epaee* clamp of fU o t eeU packed with floe plcule* of Slaee with lymphocytic Infiltration o l adjacent wane; po aabeatoafa bodlea. At 2 mo., reaction lea* lotcorc then at l mo.; falr-alzcd clump* of eion. esnadteodhrosnlaenhlttlap.baAfol cy4teaendeo8otnaola.,lurseaacptlieounleeMOa)nddlmpalortlalchllenir.ofArti-a12*:mno.o, local area* ol oncumooltl* with no flhroali or codobroochltla; moderate Dumber o( rmooth IroD-italnlc* Ubera. ^ O R (V A L D E T AL.--S T U D IE S OF A S B E S T O S IS 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 mjectipn experiments:' A , lung of a guinea pig four months after an intratracheal injection of amosite. The inflammatory reaction exhibits pronounced acctsndbtsoa 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 arn im lation 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 . F>8-_ 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 nonsilideous fibrous mineral there is peribronchiolar amimniatinn of cells and deposition of collagen similar to that shown in A and B of figure 12 (X200). . . B, lung of a guinea pig which four months before had received an intratracheal injection of glass w o o l 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 o th er figures. Glass wool fibers a re 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 - - . . - * T able 16.-- Comparison of Reactions Produced by Long Fiber and Short Fiber Dusts Injected IntratracheaUy Do m i*: T wo Injections of OS ee. oi a 6 p tr cent iu*pen*ton plven two w ttki apart. do* waa 30 me. . Anim.i used: Six tro u p i o t c u b ia pica. Periodi a t which animal! wen killed: 1, 2, 9, Sti and U month* attar Injection. Total Mineral Chrysotils (Thetford) Amoilte CroeldoUta (Bollri) Antbophylllt* Tremoli U Broclta 81 of D uit Particle* Baault* Lone fiber. 2D-S0 micron* Short fiber, 2 micron* and lee* Lone fiber, 20-60 micron* Short fiber, 20 micron* andle** Lone fiber, 20-60 micron* Short fiber. 30 micron* and lea Lone fiber, 10-60 micron* Short fiber, 3 mleron* and lea* A dlstbct flbroal*. Safer to ehrysotll* (fibroni) un heated b table H. No fibroii*. Baler to chrrtotll* (ball milled) nnbeated b table it. Typical flbroo* eodobronehlolltl* and peribronchiolitis. Refer to table U. Reaction limited to phacoertoil* with lrmpbocytle Infil tration o( adjacent wall*. Sbort fiber* packed bild* swollen obacocytes: loneer one* tree; mm* coated to form trpleal aibeitoal* bodies. At 1 mo. alter injec tion, alveoli contained cood-stxed plant cell*; moat phacoeyta* were wtthb air ipacee and bad not micrated to walla. At t mo., tree extracellular fiber* had worked tbemaelr** b t o b te n tltia l tluo*. where there wa* extenslvt proliferation ol lymphoid cell* and monocyte* b at no fibrosis. At I mo. iorelcn body reaction with aome pnenmonlUi, no bronchlolitl*. Typical aabto*i* bodb* present. - Advanced fibrous sndobrooehlolitl* and peribronehlo- Utl*. Bate* to table U. No flbroet*. At 1 m o, air spaces compreu ed and lareely fined with plant eell* packed with dust needles. Wan* heavily Infiltrated with monocyte and lymph oid cell*.. At 4. m o, a moderate decree ot cellular Infiltration o t walls; small elant cell* packed with dost splenica. At and t t t m o , ma**e* o t clant Us, containbe mineral pertIdee, in (mail bronchi but not ,, m respiratory bronchiole*; smaller one* widely n a t tered b term bai air spaa. Numerous asbestos!* - bodies. No reaction ta connective tissue. No endo bronchial proUferatlo. At It m o , many scattered small monocyte* peeked with dost. No endobron- - ehltl*. No peripheral fibrosis. In lymph node, sliebt reticulosis; no fibrods. . ' Lympboeytf* Infiltration and plant cells bnt no definite fibrosis. Rater to table IS. No fibrosis and practically no asbestosl* bodies- At X - m o , local eoOeetton* *1 duet-filled monocyte* and a lew plant cell*; a t < m o, some adenomatoid epithelial reaction; a t > m o, simple poenmonltls with phapoeytosls of sbort fibers; a t 12 m o . Isolated and sharply localised collection* ot dost cell* bslde air spaces about term bai artattoies. Reaction In wall* limited to lymphoid cell b filtntloo. No fibrosis. In lymph node, reaction limited to sUcbt prominence ot reticu Lone fiber, 30-60 micron* Short fiber, 30 micron* lum. fibrosis about bronddolae. Baler to table IS. Simple fotclpn body naetlon. No acute bfiammatlon. No accumulation ot duet b or about term bai bron.. ebbi*. No endobroaebltis. At 1 m o , scattered small Slant ceils and consumable bfiltratlon o t adjacent walls with monoeytee and lymphoid cells. At 4 m o, ' little chans* except more ceOnlar bfiltratlon ot eon - neetlv* dseoe. At I m o , lymphoid bfiltratlon and ' ' ` thicken1ns of wall* about some but not all term bai Lone fiber. 