Document 91qZd983DLYoyOEmdkrdNJb3L

r r\ * ASB3ST0SIS Experimental Studies by THE SARMAC LABORATORY SARANAC LAKE, NEW YORK Report to the JOHBS-MABVILLE CORPORATION NEW YORK, NEW YORK September 30, 1948 A_c. PLAINTIFF'S |* EXHIBIT MM PLAINTIFF'S 1$, EXHIBIT 11 2* Introduction 3* Asbestos Siinerrals I. ABSTRACT 17. SSPSiHSESTAL ASHES70SIS 5* abqperdjasntiO. Lfethods 6. Species Susceptibility 7* Peculiar Characteristics of Asbestos 8. Rate or Tissue Reaction to Asbestos Fibers 9. Asbestosis Bodies X. INHALATION EXPERIMENTS 11, King's Floats Asbestos Dust 12. Dusting Materia]. 13. Diet Composition 1U. Dust Concentration 15. Reaction in Animals 16. Guinea Pigs 17. Rate and Type of Reaction 18. Progression 19. Infection Coincident -with Dust Inhalation 20. Infection after Dust Inhalation 21. Asbestosis Bodies 22. Rabbits 23. Rats 2h. Summary and Interpretation ii 2 > *3 J. c; 6 7 *(* 8 8 8 8 9 9 9 10 10 11 12 12 12 13 o Or; 1 b 1 i i of' f'- IT 9 p V.T T* 25. Short-fiber Asbestos Dust 26. Dusting Material 27. Dust Composition 28. Dust Concentration 29. Size-frequency of Dust 30. Reaction in Animals 31. Guinea Pigs 32. Rate and Type of Reaction 33. Progression 3h. A3bestosis Bodies 35. Rats 36. Rate and Type of Reaction 37. Cats 38. Rate and Type of Reaction 39. X-ray Changes U0. Ashestosis Bodies Ul. Rabbits lt2. Rate and Type of Reaction Ii3. Asbestosis Bodies lilt. Summary and Interpretation U5. 100 Per Cent Ball-milled Asbestos Dust U6. Dusting Material U7. Dust Composition U8. Dust Concentration U9. Size-frequency of Dust ixx Page lit ill 15 15 15 15 15 15 17 17 13 18 19 19 19 20 20 20 20 21 21 21 22 22 23 W W i* X i* X r~. m i- t *tj n 50. Reaction in Animals 51. Guinea Figs 52. Rate and Type of Reaction 53. Progression 51u lymph Node Involvement 55. Asbestosi3 Bodies 56. Bata and Mice 57. Summary and Interpretation 50. Long-fiber Asbestos Dust 59. Dusting Material .60 Dost Composition .61 Dust Concentration .62 Size-frequency of Dust 63. Reaction in Animals 6U. Guinea Pigs 65. Rate and Type cf Reaction .66 Progression 67. lymph Node Involvement .68 Asbestosis Bodies 69. Cats 70. Rate and Type of Reaction 71. X-ray Changes 72. Rats 73 Rate and Type of Reaction 23 23 23 2li 22* 2k 2k 25 26 26 26 26 27 27 27 27 28 29 29 30 30 30 30 31 ?u. Mice 75 Rate and Type c:~ Reaction 76- Summary and J<;terp?eta.t:..on ;,:O07II. INJECTION U^KRIl .NTT 7Gc Infcratrae"esl ?irpt rintents 7? Comparison of fibrous and Non-fibrou:? f 60. Comparison of Various Long-fiber Dusts 81. Comparison of Long--fiber and Short-fiber fusts 62. Intravenous Eocperiments 83. Intra.perifcneal Experiments LXXXIV. OTHER EG-SEr.SMIS 35. Protective Action of Alusdn.ua Compc/unoe 36. Formation of Aabestosia Bodies UKXVII. THEORY 0? IRRITANT ACTIO*? LXXEVIII. COMPLICATIONS 39. Infection 90. Susceptibility to Tuberculous Infection 91. Susceptibility to Ncn-tuherculous Infection 92. Neoplasm EC-III. COHCWJSIOKS XCIV*. TABLES Id7. F23BRES V 'n 32 32 32 z-l1 \ 3h 55 -^c: 35 ^v' 36 37 37 .33 36 39 UO h3 _ VI I -A. 12 - 75 ABSTRACT Asbestosis is a pulmonary disease caused by the inhalation of asbestos dust. In animals it is characterized by a peribronchiolar fibrosis vhich seems to be the result of mechanical, rather than chemical, irritation of the tissue by asbestos fibers. Only the long fibers produce a typical reaction; short fibers are relatively inert. The filamented structure of the fibers is an essential factor in the mechanism of irritation. A character istic tissue response can be produced by non-6iliceous as Trell as siliceous fibrous minerals. Inhalation of asbestos dust apparently does not alter significantly the course of experimental tuberculosis in guinea pigs. The asbestosis body, nhich is a specific concomitant of asbestosis and forms soon after the entrance of the asbestos fiber into the lung, is believed to prevent further damage to the tissue by the fiber and thus to limit pro gression of the reaction -when exposure ceases* Aluminum does not exert a protective action against the tissue irritation of asbestos fibers as it does against that of quartz particles* 1. 2. Introduction OF A53E3T0SIS minerals Those characteristic feature is a structure composed of long, par allel, flexible fibers. This structure is unique because the fillers are capable of repeated longitudinal subdivision to units of molecular proportions. In length the fibers vain* from a few microns to sis or mors inches. Sons varieties are stiffer than others but many are sufficiently flexible to be spun into yarn and woven c-n modified textile machinery. 3. Asbestos Minerals The asbestos minerals are silicates of variable composition ana belong to the serpentine and the amphibcle groups. Listed below are the more common varieties. ArGphibole Group Anthophyllite (Mg, Fe) silicate (Mg, Fe, Al) silicate Amphiboie (Ca, 1%, Fa, Al, Na, K) silicate Tremolito (Ca, Mg) silicate Actinclite (Ca, Mg, Fo) silicate Crocidol: (Kaf Fe) silica/te + Fe silicate Serpentine Group Mg silicate (hydrous) -2- The bulk of the asbestos of commerce is chrysotile, 3tfeO*2Si02 2H20, which is mined in the Thetford region of the Province cf Quebec. Crocido- \ lit and amosite are also used commercially but in lEcch siailler amounts. Chrysotile occurs as veins in serpentine, a mineral sinHa.' in chemical com-position to chrysotile but which exists in massive form and is made up of microscopic fibers -without the parallel orientation characteristic of chrysctile. The massive blue black serpentine, which is smooth and soapy to the touch, is traversed by veins of fibrous chrysotile varying in width from a barely perceptible line to six or more inches. The fibers run across the vein and not lengthwise -with the formation. Attention is directed to the mineral brucite, MgO.I^O, which is often found in the same formations -with serpentine and chrysotile and may be fibrous in structure* It 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 asbesiiforsi 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 upon lung tissue. IV- EXPERIMENTAL ASBESTOSIS For many years studies have been carried on by the Saranac Laboratory in an investigation of the cause, nature and development of asbestosis. The present re port is devoted to Experimental Asbestosis. In It are described the animal ex periments -with various kinds of asbestos dust. Another report, to be prepared and issued later, 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. -3- Although asbestoais in man is a chronic disease which requires years to develop, it is possible to reproduce in one or more species of animal character istic tissue changes -which are similar to the lesions of human asbestosis. Since the life-span of the experimental animal is relatively short, it is not possible to develop the characteristic lesions in animals under the usual industrial con ditions. Consequently, to obtain a complete evaluation of the tissue response to inhaled particulate and fibrous material, it is necessary to accelerate the reaction by employing higher concentrations of dust than would ordinarily be en countered in industry. V/hile conditions of exposure are thus different, the information yielded by experiments with animals is invaluable in furnishing a better understanding of the reaction of the human organism to inhaled asbestos dust. 5. Experimental Methods For investigating the biological reaction of the experimental animal to the various asbestos minerals, two types , of technique 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, cabs, dogs, rats or mice - are kept for eight hours a day in a cubical dust room, eight feet in dimension, in which a cloud of asbestos dust is maintained, i At intervals during the experiment, a few animals are sacrificed and the tissue examined to determine the nature and extent of the dust reaction. Some animals are exposed for periods up to three years. The Injection experiments, in which the dust, either dry or suspended in fluid, is introduced into the animal by the Intravenous, intraperitoneal or intratracheal procedure, are used to determine whether or not a particular dust has a potential capacity to produce tissue reaction. long-term inhalation, experiments Romish information upon 'which great reli ance is placed 'when estimating the degree to "which a dust might be hazardous to industrial workers. Whether or not atmospheric dust, even though potentially dangerous, can be inhaled, pass the natural defense barriers and reach the pul monary tissue in quantities sufficient to cause damage can be determined only by inhalation procedures. Injection experiments are useful because in them contact between the dust particles and tissues is assured and the potential capacity of the dust to produce reaction can be estimated accurately. When dealing with fi brous minerals like asbestos, the intratracheal method is valuable since it per mits observing the effect of the fibers on pulmonary tissue. 