Document 939N4K0MLp42BE1JD83nVZ06L

IillJL Experimental Studies "by THE SARANAC LABORATORY SARANAC LAKE, BEST YORK S> ca,, Report to the JOHNS-MAMILLE CORPORATION SECT YORK, wm YORK January 31, 1949 Submitted by: Arthur J. Vorwald, W.D. Director a U PLAINTIFF'S EXHIBIT piwtQQOSttL PLAINTIFFS EXHIBIT mum CONTENTS l ABSTRACT 2 Introduction 3C Asbestos Minerals nr EXPERIMENTAL ASBES T 0 S I S 5c Experimental Methods 6, Species Susceptibility 7. Peculiar Characteristics of Asbestos 8. Rate of Tissue Reaction to Asbestos Fibers 9. Asbestos!s Bodies X INHALATION S X P E R I ff EH I s XI KING'S FLOATS ASBESTOS DUST 12. Dusting Material IS. Dust Composition 14c Dust Concentration 15a Reaotion in'Animals 16. Guinea Pigs 17c Rate and Type of Reaction 18 Progression 19. Infection Coincident with Dust Inhalation 20. Infeotion after Dust Inhalation 21. Asbeotosis Bodies 22. Rabbits 25* Rats 24. Summery and Interpretation . *5 1 2 2 3 4 S 5 6 7 7 8 8 8 8 9 9 9 10 10 11 12 12 12 IS CONTENTS 26. 27. 280 29. 30. 81. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 46. 47. 48. 49. XXV SHOOT-FIBER ASBESTOS DUST Dusting Material Dust Composition Dust Concentration Sise-frequency of Dust Reaction in Animals Guinea Pigs Rate and Type of Reaction Progression Asbestos!s Bodies Rats Rate and Type of Reaction Cats Rate and Type of Reaction X-ray Changes Asbestosis Bodies Rabbits Rate and Type of Reaction Asbestosis Bodies Summary and Interpretation XLV 100 PER CENT BALL-MILLED ASBESTOS DOST Dusting Material Dust Composition * Dust Concentration Sise-frequency of Dust * Page 14 14 IS IS IS 16 16 15 17 17 13 18 19 19 19 20 20 20 20 21 21 21 22 22 23 CONTENTS SOo Reaction in Animala 61. Guinea Piga 62. Rate and Type of Reaatiaa 53. Progression 54. lymph Node Involvement 55. Aflbeatosie Bodies 56. Hate and Mice 87. Summary and Interpretation mu LONG-FIB ASBESTOS DOST 59, Dusting Material 60. Dust Composition 61. Dust Concentration 62. Size-frequency of Dust 63. Reaction in Animals 64. Guinea Pigs 65. Bate and Typo of Reaction 66 ,, Progression 67. Lymph Node Involvement 68. Asbestosi3 Bodios 69. Cats 70. Piate and Type of Reaction 71. X-ray Changes 72. Rats 73. Rate and Type of Reaction Page 23 23 23 24 24 24 24 25 26 26 26 26 27 27 27 27 28 29 29 30 30 30 30 31 CONTENTS 74, Mice 76. Rato and Type of Reaction * 0 ,, end leterpv m,;.-? iica ucsm IB JEGTIOU EKPBKIMEHS 78. Intratracheal Experiments 79. Comparison of* Fibrous end Eon-fibrous Dusts 80, Comparison of Various Long-fiber Dusts 81 Comparison of Long-fiber end Short-fiber Dusts 82, Intravenous Experiments 83. Intraperitoneal Experiments LXXXIV OTHER EXPERIMENTS 86. Protective Action of Aluminum Compounds 86. Formation of Asbestosis Bodies LXXXVII THEORY OF IRRITANT ACTION 89. Infeotion immi COMPLICATIONS 90. Susceptibility to Tuberculous Infection 91. Susceptibility to Eon-tuberculous Infection XGII SUMMARY XCIII TABL ES XCIV FIGURES .SM& 31 m 31 32 32 32 33 34 34 35 35 35 36 36 37 37 38 38 40 43 * 71 72-75 ABSTRACT - \s. Ac Various forme of asbestos fibers produce a peribronchiolar fibrosis of the lungs of guinea pigs, rats, oats and rabbits but not of mice end dogs. B. The mode of action, of the asbestos fiber is primarily mechanical rather than chemical in nature. Ct Long asbestos fibers are essential in the production of peribronchial fibrosis; short fibers are incapable of producing this reaction, Bt. typical experimental asbestotic fibrosis was produced by the inhalation of an atmospheric suspension containing an average of 138 million particles per cubic foot of air of which only O.S per cent consisted of fibers longer than 10 microns S. 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, viz. as the concentration is increased, the reaction develops in shorter time. F,, Established experimental asbestosis ceases to progress on discontinuing exposure to the dust. G. The formation of asbestosis bodies represents a coating of the fibers by blood ancl tissue elements which results in loss of ability of the fiber to produce fibrosis. H. Aluminum hydroxide failed to neutralize the fibrosing action of the lung fiber asbestos (Section 85). I. Inhalation of asbestos dust did not alter significantly the final outcome of experimental tuberculosis in two series of guinea pigs exposed. 2. Introduction. Asbaatosia is a form of pneumoconiosis resulting from prolonged inhala tion of s^b^tos dust. The nsxas asbestos. literally "unburnable,n ia not that of a particular mineral but is a term applied to a number of different minerals whose characteristic feature ifi a structure composed of long, par allel, flexible fibers. This structure is unique because the fibers are capable of repeated longitudinal subdivision to units of molecular proportions. In length the fiberB vary from a fear microns to six or more inches. Some varieties are stiffer than others but many are sufficiently flexible to be spun into yarn and woven on modified textile maohinoiy. 