30-60 micron* Short fiber (mad* by crusbbe lone fiber* with rubber police man) bronehloie*. , .... Typical fibrosa endobroneMofitls and peribronchiolitis Uk* reaction to beate* mineral*. Refer to table 16. Inert type o t reaction. At 1 mo. after Injection, small monoeytee. widely scattered throufh air spaces; focus of atelectasis with lymphoid bfiltratlon o t eomprresed atripaca wills. Vo wdobtoodiial n tetlo o aa with cbrysotlle. At 2 m o, reaction similar to that at 1. mo.; typical beatosi* bodies eeen. At 12 m o, small clumps ot bactlve dust-filled phacoeytes; no fibrosis. No reaction In lymph nodes. Comparison of Long Fiber and Short Fiber Dusts. With quartz dust it has been demonstrated that the smaller the particles the more 36 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE intense is the tissue reaction and that particles larger than 3 microns in diameter cause little, reaction. In the case of asbestos, however, the reverse is true and apparently only long fibers have any specific effect, as was suggested by the inhalation experiments. This is confirmed by the data of table 16, in which a series of tests with fibrous minerals is reported. When the injected dust consisted of fibers 20 to 50 microns long, all the fibrous minerals tested except anthophyllite, as noted in the preceding section, produced fibrosis; when the material was prepared by first grinding the fibrous dust until the length of fibers was reduced to 20 microns and less or, in some cases, to 3 microns and less, none of the injected dusts caused fibrosis. .. ' " . -... These results differ from those of King, Clegg and Rae,11 who reported the production of reticulosis comparable to the experimental silicotic nodule in rabbits receiving monthly intratracheal injections of 100 mg. of Rhodesian asbestos fibers, 15 microns long, and the produc tion of diffuse interstitial fibrosis in rabbits receiving similar injections of short fibers, 2.5 microns in length. We believe this dose, especially in the long term rabbits, is highly excessive. In our experiments the dosage was kept low in order to minimize untoward reactions which might obscure the peribronchiolar type of fibrosis which characterizes early human asbestosis. _ . t <' '. 1 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 far different from quartz in their action on tissue. It has been repeatedly demonstrated that intravenous injection of quartz particles 3 microns and less in diameter will cause a typical tissue reaction with the development of hyalinized fibrotic lesions in extrapulmonary sites, such as the liver and the spleen. Asbestos minerals, however, on intravenous injection generally produce only an inert type of reaction, as is revealed by the results given in the table. The reason for the early deaths in the experiment with chrysotile particles is not clear. E xperim ents 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.  T able 17.--Summary of Injection Experiments by Intravenous Technic --------------------------------------------------- --------- 7--------------------------------------------------------------------------------------- Total emouot o t d u ll v i i 1.0 O n., divide! Into 20 equal dowa (each done v ia 6 ce. o | a 1 per caul anaprnelou) which were iclveu twice a week lor 10 wcoka. Maximum Survival i Size of Alter l.aat Habblta lojectlou. ii Mineral Dust P irilrln Uaod Mo. \ ' Reaulta ( | Clirym>lllo 1 utlcrone aol leva (The! lord) (hall milled 192 hr.) ` / *1*116 rabblta did uot tolerate lutravenoua Injeetlooa of finely ground chryaotlle ami 6 of the 0 died 1 after l to 0 Injection of even diluted auaiwualoua; the other animal died after 27 Injeetlooa of one* quarter atrenglli auepcitalou (6day after Arat Injection). Reaction limited to few large giant phagocytca of Inactive type In liver, eptocu ami lunge. No thrombi of dual cell area In pulioouary capil larice. No deflulte explanation for fatalltlea dlacovered, but material uiay liava been retalued In ; heart, caualng local thrombi. Ainoaite a microtia and leva t * (ground In agate mortar) . 