6. Species Susceptibility Unlike free silica, asbestos does not exert its specific effect in all organs of all species of animal (Table l). Injection of fine quartz into various organs of the guinea pig, rabbit, rat, cat, dog, chicken and even tadpole mil produce silicotic nodules. However, similar injections of long or short fiber asbestos have resulted in a fibrous reaction in the lung and, to a lesser extent, in the peritoneum but not in other organs. 7. Peculiar Characteristics of Asbestos Experience has demonstrated that most of the particulate matter inhaled into the lungs of man and animal is ID microns or less in maximum diameter. Larger particles apparently are excluded by the protective mechanism of the upper respir atory tract. In the case of fibrous materials, however, this restriction does not apply and fibers 100 and even 200 microns in length have been found in the termin al air spaces of human lungs. In small laboratory animals exposed to asbestos dust the marimum length of fiber found in the lung rarely exceeds 60 microns. Not every kind of fibrous material is inhaled with equal readiness; for example. the synthetic fibers of glass wool apparently are too inflexible to pass easily through the nose, pharynx, trachea and bronchi and seldom reach the terminal bronchioles and alveoli* Inhaled particulate matter cornss to rest throughout the terminal air spaces (alveolar duct, atria, alveoli) in all parts of the lungs; inhaled asbestos fibers are first retained in the respiratory bronchioles. These vary small tubes are immediately distal to bronchioles lined by ciliated epithelium. Their own essen tial lining is a low cuboids! type of epithelium but, as their name implies, they actually function in respiration through lateral alveoli given off as pouches along their walls. Either these pouches, or the abrupt change in the character of the lining epithelium, or the decrease in diameter of the tube, or perhaps the combination of all three factors is responsible for local retention of the inhaled fiber. Only after asbestosis is well established are appreciable numbers of fi bers carried into the more peripheral air spaces. 8. Rate of Tissue Reaction to Asbestos Fibers The rate of tissue reaction to asbestos is much more rapid than to an active dust nv quartz. Evidences of tissue response appear as soon as fibers have localized in sufficient concentration in specific areas. In rats receiving asbes tos fibers by intratracheal injection this evidence is visible as early as two weeks after injection; for quartz dust the latent period might be two months or more. The behavior of the tissue reaction to inhaled dust after the termination of exposure is not the same in silicosis as in asbestosis. In silicosis, the young nodules become larger; in asbestosis, young scar tissue,that may have formed,con tracts and becomes more dense but the area of involvement decreases in size. If exposure to asbestos dust is terminated after a brief period, the recently-inhaled fibers in the rung may cause the fibrous tissue response to continue for a short time, until the fibers have been coated. This progression is of only a slight degree and of little significance. 9. Asbestosis 3cdies The peculiar structure knorm as the asbestoais body or curious body is a specific concomitant of asbestosis. The typical body is a golden-yellcw, beaded or haustrated rod viiich may be either straight or curved. Often one or both ends are bulbous like a dumb-bell. The bodies vary considerably in length, and di mensions up to 2^0 microns have been recorded* It is believed that asbestosis bodies are due to a deposit of protein and iron pigment upon the surface of inhaled fibers. In guinea pigs they form after about 60 days of contact -with the tissue. They are abundant in man and the guinea pig (see Table 1) but are much larger in the former, probably because the largersized air tubes admit fibers of greater dimension. In cats, rabbits and mice there is an atypical coating of a few of the fibers after much longer residence in the lungs. In rats and dogs no bodies could be discovered. Although the evidence is incomplete, it appears that the formation of the asbestosis body prevents damage to the tissue by the fiber. X. INHALATION EXPERIMENTS Four comprehensive inhalation experiments have been conducted at the Saranac Laboratory with various forms of asbestos dust. In each of these investigations more than 160 animals were used and the experiments were carried on for periods ranging from 2 to more than 5 years. The four kinds of asbestos dust employed are identified as King*s floats, short fiber, 100 per cent ball-milled, and long-fiber asbestos dust. -7- 11* Inhalation Experiment with l:King *s Floats'* /asbestos Duat The first inhalation experiment conducted at the Saranac Laboratory with asbestos dust "was begun in 1928. Animals inhaled the dust for periods up to nearly three years and seme guinea pigs lived for about four years after their first exposure to dust. A preliminary report giving observations after 29 months of exposure appeared in the February, 1931 issue of THE JOURNAL OF INDU STRIAL HYGIENE.*- At that tine observations covered a period of only 2-l/li years and the conclusions as to the ultimate effects of inhaled asbestos dust were provisional. Results of the completed study show that most of the conclusions drawn in the preliminary report were substantiated. A complete review of this experiment follows. 12. The dusting material was a commercial variety of asbestos dust known as King's floats and was composed of short fibers and particles of variable size- It was obtained from, the Tnetford, Quebec plant of the Asbestos Corporation of America. 13. The dust composition (Table 2) reveals that the amount of fibrous chrysotile was only lU per cent, a rather 1aw value. However, there was suf ficient fibrous material to produce a characteristic fibrosi3. li*. The dust concentration at first was quite low and for impinger samples taken soon after the experiment was started, the average light field count: by the standard technique was only 6.0 million particles per cubic foot of air. An appreciable number of large particles (or fibers) also were present, as STUDIES ON EXPERIMENTAL FNEIM)C0NI05I5. VI. Inhalation of Asbestos Dust., Gardner, L.U., and Cummings, D.E. J. Ind. Hyg., 13: 6-8l, 97-llii, 1931. -8- shown by an average count of 0.8 million for particles greater than 10 microns. After the inhalation experiment had been under way for about two years, the speed of the rotating paddle in the dusting machine was increased and for the remain ing 9 or 10 months of the experiment considerably more dust vas dispersed into the atmosphere. Average dust counts for impinger samples collected after this change were $3.7 million for the usual light-field method, and 1,6 million for particles larger than 10 microns. 1$. Reaction in Animals to Inhaled "King's Floats" Asbestos Dust. Results of the investigation, briefly summarized in Table 3, show that inhala tion of King's floats asbestos dust produced a typical peribronchiolar fibrosis in guinea pigs but not in rabbits or rats. 16. Guinea Pigs. Seven groups of guinea pigs were used. In three groups the effect of a continuous and of an interrupted dust exposure was studied; in two other groups the relationship between infection and dust exposure was investigated. The remaining two groups were infection controls. 17. Rate and Type of Reaction. Guinea pigs inhaling thi3 dust for periods up to 33 months developed a characteristic fibrosis occurring in conical patches about the respiratory bronchioles. During this exposure the peripheral alveoli were not involved. The particulate elements in the dust were transported to the lymphatic system where they caused no signi ficant reaction; the fibrous elements remained fixed at the site of original localization and were seldom detected in the lymphoid tissue. Pleurisy and fi brosis in the septa were observed only when infection complicated the process. After exposure of approximately a year, a small amount of cellular reaction had been produced about many respiratory bronchioles. As more dust was inhaled, it continued to accumulate in the same location and later stages of the disease consisted of tensions of the original lesions. New areas were not involved. 