3. Asbestos Minerals The asbestos minerals are silicates of variable composition and belong to the serpentine and the amphibole groups. Listed below are -the more common varieties. Amphibole Group Anthophyllite (Mg, Fe) silicate Amosite (Mg, Fe, Al) silicate Amphibole (Ca, Kg, Fe, Al, 8a, K) silicate Tremolite (Ca, Mg) silicate Actinolite (Ca, Mg, Fe) silioate Grocidolito (8a, Fe) silioate + Fo silicate Serpentine Group Chrysotile Mg silicate (hydrous) The bulk of the asbestos of cossHcroe is chzysotile, 325gO.2S102.SHgQ, which is joined in the Thetford region of the Province of Quebec* Crocidolite and amoeite are also used commercially bub in much smaller amounts. Chrysotile occurs as reins in serpentine, & mineral similar la chemical com position to chrysotile but which exists in. massive fora and is made up of microscopic fibers without the parallel orientation characteristic of chryso tile, 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 aorose the vein and not lengthwise with the formation. Attention is directed to the mineral brucite, jgO,HgO, which is often found in the same formations with serpentine End chrysotile and may ba 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 asbestifom minerals, bruoite 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 SXPBRIKBUTAIi ASBESTOS! S For many years studies have been carried on by the Saranac Laboratory in an investigation of the cause, nature and development of asbeatosis. The present report is devoted to Experimental Asbestosis. In it are described the animal experiments with various kinds of aebestos dust, Another report, to be prepared and issued later, will be concerned with Human Asbestosis and will cover the health aspeotB of workers who have bean exposed to asbestos dust in an industrial environment. r'i.i'-c-.:~ stesuc-ais in man is a chronic disease *vuich requires years is develop, it is poaedbie to reproduce in one cr more ssecies of animal cha.: -- vO - clr tirouo chants which era similar to the lesions of human asbestos is, since the li fe-span of the experimental animal is relatively short, it is not poser bln to develop the characteristic lesions in anianls under the usual industrial con ditions * Consequently, to obtain . 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 vTould ordinarily bs enCDiiaherod 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 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, eats, doge, 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 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* -a- Long-term inhelnticn experiments furnish information -upon whioh great reli ance is placed when satinating the degree to which a dust might be h&sardoua to industrial workers. Whether or not atmospheric dust, even though potentially dangerous, con be inhaled, pass the natural defense barriers and reach the pul monary tissue in quantities sufficient to cause damage oan he determined only by inhalation procedures. Injection experiments are useful because in them contact between the dust particles and tissue3 ie assured and the potential capacity of the duet 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, d. Specieg Susceptibility Unlike free silica, asbestoe does not exert its specific effect in all organs of all species of animal (fable 1), Injection of fins quarts into various organs of the guinea pig, rabbit, rat, mouse, oat, dog, chicken and even tadpole will produce silicotic nodules. However, similar injections of long or short fiber as bestos have resulted in a fibrous reaction in the lung and, to a lesser extent, in the peritoneum but not in other ergons of the guinea pig, rabbit, cat and white rat. The dog and white mouse failed to respond. This variation in species is yet to be accounted for. 7. Peculiar Characteristics of Asbestos Experience has demonstrated that most of the partioulate matter inhaled into the lungs of man end animal i3 10 microna or less in maximum diameter. Larger par ticles apparently aro excluded by the protective mechanism of the upper respiratory tract. In the case of fibrous materials, however, this restriction does not apply and fibers 100 and even 200 microns in length have bean found in the terminal air spaces of human lungs. In small laboratory mireals exposed to asbestos dust the maximum length of fiber found in the lung rarely exceeds 60 microns. Hot every kind of fibrous material is inhaled with equal readiness: for example. the synthetic fibers of gjc-is word, .n flexible to pass easily through the nose, pharynx, trachea cm reach the terminal bronchioles and alveoli, Inhaled particulate natter comes to rest throughout the terminal ear spaces (alveolar