17 Advanced pulmonary Infcctton killed 1 animals a t 0, U and 17 mo. after laat Injection, ahortenln* Intended duration of experiment aod complicating picture. However, rabblta killed earlier (S aud 0 mo.) ahowed only loert phagoeytoala with no progreaalou lu the 6 mo. atilmal. The laat two (11 aod 17 mo.) w en probably the tauie although focal nccroala of tho Uver and amyloid of the apleen made Interpretation difficult. (rocMollte Tmlcrone aod lata 4 ` (ground to agate mortar) . IS Reaction waa th a t to ao loert aubetaoce with no chaoge In 12 mo. (Other obaervatlooa a t t, 1 and 0 mo.) Simple phagoeytoala of particles. No temkucy to agglomerate and no cliaoge lu adjacent tlaauee. Grinding tlie dual to alsea of 3 microna aud uuder destroyed the llhroua alruclure of tide mineral, aod the Injected material reeembled pint rather than Altera. Anthophylllte I mlerona and leaa 4 (hall milled M uuhr.) tl Reaction eeacotially that ot an Inert mineral. Obeervatlooa made a t 3, A, 12 and 24 mo. Only auggea* lion o t Irritating propertlea manlfeated In apleen ami lymph node, but n o t liver, ot the 24 mo. rabbit. In thta aolmal there had been proliferation of mononuclear and giant cella th at waa not prevent In eittier apleen or lymph node of 12 mo. animal. 11m abecnce of aaaoclated ObrobUatlo reaction In three orgaoa and of any chauge In the liver condition Juetlllea the claaalAcation o t aniliopbylllte aa ao Inert alllcate. No fiber were retalued In luog to deinonatrate whether aabeatoala bodlea would develop. Tmnollte S microna and leaa 4 (ode Iron) (hall milled HO h r .) Tremoli! (aoda) 1 mlcroua and leu 4 (ball milled 48 hr.) 1 Reaction aaaantlally th at o t ao Inert mineral. I.aat animal killed allowed a little prollfrratl'in and lympbocytle InfUtratloQ In liver, not eeeu earlier (a t 3, 1 and 12 mo.). No evidence of any activity in lealone in other orgaoa. . U An Inert foreign body reaction with no change In 24 mo. Oleervallone made a t 3, 0, 12 and 24 mo. >v. I- J .t .......... -*14 V -T: >- T able l8.-^S~uwm ary of Injection Experim ents by intraperitoneal Technic D one: Each Mineral Chrysotlls (Thetford) Chryaotlle (Thetford) Amosits Oroeldollta Antbophylllte Anthophylllle (originally . labeled tale) lYemollts i (soda-iron) Tremollta (aoda) Anthophyllltc PyrophylHU (fibrous) Pyroptiylllte (crystalline) animal received a Ingle Intraperitoneal Injection Size of Duat Partldea Guinea P its Heed Maximum Survival Attar Injection, Mo. 1 mlerona and leaa 1& Sfi (ball milled 21 hr.) Loot fiber (throuch 100mesh) 1 mlerona and leee (ground In agate mortar) 8 mlerona and leaa; alao eome long eplculee (ground In a t at mortar) 8 mlerona and leaa (ball milled 1,400 hr.) Moatly 1 mlerona and leaa; aome libera 80 mlerona or more long (ground In agate . m o rtar) 8 mlerona and leaa (ball milled I t hr.) 8 mlerona and leaa (ball milled 48 hr.) 100 mlerona and leaa 100 mlerona and Irfll 100 mleroos sod less 4 1 0 It 7 It ft tt s It 6 It 8 . IS 6 12 6 It 6 It of 1 cc. of a 10 per cent duet suapenalon. Total amount of duat Injected waa 0.J Om. Reaulta . " S ! " ' ? SU Zt p*.nlcle*. b* Phagoeyteo, chiefly multlnucleated !T| \ **?? toJ*ct,on flbrou* etemrut* of duat appear to bav dlaaolved leaving only the Inaoluble magnatlt, a eontamlnanL No reaction In aurrouodlni fa t or areolar lim it. No t i u r rajeetloo.* ^ mflbmal lymph nodea. Obaervatlona made a t Intarvala# from 1 ^to M mo. P*,f!"lt* l>br u* " * tU" n P r Ju J. ddleate and nonhyallne. Atypical aebeitoala bodice developed, but all wert unusually smalt. iNo evidence of extra long fibers seas. Observations only e t | mo. Infection Interfered with Interpretation. Duat reaction appeared to bo of Inert typo aod limited to pbagoeytoala with a moderate tendency to lymphocytie Infiltration. ObewaUona a t I. 1. 