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 were phagocytized and many of them were carried into the wnll by migratory ceils. Mononuclear leucocytes attracted to the area caused an appreciable thickening of the bronchial wall. After 16 months a delicate fibrosis made its appearance. The process evolved 30 gradually that mitotic division of fibroblasts could rarely be discovered; nevertheless, the number of fine intercellular collagenous 1 fibers (fibrosis) steadily increased. As this fibrosis contracted, it partially closed the alveoli, and with this atelectasis the lining epithelium assumed its embryonic cuboidal form. The result was the adenoma-like appearance that Vfillis described in guinea pigs inhaling silicon carbide. The longer exposures resulted only in more thickening of the walls of the air spaces, largely due to an in crease in the amount of fibrosis. The fibrous tissue always remained cellular and never showed the hyalinization characteristic of silicosis. 18. Progression. The reaction produced in exposed guinea pigs did not pro gress significantly during a subsequent period of 37 months when the animals lived in a normal atmosphere. Between 8 and 11 months after exposure ceased, the cellular reaction had been completely replaced by thin strands of fibrous tissue. Observed still longer, the scar tissue decreased in amount but in the last animal sacrificed, 37 months after discontinuing dust exposure, some fibrosis was still visible. 19. Infection coincident with flust Inhalation. Of the group of liO guinea pigs infected with atten uated tubercle bacilli (R^ strain) 31 died or were sacrificed before two years of dust exposure and were reported in the paper by Gardner and Cummings mentioned above. Seventeen of these died from intercurrent pneumonia. Briefly, the re sults were as follows: 10 revealed some evidence of spread of the tuberculous processj in 6 of these it was confined to the lungs and in the other U the abdominal viscera also were involved. Usually a slight local extension of the tuberculous infection had occurred but subsequent healing had resulted in fi brosis of both the pulmonary lesions and the secondary lesions in other organs. The healed pulmonary lesions showed acre fibrosis than is characteristic of either tuberculosis or asbestosi3 alone. The 9 animals which were still alive after two years of dust exposure were sacrificed at intervals during the following year. In U of them the primary foci of infection had healed with fibrosis and even calcification and there was no evidence of progression. In the other 5> the tuberculous foci showed evidence of having previously spread locally: in U of them it had healed, by the time of autopsy, with excessive fibrosis; in the other animal there was a generalized chronic tuberculous pneumonia in one lobe and isolated primary tubercles, vhich were still active but had not spread, in the other lobes. Evidence of extension of the infection was first seen after 7 months of dust inhalation; during the next 20 months more than half of the animals showed an actively spreading tuberculosis and in 3 of them small cavities had developed. During the last 8 months no animals exhibited any evidence of active infection although in half of them the healed fibrous scars of previous extensions were ob vious. Sixty per cent of the guinea pigs with spreading pulmonary tuberculosis showed tuberculosis of the spleen and liver, 20. Infection Superimposed Upon an Established isbeatosis. Twelve guinea pigs, after in haling asbestos dust for nearly 26 months, were infected with tubercle bacilli and then removed to normal air. The subpleural tubercles in the dusted animals were no .more numerous than in non-dusted controls , but a considerably number were found in the depths of the lung about foci of asbeatosis. The reaction to infec tion showed only slight local extension about the origiral sites in the lungs and tracheobronchial lymph nodes. The abdominal viscera were involved in only one animal. Caseation was found in tubercles 1-1/2 months old but by 5-1/2 months it had completely disappeared, leaving only scar tissue. The latter still persisted in the last animal, which was killed lii iiionths alter infection. 21. Asbestosis Bodies. Moderate numbers of asbestosis bodies occurred in the lungs of the guinea pigs, becoming mox-e numerous and more distinctly segmented in later months. 22. Rabbits. Rabbits exposed to the asbestos dust for periods up to 19 months developed a low-grade foreign-body type of reaction 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 6 and 18 months, lived in normal air for more than two years* At autopsy neither animal showed any evidence of cellular reaction or fibrosis in the term inal bronchioles nor were there any a3bestools bodies. 23- Rats. All the white 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 their lungs. In a few of the rats, an occasional asbestosis body was discovered but there was no fibrosis. -12- 2lu Summary ana Interpretation of Inhalation Experiment with King's Floats Dust. The findings in the experiment with iting * s floats oust can ce suiomar- ized under three headings. A. Effect of the inhaled dust on normal animals. The Sing's floats dust caused a charac teristic peribronchiolar fibrosis in guinea pig3 but not in rabbits or rats. The fibrosis did not progress after the dust exposure was discontinued and the guinea pigs transferred to normal air. B. Effect of the Inhaled dust on tuberculosis in guinea nigs. In guinea pigs in fected with attenuated tubercle bacilli and then placed in the dust room, the results were more variable than is usual in an experiment of this type. A few animals showed no sign of progression; in most of them there was evidence of tem porary progression with subsequent healing; in one animal there was continuous progression to death. In contrast, when guinea pigs, after being infected, are exposed to quartz instead of asbestos dust, the infectious process continues to progress and eventually causes the death of the animals. On the other hand, ex posure of infected animals to a harmless dust like calcite or gypsum does not lead to any progression of the infection. Guinea pigs infected with attenuated tubercle bacilli following the termination of about two years' exposure to asbestos dust did not develop progressive disease. The only modification of the infection was in its localization, a few bacilli being retained in the fibrous terminal bronchioles and forming tubercles there in addition to the usual foci beneath the pleura. In view of this variability, the unusual nature of the response and the high proportion of deaths from intereurrent pneumonia it is felt that definite conclu sions as to the influence of this dust on the course of tuberculous infection are not justified. -13- C. Effect of tuberculous injection on the reaction to inhaled dust Infect-5.^ r f.v^p t!p*Tv^*ncv' of fcH dust to produce fibrous tissue. This change occurred whether or not the bacterial lesion was in contact with the area of dust reaction. 25* Inhalation Experiment with Short-fiber Asbestos Dust. Since hazardous dusts like quarts are most effective in producing fibrosis when the particles are 3 microns and less in size, an inhalation experiment was carried on to determine whether this condition is true also for asbestos dust. It was thought that by using a short-fiber asbestos dust consisting almost entirely of fibers and particles smaller than 3 microns an accelerated tissue response might be initiated and an advanced reaction obtained in a short time. The previous inhalation experiment with King's floats asbestos, which contained fibers from 1 imn. to 1 micron or less in length as well as a great deal of particulate matter and which produced a typical peribronchiolar fibrosis in exposed guinea pigs, served as a basis of comparison. 26. The dusting material for this experiment was forwarded from the Manvllle plant of the Johns-Uamrille Corporation. It iras the remains of fibers collected in dust bins after a carding operation and screened to pass 200 m83h. Since the material as received contained many long fibers, it was ground in a steel ball mill to reduce practically all the particles to 3 microns or less in size. When used alone in a standard dusting machine, this finely-ground asbestos tended to pack in the hopper and it became necessary to mix one volume of the un ground material with three volumes of the ground to generate a satisfactory dust cloud. The addition of the small quantity of unground asbestos was unfortunate because it confused the interpretation of results. Probably the minor amount of reaction that developed was due to the long fibers in the mixture although the data of this experiment do not prove the point. 27. The composition of the short-fiber asbestos as received is disclosed by the chemical and petrographic analyses given in Table 1. Samples taken before and after grinding yielded about the same values on analysis, indicating that there was no contamination from the mill or loss of water content. 