duet, atria, alveoli) in all carts of the lungs; inhaled asbestos fibers are first retained in the respiratory bronchioles. These very small tubes are immediately distal to bronchioles lined by ciliated epithelium* Their own essen tial lining is a low cuboidal type of epithelium. tut, 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 "seal 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 like quart. Evidences of tissue response appear as soon as fibers have localized in sufficient concentration in specific area3. In rat3 receiving asbes tos fibers by intratracheal injection this evidence is visible os early as two weeks after injection; far quarts dust the latent period might be two montlis 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 L fibers in the lung nay cause the fibrous tissue response to continue for a short tine, until the fiberB have been coated. This progression is of only a slight degree and of little significance* 9, Asbestosis Bodies The peculiar structure known as the asbestosis body or curious body is a specific concomitant of asbestosis. The typical body is a golden-yellow, beaded or haustrated rod which nay be either straight or curved. Often one or both ends are bulbous like a dumb-bell. The bodies vary considerably in length, and dimensions up to 250 microns have been recorded. It is believed that asbestosis bodies ore 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 end 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 IK HALATION EXPERIMENTS Four comprehensive inhalation experiments have been conducted at the Saranao 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 ore identified as King's floats, short-fiber, 100 per cent ball-milled, and long-fiber asbestos dust. XI INHALATION E3CPERD2EHT WITH "KINO* S PLOATSn ASBESTOS DU8T The first inhalation experiment conducted at the Saranac Laboratory with asbestos dust was begun in 1928. AnInals inhaled the dust for periods up to nearly three years end 8case 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, ienu of THE JOURNAL OF INDU STRIAL HTfllENE.* At that time observations covered a period of only 2-1/4 years and the conclusions as to the ultimate effeots of inhaled asbestos dust were provisional. Results of the completed study shoar 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 Ring's floats and was composed of short fibers and particles of variable sise. It was obtained from the Thetford, Quebec plant of the Asbestos Corporation of Amarioa. 13. The dust composition (Table 2) reveals that the amount of fibrous chrysotile was only 14 per cent, a rather low value. However, there was sufficient fibrous material to produce a characteristic fibrosis. 14. The dust concentration at first was quite low and for lmpinger 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 KETMJCCNIOSIS, VT, Inhalation of Asbestos Dust. Gardner, L.U., and Cummings, D.E. J. Ind. Hyg., 13: 65-61, 97-114, 1931. shorn by an average count of 0 b r-iiliion ion 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-" lug 9 or 10 months of It n.'O vJufQ1?JLl l is considerably more dust was dispersed into the atmosphere. Average dust counts for impinger sampO.es collected after this change ware 53.7 million for the v.v!-.1. 1:5arid and 1.6 million for particles larger than 10 microns* 15. Reaction in Animals to Inhaled l!Singrs Floatsn Asbestos Dust. Results of the investigation, briefly summarised in Table 3, show that inhala tion of Sing's floats asbestos dust produced & 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 du3t exposure was investigated. The remaining two groups were infection controls. 17. Rate and Type of Reaction. Guinea pigs inhaling this 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 extensions of the original lesions, Kew 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 assay of them were carried into the wall by migratory cells ,, ifononuclear 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 so gradually that mitotic division of fibroblasts could rarely be discovered; nevertheless, the number of fine intercellular collagenous fibers (fibrosis) steadily increased. As this fibrosis contracted, it partially closed the alveoli, and with this atelectasis the lining epithelium assumed its embryonic cuboical form. The result was the adenoma-like appearance that `.Mllis 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 uO guinea pigs infected with atten uated tubercle bacilli {Rp 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 inx or current pneumonia. Briefly, the re sults were as follows: 10 revealed some evidence of spread of the tuberculous process; in 6 of these it was confined to the lungs and in the other i; 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 more fibrosis than is characteristic of either tuberculosis or asbestosis alone. The 9 animals -which were still alive after two years of dust exposure were sacrificed at intervals during the following year. In I* 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 locallyt in h 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 pig3 with spreading pulmonary tuberculosis showed tuberculosis of the spleen and liver. 