8 and 11 DlOe * Duat tod eonalated only of larae mononuclear and flo a t phayocytea aarrouaded by a mlolmum amount of eeUular connective tlaaue. The Injected duat contained not only One material th at In grinding bad been maahed Into In efu lar plataa b ut alao many lo n f aplenlaa 10 mlerona o r more In teofth. No aebeatoela bodlea ecen. althouah the looter aptaolea appeared lightly earollen and greenlah. Obaervatlona a t 1, l, and I t mo. _ . EaaentlaDy Inert foreign body reaction. In early anlmala ft, t aodI mo.)foctia of monocyte and email giant ceOa and a little central oecroela. In the I mo. animal tb e n waa alao alight peripheral flbroala. A t I t and t t mo., nonprogreealve maaa of monocyte and giant ealle; no ftbroela. Reaction, Which eonalated of very largo giant eella aurrounded by a variable number of lympbo- . cytee, waa much heavier to the unintentionally long Sber* than to the One duat In tbo experiment . above. There waa more or leva proliferation of flbroblasta producing cellular connective tlaaue . vlelble In arena where tha quantity of foreignparticle waa not aog n a t th a t It obacured the reaction. Obaervatlona a t I, t, S and 11 mo. .*.. -> . i 1 Inert type of reapone never progreaalng beyond the atage of very alight lymphocytic reaction about manner of duat-Ailed phagocyte. No flbroala. Obaervatlona n t 1, *, 8 and 11 mo. Inert nonproficgalva foreign body typo of nactlpn. No flbroala. Obaervatlona a t I, f, 8, II and 18 mo. 1 . . ' ( - Dlatloet aarly flbroala produced by antbophylllte and flbroua pyrophylllte with aubaequent refreealoo; cryatalllne pyrophylllte Inert throughout. At l mo., giant cellc about long thick apllntera; a t I mo., definite flbroala replacing giant cell of antbophylllte and flbroua pyrophylllte reaction; a t 8 mo., flbroala which atarted a t 4 mo. bad decreaaed, eapedally with flbroua pyrophylllte. At 12 mo., reaction to all three duata eonalated of foreign body giant cella with lymphocyte but without neeroela or flbroala. No aabeatocla bodice. - ' * Each animal receiving long liber ehryaotlle waa given ao Injection of 1 ee. of a OA per cant duct auapenalon. \ FORWALD ET A L --STUDIES OF ASBESTOSIS 39 . . "Paoracrtv* Aenow or A l u u in u v Compounds "W hen colloidal aluminum hydroxide had been added to a suspension of long fiber chrysodle 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. ......... F ormation o r 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 is su e R eae n o w to A sbestosis B odies 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 F 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. f. 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 . / . ______ _______ T able 19.--Analyses of Fibrous Mineral* ~ : Plbrooj Minerali 8IOt T ttO t % % TtO % AltOi CaO % % Amorite-................. 4BS* 4M U M IM i.m Amphibole............... 45.04 4.06 1 .0 ILZt AntbopbrUte-......... U M OST 0M 0.44 Brodtc..................... OSO o .n 0JS 0.40 0.04 OhmotDe................ M LSS 0M 0M CroeMollte.............. U M 16S7 4M L01 0M Tremolile................. U M 7J1 ..... OM ', 4*44 MrO % (SB SSO ' 1IST . USO USB USS KM NliO. % 0M 0.41 OlU OSI 0M SSt 0.1$ In itia l lom X0 < io*c. > io s a % % % OSO . 0.BB SM 0.11 OJt ISO' 0.U ; o s i . 4.QB 0.1B < OSI MSI oss 4J0 14JP OST 0.02 t u 0M . 0S4 1.71 Tots % nx MX UM1 M.3 WM WM W. 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. COMP U CATIONS . . 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. SuSCDTTBtUTY to T ubebculous I kfechon 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 animal.? 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. SUSCEPTHtLITY TO No.NTUBHtCULOUS iNnCTTOX 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 ' -* *V .' ' . * ~. - COMMENT yAND SU'MMAKY . 