28. The dust concentration varied somewhat during the experiment and light field counts for atmospheric samples collected inside the animal cages with the impinger apparatus ranged from 83 million to 182 million. The average of counts was 130 million for the first year of the experiment, 13U million for the second year and lliO million for the third year. 29. Size-frequency measurements of air-floated dust from inside the cages at a magnification of 1300X revealed a great preponderance of fine par ticles (Table 5). Nearly 90 per cent of the particles seen were smaller than 3 microns. 30. Reaction in Animals to Inhaled Short-Fiber Asbestos Dust. Foot species of animals - guinea pigs, rchi.te rats, cats and rabbits -- were used in this experiment. The results of the dust exposure, which are summarized in Table 6, -will be considered more in detail below. 31* Guinea Figs* Eighty guinea pigs were originally placed in the dust room but 21 of them were later eliminated from the experiment and killed because of enlarged lymph nodes. Of the other 59 animals, U6 remained in the dust room until they were sacrificed or died from natural censes and 13, after being exposed to dust for 20 months, were transferred to a normal atmosphere. 32. Rate and Type of Reaction. The type of tissue reaction to the inhaled short-fiber asbestos rsac essentially the same as that already observed in the experiment "with King's floats asbestos. The rate of reaction also was approxi mately the same but the extent of involvement with the short-fiber dust was very much less and after 16 to 2h months of exposure only a very fen small foci of reaction, which generally required microscopic examination for detection, were produced in the guinea pigs. Until exposures had continued for approximately one year, there was little tendency for dust-containing phagocytes to collect into clumps. By 16 months phagocytes had begun to collect about the walls of a few of the respiratory bron chioles with a little proliferation or infiltration of mononuclear cells in these walls. There were also some multinucleated cells but they were always of the inert foreign-body type. At 20 to 2la months the cellular clumps were sometimes quite marked and sometimes changes in the epithelium resulted in the adenoma-like or "adenomatoid" appearance previously described in Section 17. In most of the subsequent members of the series, the reaction remained cellular in type. In a few, however, fibrous elements dominated the picture. In the latter case, the collagen was pale in color and tenuous with no heavy swollen hyali.nl zation. As in the rats described below, the alveolar walls might be made up of a bind of collagen supporting a layer of epithelium, but with no contained capillaries. In the tracheobronchial lymph nodes the reaction was more pronounced in this experi ment than in the previous one with King's floats asbestos, probably because of the transportation of an excess of fine particles to the nodes in animals inhaling short-fiber asbestos. The reaction was essentially an increase in reticulum, rather than a fibrosis, with preservation of the original cells between the thickened reticular fibers. Diffuse chronic pleurisy without evidence of pulmonary infection was present in a few animals. _1 A- 33. Progression In the 13-3/4 months following tho cessation of 20 monthsr exposure to dust, progression of disease was not definitely demonstrated but neither could it be absolutely disproved; owing to the varia bility of the response in different animals. At the end of the dust exposure of 20 months tiro pigs were read as + and one as 2*. Among the 13 removed from dust the findings were variable: in 2 the reaction was i; in k it was + : in 3 it was 3+| in 3 it was U+J and in one animal it was 3>+. It is quite possible that those with the most marked changes had already developed more reaction than the remain der by the time exposure ceased. Since ihs more severe reactions occurred spora dically and bore no relationship to the length of time after cessation of expo sure the differences were attributed to variation in individual susceptibility. This view received support from the chemical analyses (Table 7), which often re vealed comparable amounts of ash and silica in lungs with widely different amounts of tissue change. For example., the ash and silica values were quite similar for three animals in dust 20 months and then in normal air 13-3Ai months, yet the tis sue reaction for one animal was k+; far another, +; and for the third, only . 3k. Asbestosis Bodies. The formation of asbestosis bodies was at first ex tremely limited. After 5 months' exposure only a very rare short body could be found, usually inside of cells. Around the finest intracellular particles there were yellow deposits having the same color as the asbestosis body. Exposure of one year had permitted an accumulation of many longer fibers about which the asbestosis-boay coating developed. Most of these were still short enough to be partially or entirely within phagocytic cells. By the 20th month and thereafter, they were comparatively numerous although still rare in comparison with the findings in the King's floats experiment. 35. TKhite Rats. Seventy-three white rats were exposed to atmospheric shortfiber asbestos dust for periods up to 32 months. Saeri- ficings during the first 10 months were made bimonthly and for the remainder of the experiment at less frequent intervals. 36. Rate and Type of Reaction. The dust cells until 8 months were widely scattered and existed in foci only sporadi cally,. Reaction wa3 limited to occasional slight thickenings of the septa about small accumulations of dust cells. In a few rats at 10 months, there was a sugfestion of early fibros3 but the change was so slight that it would probably be overlooked without the clump of dust cells to attract attention to the area. Only 10 animals were exposed from 12 to 32 months. In each of them the lungs showed minute patches 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 alveolar ducts in which the walls of the air spaces were very thick, due to swollen collagen framework. Connective tissue and Foot--Bielachowski silver preparations revealed complete loss of capil lary bed locally. Outside the collagen was a thin layer of epithelial cells. This did not resemble the "adenomatoid" change characteristic of guinea pig as bestosis. No pleurisy was present. 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 asbesto sis body. The tracheobronchial nodes showed compact focal collections of mono cytic 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 and extending into the mediastinal areolar tissue. Compared with the response to active dusts like quartz and chert the reaction to short-fiber asbestos was negligible. Results of chemical analyses made on the -white rats are given in Table 8 and the average values have been tabulated in Table 9 for comparison -with similar values for rats inhaling other dusts. The concentration of atmospheric particles to which the animals were exposed was approximately the seme for asbestos and quartz; for the gypsum-quartz mixture; it was about twice as high and for chert five times as high. It will be noted that the percentages for asbestos are lower than those for quartz or chert but are similar to those for the gypsum-quartz mixture, in which atmospheric agglutination tended to reduce the amount of dust inhaled. It might be inferred that the total.quantity of asbestos dust inhaled was low or that it had been eliminated from or dissolved within the lungs. In the present state of our knowledge evaluation of these hypotheses is not possible. 37. Cats. Twenty- cats were U3ed in this inhalation experiment v;ith the short-fiber asbestos. Eighteen were kept in the dust room until death, the exposure period ranging from one month to nearly h-1/2 years, and two, after a dust exposure of 31-1/2 months, were removed to normal air. One of these was sacrificed 5 months, and the other 2h months, later. 38. Rate and Type of Reaction. The reaction was essentially that to an inert dust, even after more than U years of expo sure. The tissue response in this species was confined to microscopic foci of fibrosis in the walls of groups of subpleural alveoli, rather than in the peri bronchiolar areas. In one animal the change was extensive enough to be visualized cn gross inspection of the section. 39. X-Ray Changes. Only in the animal with the longest exposure did the X-ray reveal definitely abnormal shadows. After 29-3/b months the picture was negative; after months a faint mottling could be detected throughout both lungs. At autopsy, 8 months later, there was only microscopic fibrosis in the subpleural zone plus heavy lymphocytic infiltration about small bronchioles- 110. Asbeatosis Eodiss* Cn prolonged search a few yellow atypical asbesto3is bodies, smooth and without haustrations, were found in two animals exposed for more than a year. 111. 