20. Infection S uperimposed Upon an Established Asbestosis. 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 -11- l no more numerous than in ncn-dusted controls, but a considerable number were found in the depths of the lung about foci of asbestosis. The reaction to infec tion showed only slight-local extension about the original sites in the lungs and tracheobronchial lymph nodes. The abaoa-inal viscera were involved in onlyone animal. Caseation was found in tubercles 1-1/2 months old but by -1/2 months it had completely disappeared, leaving only scar tissue* The latter still persisted in the last animal, which was killed lu months after infection,. 21. Asbestosis Bodies* Moderate numbers of asbestosis bodies occurred in the lungs of the guinea pigs, becoming more 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 ox* fibrosis in the term inal bronchioles nor were there any asbestosis bodies. 23- Rata. 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- ized under three headings. dust caused a charae teristic peribronchiolar fibrosis in guinea pigs 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 pigs. In guinea pigs in- fected with attenuated tubercle.bacilli and then placed in the dust room, the results were more variable than in 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 healings 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 intercurrent pneumonia it is' felt that definite conclu sions as to the influence of asbestos on the course of tuberculous infection are not justified. 1n tt*t INHALATXOH EKPSRIfflKEl WITH aBOKT-FIBSS ASBESTOS DUST Since hazardous dusts like quartz are most effective in producing fibrosis when the particles ore S microns and less in size, an inhalation experiment was carried ca to determine whether this condition is true also for asbestos dust* It was thought that by using a short-fiber asbestos dust consisting almost en tirely of fibers and particles smaller than 3 microns an aocelorated 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 mm. to 1 micron or less in length as well as a great deal of par ticulate matter and which produced a typical peribronchiolar fibrosis in exposed guinea pigs, served as a basic of comparison. 26. The dusting material for this experiment was forwarded from the Kanvtlie plant of the Johns-Kanville Corporation. It was the remains of fibers collected in dust bins after a carding operation and screened to pass 200 mesh. 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. Then used alone in a standard dusting machine, this finelyground asbestos tended to pack in the hopper and it became necessary to mix one volume of the unground material with three volumes of the ground to generate a satisfactory dust cloud. The addition of the saall quantity of unground asbestos was unfortunate beoause 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. -14- 0*7 - { The composition of the short-fiber asbestos as received is disclosed by the chemical and petrographic analyses given in Table lu Samples taken before and after grinding yielded about the same values o: 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 ajximal 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 lUO million for the third year* 290 Size-freguency 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. Four species of animals - guinea pigs, white 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 Pigs. Eighty guinea pigs were originally placed in the dust room but. 21 of them were later eliminated from the experiment and killed because of enlarged lymph nodes. Of the other 59 animals, U6 remained in the dust room until they were sacrificed or died from natural causes 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 inlialed short-fiber asbestos ^ essentially the same as that, already observed in the experiment with fling'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 2b months of exposure only a very few amaii 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 2k months the cellular clumps were sometimes quite marked and sometimes changes in the epithelium resulted in the adenoma-like or nadenomatoid" 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 hyalinization. As in the rats described below, the alveolar walls might be made up of a btod 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 fihrosis, 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. --16-- 33. Progression. -0"%? / 44 *'.0* i. %rCs. > the cessation of 20 months' 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 two pigs were read as * and one as 2*. Among the 13 removed from dust the findings were variable: in 2 the reaction was : in a it was +j in 3 it was 3+J in 3 it was t+j and in one animal it was 5+. 