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 intcatracheal injection to long chrysotile asbestos fibers. *- * *'' . , Both inhalation and injection experiments provide ample support for this statement Figure 8 A reveals the cellular fibrosis that occurs in guinea pigs following inhalation of long fiber asbestos; figure 9 shows the fibrosis caused in the cat by inhalation of long fiber asbestos dust. Similar but less extensive fibrosis occurred also in rats and rabbits (table 1). Mice and dogs failed to respond. This variation in response of different species to identical dust exposures is still to be accounted for. B. Long asbestos fibers are essential in the production of the'peribron chiolar fibrosis; short fibers are incapable of producing this reaction. - 42 INDUSTRIAL HYGIENE AND OCCUPATIONAL MEDICINE ' . . * Inhalation experiments with asbestos dust suggest, and intra tracheal injection experiments confirm, that peribronchiolar fibrosis is produced by asbestos fibers between 20 and 50 microns in length but not by particles shorter than 20 microns (tables 16 and 18).. This indicates that the minimum length of fiber possessing the capacity to produce the typical peribronchiolar fibrosis in animals is somewhere between 20 and 50 microns. . Pointed studies have not been carried out .to determine the upper limit of effective fiber length. It'appears, however, that that limit will be determined by the inhalability of the fiber. r.j ,,: -t - j- _ . G The mode of action of the long asbestos fiber in the production of . -vv asbestosis .is primarily mechanical rather than chemical in nature. A ` \ . \ ' The evidence for this conclusion has been reviewed in a preceding section, page 3 9 .` 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 v t - : ' ;"r " ' D. Typical experimental asbestosis was produced by the inhalation of\ an atmospheric suspension containing an average of 138 million ^ asbestos particles per cubic foot of air by light field count, of which. less than. 4 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. . -. 1 / E. The duration of exposure required to develop the pulmonary reaction . to inhaled asbestos dust is inversely proportional to the concentration. _ of long fibers in the atmosphere; as the concentration is increased, the reaction develops in shorter time. ..... ....... * .. ' The basis for this statement appears in the data of the inhalation experiment with long fiber asbestos. For that experiment the average concentration of the atmospheric dust was about 40 million particles per cubic foot of air, and size-frequency determinations disclosed that 6.7 per cent of the air-suspended material consisted of fibers longer than 10 microns (table 11). Thus, 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. . -,,.s vj , - .' ' The' experimental investigation shows, in fact, that~on~di^litinuance ~ of exposure there was an appreciable clearing of the mature pulmonary V ' 'V'hv VORWALD E T 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 been retained in the lungs. After 24 months of continuous exposure the average J.\ *- } * .t m j-.v f . ' ` / ..\ e- i m.-* ..... V/ --y),' -A. i&* ; :- ft- , ?v v U ^ r -- *-* ~ * ~ 'i . ' ' > '*** S-Ktr1'** *i. ^ --~ ^ ^ 4' v \ \ Jc : ^ V * "j , - i ^ v > * S > 6 -- Ball-milled asbestos inhalation experiment: A , lung of a guinea pie with 24 months dust exposure. A bronchiole is shown at the center, with a slight accumulation of phagocytic.cells but without the formation of collagen ( x 2 0 0 * tnh^atlrm^A^n * P " "" pig with 28 months' dust exposure and then 12 months' "rmal. air;. The reaction is much like that shown in A . but th e r e b a slight deposition of collagen, most apparent at the left ( x 200). lu e 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 i l l i o 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 tnillioa far the short fiber experiment . - Guinea pigs, rats and mice were used in the inhalation experiment with the 100 per cent ball-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 Asbettoe Dust Mature of Experiment Dust exposure con tinuous tbroogbout llte Duet exposure fol lowed b r pro tonted residence In normal air - Animate M fu lata pic Meats tlm lee Ucutnea pics f Mari ni urn Duet Expoinn. Mo. Ha> u a Maximum Survival Alter