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 loft in normal air 6 months before being sacrificed. 112. Rate and Type of Reaction. There was never enough fibrosis to be de tected grossly and there was no chronic adhesive pleurisy. Microscopic evidence of alveolar wall thickening was first detected after about 3 years of exposure and was seen in all five animals examined thereafter. In one animal that died of paralysis after nearly four years of ex posure the reaction was extensive enough to be visible on gross inspection of tissue sections. The possibility of pulmonary infection in this animal could not be excluded. However, in another animal dying two years later the focal fibrosis was not nearly as obvious or as advanced. Areas of involvement, which were largely visualized because of phagocytic reaction within the air spaces, tended microscopically to become more fibrous with the passage of time but there was never much encroachment upon the lumen of air spaces and the architecture of the lung was prese*ved. 113. Asbestosia Bodies. Asbestos bodies were not detected in rabbits that died early in the experiment but were seen in all animals that had been exposed to the dust for more than three years. Ui. Summary and Infcerpretatlon. The original purpose of the experiment -was to evaluate the chemical theory of the pathogenesis of asbestosis. It was felt that if the tissue reac tion to asbestos were chemical in origin an accelerated or accentuated response would result from exposure to finely-divided asbestos, as is the case with quartz. Thi3 experiment, in which the reaction was slower and less extensive than with King's floats, indicates that the reaction probably is not primarily chemical in nature. Of the four species exposed in this experiment only the guinea pig and rat reacted with characteristic peribronchiolar fibrosis. The cat reacted with atypical sub-pleural fibrosis and in the rabbit the fibrosis which occurred could not be positively attributed to the dust because of a strong possibility of pulmonary infection. US- Inhalation Experiment -with 100 Per Cent Ball-j&Hed Asbestos Dust. In the inhalation experiment with short-fiber asbestos dust a small quantity of unground asbestos was mixed with ground material in order to produce a suitable dust cloud. When evidence of a dust reaction appeared in the guinea pigs during the experiment, it was not clear whether this was a tissue response to th9 small number of long fibers in the unground asbestos or was a delayed effect of the more abundant fine dust. Consequently, another inhalation experiment was started in which no unground material was used. U6. The dusting material was the ground short-fiber asbestos used in the previous inhalation experiment but no unground material was mixed with it. To obtain a sufficient amount of atmospheric dust, the design of the dusting apparatus was changed to an open type of hopper and fresh dust was added daily. Owing to the tendency of material to form small spherules which '.prevented much of the fibrous portion from floating out of the hopper, the dispersal of the dust -was not entirely satisfactory and after 7 months of operation, the dust ing machine was reconverted to its original design. To prevent ''pilling:i or the' formation of spherules of asbestos, steel -sire brushes T?ere attached to the in side surface of the hopper and to the rotating paddle* This arrangement gave satisfactory results and was used for the remaining 21 months of the experiment- hi* The composition of the rarer material (shoz-t-fiber asbestos) and cf atmosplieric durst liberated from the ball-milled product in the dusting ma chine is given in Table 10. These values are based upon petrographic study and X-ray diffraction analysis. The atmospheric sample was collected with an elec trostatic precipitator after wire brushes had been installed in the dusting machine* Previous to this- the chrysotile content of the air-auspended material was undoubtedly less than the 1? per cent value given in Table 3.0* In an inter im report, it was stated that the air-borne dust contained about 5 per cent of chrysotile before the wire brushes were used and up to 8 per cent afterwards, but these values were probably low. Quantitative estimates on ball-milled asbestos dust may be somewhat inaccurate because it is difficult to determine how much of a dust sample is fibrous chrysotile and how much is non-fibrous serpentine. I48. The dust concentration for the first 7 months of the experiment was about 100 million particles per cubic foot of air. .After the wire brushes had been installed, the dust counts were a little higher and the overall. average for the first year was 106 million. The average of counts for the second year was 163 million and for the third year 1U5 million. 2ha slae-frequency of the components of atmospheric dust collected inside the animal cage3 with the electrostatic apparatus is reported in Table 11. Tvo camples were tsirea, or*e before the wire brashes were installed and one after. It will bo noted that after the -sirs brushes were in use a greater proportion of very fine particles and also of longer fibers was released into the air. 50 Reaction in .Is to j.nhrUed ICO Per Cent Ball~5il2ed Asbestos Dust. Guinea td.es. a were used in the inhalation experiment with the 100 per cent ball-milled asbestos dust. The results are sumsarized in Table 12. 51. Guinea. Pigs. The erperins-nt was started with 100 guinea pigs. As the dust exposure proceeded, there were 39 accidental deaths, 32 of pneumonia in an epidemic. After 28 months of dusting the 16 surviving guinea pigs were transferred, to normal air. 2. Rate and Type of Reaction. For the first year of exposure practically the only reaction to the dust was the pre sence of scattered phagocytes and an occasional minute ashestosis body. At 16 and 20 months no gross response was visible on the tissue section but microscopically peribronchiolar foci of inflammatory cells could be seen. At 2U months 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. Chemical analyses of the lungs (Table 13) reveal that in spite of the limited tissue reaction considerable dust had been retained in the lung. 53. Progression. The lungs of animals exposed for the full dusting period (28 months) and then living in normal air for 2 months revealed the changes described above and also very slight, peribronchiolar fibro sis. After 0 months in normal air the findings rrcre similar but at 12 months 3 of U animals showed gros3ly-visible characteristic peribronchiolar fibrosis with adenomatoid change. 5U. Lymph Sbde Involvement. The tracheobronchial nodes were essentially negative until exposure had been continued for more than a year and a half. Animals sacrificed at 12 months and 16 months revealed a few minute collections of phagocytes containing particles but prac tically no fibers large enough to be recognized as such. After 20 months of exposure many monocytes filled with yellow granules were present. At 30 months there had been a slight increase in reticulum but no fibrosis. No further changes occurred in the nodes. Asbestosis bodies were not seen in the nodes of any of the guinea pigs. 55. Asbestosis Bodies, ilinute asbestosis bodies were observed as early* as 3 months after exposure began, but they did not be come numerous until 16 months had elapsed. The bodies were short and practically an were intracellular, although at 20 months some were long enough to project beyond the cell borders. It is important to cote that in the later months of exposure there was i a distinct increase in the number of long fibers (up to 70 microns in length in the lungs and that after exposure ceased characteristic long asbestosis bodies were seen. 56. White Rats and Mice. In this experiment hO rat3 were exposed for periods up to 20 months and 2h mice for periods up to 12 months* Keithor speci developed even a suggestion of asbestosis 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* lib asbestosis bodies were found in the rats but in the mice there were a very few small non-haustrated forms within phagocytes* In 21 mouse lungs sectioned there were 3 instances of pulmonary adenoma (litfa). 37 Summary and Interpretation* The tissue reactions observed in this experiment ware much less extensive and slower in development than in the previous investigation with short-fibar asbestos* Since presumably there were fewar fibers longer than 3 microns in the material used in this experiment, the results tend to confirm the interpretation made in Section Ijli of the short-fiber experiment that the reaction probably is not primarily chemical in nature* The finding of long asbestosis bodies in animals inhaling the ball-milled material is an example of the difficulty of completely eliminating long fibers from an asbestos preparation* In regard to progression of reaction after removal from dust, -which was ob served in this experiment but not in the others, the following interpretation is offered: TShen the reaction is well-developed at the termination of exposure,the contraction of the fibrous tissue would obscure any possible progression. In this experiment, however, since only the earliest stage of reaction was present at the time of removal from dust, its subsequent progress was apparent* It should be noted that the degree of progression was so slight that it can have little, if any, practical significance. 58 v Inhalation Experiment with Long-fiber Asbestos Lust. After animals inhaling short-fiber asbestos dust for more than a year had failed to develop significant reaction, the hypothesis that asbestosis is pro duced by the mechanical irritation of long fibers was given added support. Since the King's floats asbestos used in the first inhalation experiment had a rather Ion? content of fibrous clirysctile and contained considerable serpentine and other impurities, it was decided to conduct a nsrrr inhalation experiment with a purer form of chrysotile which would be richer in long fibers. 59. The dusting material employed in this investigation was obtained from the Manville plant of the Johns-lfianville Corporation. Samples of se veral varieties of asbestos dust were first submitted to the Saranac Laboratory for examination and one kind, identified as Lot D, which was low in magnetite and chromite and had a fibrous content estimated to be about 7 per cent, was selected as most suitable. Steel wire brushes were fastened to the inside sur face of the hopper and to the rotating paddle in order to open up the bundles of asbestos and liberate more fibers into the atmosphere. 60. The composition of the long-fiber asbestos used in this experiment is indicated by the chemical and petrographic analyses given in Table lU. It appears that this material was a much purer form of asbestos than the shortfiber dust used in other experiments. This is borne out by comparing the approx imate analyses of the long-fiber and short-fiber idust in Table 15>. 61. 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; for -26- the second year, 2*8 million; for the third year, 3? million; and for the fourth year, 2*3 million. Examination of the impinger samples with dark field illumin ation disclosed that many fine particles less than one micron in size accompanied the larger particles and dark field counts vrere, on the average, about % or 6 times larger than the light field counts. 62. The size-frequency of atmospheric samples of the long-fiber asbestos dust and of the ball-milled dust is shewn in Table 16. Both samples were collected with the electrostatic precipitator. It will be noted that there was far more fibrous material in the long-fiber dust. 63. Reaction in Animals to Inhaled Long-Fiber Asbestos Dust. Guinea pigs, cats, rats and mice were employed in the iniialation experiment with long-fiber asbestos. PesuXts of the experiment, summarized in Table 17, are described in greater detail below. 62*. Guinea Pigs. The experiment was started with 100 guinea pigs. After exposure had been carried on for a year, a severe epidemic of pneumonia arose in the dust room and about one-third of the animals died or were killed. To replace them, 38 more guinea pigs were added to the surviving group in the dust room. 6. Rate and Type of Reaction. Histological examination revealed grossly visible lesions in the lungs after 8 months of exposure to dust, consisting of cellular infiltration about the terminal, bronchioles. At 12 months, there were adenomatoid changes in the air spaces and by the 16th month a definite fibrosis was present in these areas in half the animals. The fibrous lesion could be seen macroscopically at 20 months. From this time on the reaction increased in extent and in the amount of collagen and by the 3Uth month, it had fanned out into the parenchyma The lesions were rather sharply localized and the extensions from different bronchioles showed no tendency to fuse, even in animals exposed for the maxi-mum period (3 years). Although the intra-pulmonsry reaction sometimes reacn.su the pleura, there was no involvement of that membrane. No emphysema was visible -at any point. Some thickening of the larger bronchi rath a chronic inflammatory infiltration was re vealed, but it probably was no more than would be produced by a similar exposure to any dust. For the first months the phagocytes consisted of monocytes or very small giant cells; later, giant cell formation was more prominent. After 16 months the giant cells were large, filled with yellowish-brown pigment and sometimes vacuolated. An occasional animal showed an Admixture of polymorpho nuclear leukocytes and, in guinea pigs exposed fcr a considerable period, eosinophiles. The reaction was at first entirely cellular' but by 16 months fi brous tissue formation was definite. However, it never attained a stage of hyalinization suggestive of silicosis. A moderate individual variation occurred among the exposed a: Inals, both in the rate of developing lesions and in the stage of development attained at the end of exposure. Analyses of the lungs (Table 18) disclosed that although the tissue response was much greater in these guinea pigs than in those exposed to either shoit--fiber or ball-milled asbestos, the amount of mineral matter in the lung ash was Les. 66. Progression. In guinea pigs exposed to the dixst for 20 months and tL-.n removed to normal air, there was a marked tendency for cellular inflammatory reaction to clear. This effect, accompanied by contraction of the fibrous tissue, resulted in a diminishing size of the focal lesions. Hone -28- of these animals, killed at various periods up to lit- months after exposure, revealed lesions as large as those in the group sacrificed at the end of the 20-month exposure period or those in animals tmich remained in the dust room in four of the six remaining guinea pigs were so small that they were visible only with a hand lens. Reaction in the group exposed for 27 months and then transferred to a normal atmosphere was quite similar to the response in the 20-raonth exposure animals mentioned above. However, 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 extension of the reaction. 67. lymph Node Involvement. Reaction in the tracheobronchial lymph nodes was first visible at the third month of ex posure. At the 8th month patches of cellular connective tissue began to appear in the medulla and by the liith 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, as a variant, showed heavysheets of diffuse monocytes and large active giant cells but there was never any necrosis or hyaline formation. The spindle-shaped new cells were yellowish in color from fine pigment granules that stained for iron. No fibers or asbestosis bodies were seen. 68. Asbestosis Bodies. Although asbestosis bodies were seen as early as one month after exposure began, they were rare and hard to find. At 5 months more were visible, chiefly coiled inside giant cells, and at 8 months many bodies were free in connective tissue. They became fairly abundant as exposure progressed although in some later animals th9 asbestosis bodies were only moderately numerous. 69. Cats. Four cats inhaled the long-fiber asbestos dust for periods of 111, 25, 33 and h2 months, respectively, and were immediately sacrificed. Two other cats, after being exposed to dust for 18 months, lived in a normal atmosphere for an additional 2k months. 70. Rate and Type of Reaction. Exposure for Hi months wa3 sufficient to produce cellular accumulations of phagocytes around terminal bronchioles and peripheral arterioles together with compact col lections of similar cells in the tracheobronchial lymph nodes. At that time there were no typical asbestosis bodies, but smooth pointed yellow fibers were seen very rarely. With continued exposure, up to U2 months, reaction in the locations noted progressed to the formation of cellular connective tissue which made well-defined sheaths about the respiratory bronchioles and arterioles, marked lymphoid hyper plasia and lymphoid infiltration of bronchiolar walls. The bronchiolar epith elium was low and flattened, giving the tubes a smooth contour. Typical asbesto sis bodies were not formed although there was an occasional yellow, smooth, pointed fiber. No pleurisy was present. The reaction was similar in location to that in Idle guinea pigs, but fibrosis was much slower in development and had not readied the same degree of maturity. (. X-Ray Changes. Roentgenograms of three cats were made after exposure periods of 2?, 33 and U2 months, but tissue changes were not dense enough to be seen on an X-ray film. 72. Rats. Although 20 rats were placed in the dust room, many died from pneu monia and were not suitable for study. Five animals, of which one was exposed for 19 months and four for 25 months, were free from pulmonary infec tion and offer a basis for conclusions* _in_ 73 Rate and Type or Reaction. All four animals sacrificed at 25 months showed a well-marked peribronchiolar fibrosis. In the 19-month animal, reaction was just beginning- Asbestosis bodies were practically absent at both 19 and 25 months although two small smooth bodies ware found in the 19-month animal after a long search. Thus, these animals exhibited fibrosis without asbestosis bodies. 