Dust Expo rare. Ho. 0 0 0 IS f , Besultr So appreciable pulmooary reaction Mo racceatloo ot eabejtoets Mo eucgeatlon ol asbestosis Etbroals typical o t aabeatoeia was present i t mo. after exposure eeaeed In an amount sufflelent to be visible croaslr; smaller tod ' eould be seen mlcroacopleaOr a t t mo. and 8 mo. alter termina tion ot exposnra could be seen. At 24 months (fig. 6 A ) there was still no change large enough to be seen with a hand lens, although microscopic examination revealed cellular accumulations about terminal bronchioles and many more asbestosis bodies, chiefly within cells. The lungs of animals exposed for the full dusting period of 28 months and afterward living in normal air for two months revealed the changes described above and also very slight peribroochiolar fibrosis. For exposed animals living eight months in normal air the findings were similar, but at 12 months three of four animals showed grossly visible characteristic peribronchiolar fibrosis with adenomatoid change (fig. 6 B ). . . , . . The tracheobronchial nodes were essentially normal until exposure had been continued for more than a year and a half. Animals killed at 12 months and at 16 months revealed a few minute collections of phagocytes containing particles but practically no fibers barge 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 been a slight increase in reticulum but no fibrosis. N o 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 eva uate 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. . ; / , ' ^ ^ *0Ur sPec,es exPoscd in this experiment, only the guinea pig and to a lesser extent the white rat responded with characteristic peri bronchiolar fibrosis. The cat reacted with atypical subpleural fibrosis and the rabbit with only slight parenchymal fibrosis. ' , Ball-M illed A sbestos D u st . In the inhalation experiment with short fiber asbestos dust a small quantity of unpound short fiber asbestos was mixed with the ball milled 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. ` - Composition and Atmospheric Concentration of the Dust.--The dusting material was the ball-milled, short fiber asbestos used in the previous inhalation experiment but unground material was not mixed with 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 IS 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 IS per cent, but reliable values were not obtained. i .. . . The dust concentration during the first seven months of the experiment was about 100 million particles per cubic foot of air. After the wire brushes were VORWALD ET AL.--STUDIES OF ASBESTOSIS 17 alveolar ducts in which the wall* 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 o f 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 Cats.--Twenty cats were used in this inhalation experiment with the short fiber asbestos. Eighteen were kept in the dust room continuously until put to death, the exposure period ranging from one month to nearly 54 months. The other two were removed to normal air after a dust exposure of 31 months: one of these was killed five months, and the other 24 months, later. In general, the tissue response was confined to microscopic 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 early in th e ex p erim en t b ut w ere seen in atl an im als th a t had been ex p o sed 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 - ' Total - Duration--. Asb.% - 810a. % . ol Expo- ol Dried of Dried sura. Ho. Lune lun . - _ Total SlOa. % o lA ab _ SB 0.07 - 2.1 . 8 S.l . 0.05 . .. IB S.7 0.04 - - 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 R ats 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 2B 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) Short Fiber Asbes tos _ 0.0 ; 0.0 0.08 0.15 0.15 610, % ot Dried - Quarts 0B1 0B1 2.94 1.44 4.40 Ferroelnous Cbert OBS 0B2 3.45 2.40 8.0 Lons . GrpenmQuarti Mix ture 0.08 0.07 0.11 0.32 ' 0BS i Total 6IOt. . - Short Fiber Asbes tos Quartz 2.8 a .: 2.5 11.4 L8 41.5 3. 29.4 3B 66.6 % of Asb GypsumFemi- Quarti elnous Mix Cben ture 3B 2.8 3.8 2.0 34.4 3.1 28B 9.1 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. A t 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 VORWALD ET AL.