7U. Mice. Out of 20 white mice used in this experiment, 11 lived a year or more in dust and died or were killed without showing an appre ciable degree of pulmonary infection. 75* Rate and Type of Reaction. Reaction was limited to phagocytosis by mononuclear cells. Usually these were wide ly scattered through the air spaces; a limited number were grouped about the ter minal bronchioles producing some thickening of their walls. There was no sug gestion of fibrosis. The striking feature of the experiment was that 9 out of the 11 mice (82 per cent) exposed to dust for a year or more showed pulmonary tumors, usually adenomatous in type'. These lesions did not contain dust or asbestosis bodies. Numerous asbestosis bodies were observed in anl male killed late in the experi ment. Thus, these animals exhibited asbestosis bodies without fibrosis. 76. Summary and Interpretation. The purpose of this experiment was to evaluate the importance of long fibers in the tissue response to inhaled asbestos. The results indicate strongly that long fibers are chiefly responsible for the reaction. Thus, in guinea pigs reaction developed earlier and became more extensive than in previous experiments in spite of a smaller concentration of atmospheric dust and a lower mineral con tent in the lungs. Furthermore, a typical peribronchiolar fibrosis was produced in cats although in a previous exjjeriment -with short-fiber dust it 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. xxxra. INJECTION EXPERIMENTS In order to determine to what extent the various fibrous minerals possess the capacity to produce tissue damage, numerous injection experiments were per formed. In these experiments guinea pigs and rabbits were used and the mineral dust was injected by the intratracheal, intraperitoneal and intravenous tech niques. For the purpose of simplification the findings in each series of test have been condensed and reported in tables, to which reference will be'made later. 78. Experiments Using Intratracheal Technique. Since the asbestos minerals do not cause a typical advanced fibrosis in extra-pulmonary tissue, the intratracheal technique is the perferred way of in troducing 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. 79. Comparison of Fibrous and gon-Fibrous Dusts. To demonstrate that the ability of asbestos to produce fibrosis resides in its fibrous character, the series of injection expert ments reported in Table 19 were performed. The tests were made with unheated long-fiber chrysotile and iTith chrysotile that had been ignited to destroy its "JO flexible structure or ball-milled to reduce the length of fiber to 3 microns and less. At the same time control tests were made frith serpentine, which has the same chemical composition ss chrysohlle but .is non-fibrous. A review of the findings i-ovealc lie;;- only th-o vvihe.-v'-. "t " ' :Av':'-rti-.c -p.rodu.crd fibrosis. Fibers subjected to ignition or short-3ned by ball--milling liad lost their capacity to cause serious tissue damage. Ignition produced Important changes in the chry- sotile fibers, among them being loss of rater* an ; Iteration ii'e-m a flexible to a brittle structure and possibly other changes* 30. Comparison of Various Long-Fiber Dusts. Some very interesting findings ore disclosed by the results of the experiments included in Table 20. First, all. the long-fiber asbestos minerals tested, with the exception of anthophyllite, produced a typical fibrosis. It is not entirely clear why anthophyllite behaved differently from the other asbestos minerals. Unfortunately, f of 8 animals died of pneumonia within the first two weeks of the experiment and the remaining animals were sacrificed at- 1, 8 and 12 months; thus observations were not made at the optimum periods of 2 and U months. Second, with the mineral brucite, which is not a silicate but is a fibrous form of magnesium hydroxide, a characteristic fibrosis like that of the asbestos minerals was obtained. 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 acbe3tosis. Third, no fibrosis resulted from the injection of glass wool fibers, even m though glass wool resembles asbestos in some rays. There are fundamental differ ences, however. A glass wool fiber 3 microns in diameter is a solid rod and, 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 flex ibility. It would seem that thi3 structure and the associated fle>d.bility are important factors governing the capacity of a mineral to produce peribronchiolar fibrosis. 81. Comparison of Long-Fiber and Short-Fiber Dusts. With quartz dust it has bean demonstrated that the smaller the particles, the more intense is the tissue reaction, and that there is little reaction to particles larger than 3 microns in diameter. In the case of asbestos, however*, the reverse is true and apparently only long fibers have any specific effect. This is confirmed by the data of Table 21, 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 minerals tested (except anthophyllite, as noted in Section 80) produced a fibrosis; when the material was prepared by first grinding the fibrous dust until the length of fibers was reduced to 20 microns ani less (or, in some cases, 3 microns and less), none of the injected mineral dusts caused fibrosis. 82. Experiments Using Intravenous Technique. The experiments, described in Table 22, in which the Intravenous method of injection was employed, show that the asbestos minerals are far different from quarts in their action on tissue. It has been repeatedly demonstrated that intra venous injection of quartz particles 3 microns and less in diameter will cause a typical tissue reaction with the development of fibrosis in extrapulmonary sites, such as the liver and 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; it may have been caused by silicic acid liberated by the finely-ground mineral. ^1. 03. Experiments Using Intraperitoneal Technique. The results of injection experiments with the intraperitoneal technique are given in Table 23. It Trill 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 indicate also that the fibrosis initiated by the irritation of asbestos fibers is not restricted to the lungs, as was formerly assumed, but can be produced in the peritoneum as well. LECm. OTHER EXPERIMENTS WITH ASBESTOS MINERALS A number of additional experiments were conducted to throw more light on specific phases of the asbestosis problem. 8. Protective Action of Aluminum Compounds. Intratracheal injection of a suspension of long-fiber chrysotile to which colloidal aluminum hydroxide had been added revealed that the addition of the aluminum compound did not prevent the tissue irritation produced by chrysotile. If anything, the acute Inflammatory response to the injected fibrous mineral was accelerated. One month after the last injection of the dust suspension the bronchiolitis was becoming fibrous. 86. Formation of Asbestosis Bodies. 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. Following subcutaneous injection of two kinds of chrysotile into the groin of guinea piga - one kind containing 2 per cent and the other 0.2 per cent Fe20;jthe asbestosis bodies were equally numerous at both sites of injection. An attempt to produce asbestosis bodies in guinea/pigs by implantation of three silk bags containing fibrous chrysotile was unsuccessful. One bag planted subcutaneously in the abdominal trail disappeared; the other t-vro bags, placed in the peritoneal cavity, produced a little foreign body reaction but no asbestosie bodies in a year. Intratracheal injection into guinea pigs of asbestosis bodies recovered from human lung tissue failed to produce the typical tissue reaction to asbestos fibers. The injected material was obtained by digesting with sodium hypochlorite .solution 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 and may be maintained in vivo for a year or longer. LXXX7H. THEORY OF IRRITAHT ACTION OF ASBESTOS MINERAIS 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 upon 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 irtitating tissues. Accord ing to this hypothesis, asbestosis would be merely an indirect silicosis. Several facts make the chemical theory untenable: (1) intratracheal injections of brucite