--STUDIES OF ASBESTOSIS 15 Only aiter exposures had continued ior approximately one year was there an appreciable tendency far dust-containing phagocytes to gather into dumps. 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 clumps were sometimes quite prominent, and sometimes changes in the epithelium resulted in the adenoma-like or "adenomatoid" appearance (fig. 3 B ) previously described in the section review ing the experiment with the King's floats dust. In most of the subsequent members 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. 1! IS to . 14 SO 34 US' to to Period In .Normal Air, Mo. Amount of Asb, % of Dried Lunt Total 310,% ot Dried Lune Total SIO;, % of Aeh Dust Exposure Continuous Durln Life. s.ot 0.51 10.33 0 4.58 0.4 10.08 5.10 044 1044 5.00 0.40 0.0 0 4.76 0.4S 0.00 * 4.06 043 10.00 0 SM 0.85 14.4 6.4S 0.90 14.07 0 $.42 0.78 14.48 5.50 . 0.78 1440 0 5J3 OX 1740 4i5 . 1.37 19.40 0 606 0.75 11.37 6J5 0.00 . 15.11 Dust Exposure Followed by Prolooted Residence In Normal Air 4 * 5.16 5.11 0.48 ' 0.30 OJO 740 10 6.11 ' " 0.03 lo.ti : 1 .. 044 841 4.77 ' . 0.15 541 li 5.16 o.to 5.00 4.77 041 440 Tissue Reaction * * 1+ 3+ 4+ 4+ 1+ 8+ 1 + The symbols sversflne tbe tissue reaction in each croup of rulnes pics represent merely the relative derive of reaction, ranrlnc 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 n lungs with widely different amounts of tissue change. For example, the ash / 14 INDUSTRIAL HYGIENE AND OCCUPATIONAL- MEDICINE 'iSjsg ^ " rece,yedcontained "*ny long fibers, it was ground in a steel ball used *! th* PartideS * 3 raicrons or Iess m * W hen S ^ e h ^ L ^ i I T " 8 maChine> *" fine* ^ ound asbestos tended to TM tr^ l ? beT ,C n<?Ce5" r7 t0 ** ne volume of the unground 'J T f ,th' *round * " * satisfactory dust cloud. a s t ^ T ^ " " Lh' re that the addition of the small quantity of unground a sb ^ o s was unfortunate, because h confused the interpretation of r e s u l t s . ^ of. .*** ,hort fibcr asbestos as received is disclosed by the *TMlr 5 a TM tables 2 and 3. Samples taken fo r e was IIo ,, ^ " g yi rdtd abot the same values on analysis, indicating that there was no contamwabco from the null or loss o f witter content > atm ol'JriT T T Aurin* he' e x p ir iie n t, the light field founts for atmospheric samples coUected inside the animal cages with the impinger apparatus' ^ T r f t ^ r ^ T l ! 0" ,0 ^ milUOa Th* *Veraie ^ ""ts was 130 m illbn for for i , e tw" y l r 134 miIIionfor * e * c o n d y e a r jn d 140 million S" e' fr*<luenp ; measurements of air-floated dust from inside the at a magnification of 1,300 x revealed a great -preponderance o f fine partidtJ, nearly V T able 5.-- N um m ary o f In h a la tio n E x p e rim e n t w ith S h o r t F ib e r A s b e sto s D u s t V N'aturt e t Experiment . Dwt^ exposure eontlnooua througb- Animals ss ruiues pits ra ts IS eats 7 rabbits Dust .exposure lollowed by Prolonged residence In normal air U mines pin 1 cats 1 rabbit * * " M ixlnon' Usai. Survival mum Alter Dust Ex- Dust ExPOSUTC, POSUK, Mo... Mo... r* , I EesuluTp'. M 0 . Bats ot resetion sbout th asme ss In exiMt I tnent Itb Klnt's floats asbeatos but txteatl St - of Involvement very mueb lesa t I 0 Cbaraeteristle patebts ot perlbronehlolar flbi* I _ sU; no aibeatola bodica----- I 54 * 0 ' Huboteural reacikm only > I 47 * : Ho ibroals sesti grosaly; microscopie erldetwl . coxfpaolivusro*la;r .all tb-leke. n!lng after se tnootu-Il U ' pf!uS^!-!n!eIU}fbe_re, l*ea,,-r.l^yrae,s0uvb*U, s<b"e>d"nomr dieldlnoulibetbj SI S4 Said as tor continuoui exporare 8 w i 1* to oottauoui xporure; evideocf ef| i Ufbt rerretaion * xpootb the animals were exposed to 100 oer cent bau.m iu shstn. .O P ^ e i ' i n ^ l V c ^ S M d .*0 l0 n , flbOT 10 & mixed Itb tbe ground srbesto. , 90 per cent of the particles seen being smaller than 3 microns. It was estimated 1lu0 LmvicPrr^onsX!in" lTengythi!. P*r Ce"` .. f th* duS` W" " the form o{ fiber* P la te r than Whi,e n U t " ** " d ^ b b its--were used in Us experiment The results of the dust exposure, summarixed in table 5 are presented in greater detail below. , ' e r o o ^ T m ^ l" fC*rW a P,Vr Eigl:ty guinea pigs were riP "y placed in the dust o ^ l i 21 th.em. were la,er ehminated from the experiment and killed because fa f c S fa ^ T S T * CerV,Cal lymph nodes `housht to be due to imercurrent m fe on of the upper respiratory tract Of the other 59 animals, 46 remained in the dust room until they were killed or died at periods up to 34 months and l" animjJs were transferred to normal air after being exposed to the dusi la f tiMUe r" Cti n provoked hy the "haled short fiber asbestos was n *an,e tha` alr" dy 0bserv* 1 fa the Pdrim ent with King1, float, bestos. The rat of reaction also was approximately the same, but the extent .. 7 -- `J r A t e 14 " "* >' p i " " ddeetLectt^ionn, had been prC^odunc`ed7 in thegegnueirnae'aly prigesq.uired microscoP'c examination for 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. *7 T T T ^ --- .y j - " ' "'--.'.r-T .......................................... 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 Ftsut 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 asbesfos- dust. . I t 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. Since 10. Vorwald. A. J .; Pratt, P . C : Durkan, T . M .: Delahant. A . B.. and Bailey, D. A .: Siderosis: A .B en ig n 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 2 0 0 ) . VORWALD ET A L --STUDIES OF ASBESTOSIS 11 Asbestos bodies (fig. 2 B ), first seen in the lungs of the guinea pigs that had h-ii-i 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 P ig s Infected with Tubercle Bacilli at the Ontet 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.1* Seventeen of these died from intercurrent pneumonia. Briefly, the results were as follows: Ten revealed some evidence oi spread of the tuberculous process (fig. 5 A ) ; in 6 of these it was confined to the lungs, and in the other 4 the abdominal viscera also were involved. Extension of the infection was first seen after seven months of dust inhalation; during the next 20 months more than half of the animals showed actively spreading tuber culosis, and in 3 of them small cavities had developed. During the last eight months no animals exhibited any evidence of active infection although in half of them the healed fibrous scars of previous spreads were obvious. The scars 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. S B ) . 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. . \ ' ."'5 ""l! ' 5 ' ' Reaction in Guinea Pigs Infected w ith 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 nontubereulous 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 5H months it had completely disappeared, leaving only scar tissue. Foci of fibrosis still persisted in the last animal, which was llled 14 months after infection. - . ' - ------ ------ Reaction in Rabbits.--Rabbits exposed to the asbestos dust for periods up to 19 months showed a foreign body type of reaction of low grade, but no fibrosis. Although their lungs contained particulate elements of the dust, fibers were not present, indicating that the upper respiratory mechanism of the rabbit is adequate to exclude fibrous foreign bodies. Two rabbits, after inhaling-dust for six and 19 9. Steenken. W., Jr., and Gardner, L. U .: R Strain of Tubercle Bacillus: Its Dissociation and Virulence of Variants in Normal frnd Silicotic Guinea Pigs. Am. Rev. Tuberc. 54:51, 1946. ' 10 1S D V STRIAL HYGIESE AND OCCUPATIONAL MEDICINE chronic pulmonary inflammation resulting from many causes. W illis* 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. Flg' * float* '"halation experiment: A , lung of a guinea pig witf six months dust exposure followed by 35 months' inhalation of normal air? Th< reaction u rather slight, but distinct fibrosis is present (X 2 0 0 ). Note that 2f months of continuous exposure (fig. 3 5 ) 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 tha that in A but much less than the reaction in figure 3 B ( x 200). 8. W illis, H . S., and Brutsaert, P .: Tumor-like Structures in the Lungs of Guinea Pigs Artificially Exposed to Silica Dust, Am. Rev. Tuberc. 17:268 1928. '