fibers, which had a silica content of only 0.90 per cent, caused a typical fibrosis like that produced by the asbestos minerals; (2) free-eilica particles increase in potency as the particle size becomes less, but asbestos fibers shorter than about 10 to 20 nrLcrons are relatively innocuous; (3) clurdnum hydroxide neutralises the irritating effect of quartz but not of asbestos $ (h) serpentine has the sane chemical composition as long-fiber chrysotils but it does not produce the same kind of tissue reactionj (5) there is a wide range in the chemical composition of the minerals which do cause asbestosis (see Table 2lj). In view of this evidence it seems more likely that asbestosis is caused by an unusual mechanical irritation from long asbestos fibers. Probably this irritation is related to the peculiar filamsnted structure of the fiber and > -- - -- ~ ~-- -- --1 m--m <- the associated flexibility, which are possessed by no other foreign body. For example, ignition of chrysotile fibers changed their structure and made them inert while the same fibers, before being heated, would produce fibrosis (see Table 19). Further support for the theory of mechanical irritation is that asbestosis occurs in an organ of liigh mobility- - the lung - and that a fibrous reaction can be produced by injection of asbestos fibers into the peritoneum, where there is also a degree of mobility, but not in other extrapulmonary organs, o LmVIII. COMPLICATIONS The experimental investigation with asbestos minerals was concerned primarily with the effect of the dust on normal tissue but some attention was given to other phases, such as susceptibility to infection and occurrence of malignancy. 89. Infection* The only experiment in which the effect of inhaled asbestos dust on a pul monary infection was studied was the first inhalation experiment, carried on with "King's floats" dust. It is, perhaps, unfortunate that infection studies were not made in the other inhalation experiments also. 90. Susceptibility- to Tuberculous Infection. The development of a tuberculous process initiated at the beginning of exposure to asbestos dust, and also of an infection superimposed upon an established asbostosis, tras described in Sections 19 and 20 of this re port. It "will be noted that asbestos, when classified according to the effect of a dust on tuberculous infection, would be placed below an active dust like quartz but above inert dusts, 3uch as calcite and gypsum. In animals infected with attenuated tubercle bacilli, quarts frill cause the infectious process to progress until the animal dies of tuberculosis. Inert dusts will have no effect on the infection and the lesions will usually heal and the disease disappear. Asbestos dust is in a different category, fcben the fibrous dust was being in haled during the evolution of the infection, there was a spreading of the tuber culous 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 following the completion of nearly three years of exposure to asbestos dust, progressive disease did not develop* The only modification of the infection was one of localisation, a few bad 111 being retained in the fibrous terminal bronchioles and farming tubercles there in addition to the usual foci beneath the pleura. Such tubercles healed in a few months and there was nothing to suggest any influence on the course of the disease. 91. Susceptibility to Mon-Tuberculous Infection. There was no pointed ex periment concerning the effect of inhaled asbestos dust on non--tuberculous infection. Intercurrent pneu monia. among animals exposed to asbestos dust was rather common, 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 non-tuberculcus infection* 'Nevertheless, since such epidemics are not uncommon in inhalation experiments frith 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 non-tuberculous pulmonary infection. 92. Neoplasm* No specific experiment was conducted to determine whelher the inhalation of asbestos favors the development of neoplastic disease but certain observations on this subject were recorded in the outline of the proposed monograph on asbestosis submitted by the late Or. L. 0. Gardner in February 19lt3. In it he called atten tion to the high incidence of lung cancer among mice inhaling lcog-fiber asbestos. In his experimental notes, however, he referred to these lesions is adenomas. There is an important distinction between adenoma and cancer rhich should be made clear. A cancer' is a tumor, or neoplasm, capable of local invasion and de struction of tissue, which can distribute cells through the lymphatlcB or blood stream to produce isolated foci, from which new tumors develop* This phenomenon of dissemination is known as metastasis and any tumor which exhibits it is a malignant growth, of which cancer is one type* An adenoma, on the o.her hand, is a so-called benign or non-malignant tumor (neoplasm) which may.or mr not be capable of local invasion but which does not metastasize* In order to clarify the exact nature of these lesions the path >logical mater ial is being carefully examined. Since it is felt desirable to hern the benefit of Doctor Vorwald's judgment, a review of the data on this subject is being post poned until after his return from Europe. Rether than delay the entire report, further discussion will be reserved for a supplement to be issued lat v. Owing to the vast ar,cunt of data included in this report it seems most convenient to state the conclusions derived from the investigation and. when necessary, follow each one -slth a brief resume of the evidence. A Various forms of asbestos fibers produce a peribronchiolar fibrosis lungs of guinea pigs, rats, cats and rabbits but not of mice and dOgS. Both inhalation and injection, er-cperiments provide ample support for this conclusion. Figures and 6 show the reaction to two different kinds of asbestos mineral. B* The mode of action appears to be primarily mechanical rather than chemical in nature. The evidence is given in section LiXiVIT. Figures 1, 2, 3, U and 7 illustrate the important points. The fibrous filamented structure of asbestos appears to play aa essential part in the irritating action, since the solid fibers of glass wool do not produce fibrosis (see Figure 8). C. Short asbestos fibers do not produce fibrosis. The conclusion is implied in the evidence mentioned in para graph B above. Experiments which further support this finding are reported in Tables 21 and 23. D. Typical fibrosis can be produced by an atmospheric suspension of asbestos dust containing only an extremely small proportion of long fibers. In the inhalation experiment with 100 per cent ball-milled asbestos dust a typical, though delayed, fibrosis mas t obtained 'see; Tabic; 22.)* although iocs than 1 per cart of the atmoapharic dust consisted of fiber? longer than 10 microns, as is shown in 'fable 17.. In contrast* the intra tracheal injection xperimsnt with fine asbestos duct con taining no long fibers failed to produce fibrosis, (see Table 21). E, Inhalation of asbestos dust apparently does not alter significantly the course of experimental tuberculosis in guinea pigs. This conclusion is tentative since the evidence on which it is based does not conform with cur usual experience. Refer ence to Table 3 will shew that when, infection was coincident with onset of dust exposure there was temporary progression of the disease with subsequent healing; when infection was initiated after 25 3/h months of du3t exposure the course of the tuberculous disease was not appreciably altered. In contrast,it has been observed in experiments with mixed dusts containing quartz that if there is a slight progression of the tubercu losis when infection and dust exposure are coincident, this effect is more marked (instead of less, as with asbestos) when infection is initiated after a period of dust exposure. This conclusion, concerning the effect of inhaled asbestos dust on tuberculosis seems justified because in the more sensi tive test (infection initiated after a period of dust exposure) there was no appreciable increase in susceptibility to the tuberculous infection. However, since the findings in the asbestos study do not- coniform with previous experience, and b` dealing -with infection. contained ocb.y a- relatival;* snrj.l CiJ'"'OV'T^ '^X '* *** *"' { '-'^A :`,0'"* l '. '1 "* di;" r j` J T.'C'r'f?'` ?.*> further irxvo:5t>3.gr.tioB of this phase of us'bsstoois cs cor:The fcraation of asbestosis bodies ssei"s to represent a coating of the fibers end results in loss of The ability to produco fibrosis. Intratracheal infection of asbe^tosis bodies failed be prechics i.'ae typical, tissue reaction (sea section. 36). The cessation of progressive reaction to inhaled asbestos dust soon after socposuro terainatee nay be due to the .formation of asbestcsis bodies. -42-