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 the 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 0 three animals in dust 20 months and then in normal air 13-3A months, yet the tis sue reaction for one animal was U+j for another, and for the third, only . 3U. Asbestosis Bodies. The formation of asbestosis bodies was at first ex tremely limited. After 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-body 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. 3?r Waite Rats. Seventy-three white rats were exposed to atmospheric shortfiber asbestos dust for periods up to 32 months* Sacri- 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 was limited to occasional slight thickenings of the septa about small accumulations of dust cells. In a few rats at 10 months, there was a sug gestion of early fibrosis 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 130 diameters or more, consisted of patches along alveolar ducts in which the walls of the air 3paces were very thick, due to swollen collagen framework. Connective tissue and Foot--Bielschowski 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. Kb 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 rn&nocytic cells at 12 months and, at 20 months , some diffuse thickening of the reticulum. In a few rats there wa3 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. -18- luvulls of els;atcal analyses luio.de on the whit: r.-.is are given in Table 3 and the average values bavc been tabulated in Table 9 lor comparison with similar values lor rats Inhaling other dusts. The concentration of atmospheric particles to which the animals were exposed was approximately the same for asbestos and quarts;: for the gypsum-quartz mixture, it was about tid.ee as high and for chert five tires as high. It Td.ll be noted that the percentages for asbestos are lower than those for quarts or chert but ere 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 lor/ 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 used in this inhalation experiment with the ehort-fiber asbestos. Eighteen were kept in the dust room until death, the exposure period ranging from one month to nearly U-->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 i* years of expo sure. The tissue response in this species liras 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 on 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 29-3A months the picture was negativej after U5 months a faint mottling could be detected throughout both lungs. At autopsy, 8 months later, there was only microscopic -IQ- fibres!? in the subpleural zone plus be bronchioles iiO. Asbestosis Bodies. in prolonged nocaas. /ii.OOOrl in two animals exposed for more than a year. 0.OO!*. L' yellcs? atypical asbestosis Lt U J.li ,'C* 'e found hi* Rabbits. Sight rabbits were exposed to oust for periods extending from one to more than firs ynars. The last animal was removed from the dust room and left in normal air 6 months before being sacrificed. U2. 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 vail thickening was first detected after about 3 years of exposure and was seen in ell 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 pulnwnary 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 preserved. U3. Asbestosis 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. -20- 44. Smamfiry and Interpretation Tho 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 quarts. This experiment, in which the reaction was slower and less extensive than with King's floats, indicates that the reaction 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 cub-pleural fibrosis and in the rabbit the fibresis which occurred could not be positively attributed to the dust because of a strong possibility of pulmonary infection. XLV INHALATION EXPERIMENT WITH 100 PER CERT BALL-MILLED ASBESTOS DOST 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 the 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 imground material was used. 46. The dusting material was the ground short-fiber asbestos used in the previous inhalation experiment but no imground 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. s notorial tc forts small 1 spherules which prevented much ex' the fibrous; portion floating out of the hopper, the dispersal of the dust- r.as not entirely s- stisfaciory ana after ? months of operation, the aust- ing machine was j eecnvertea. its original design. To prevent "pilling" or the formation, of spherules of a, ibestos, steel wire brushes sere attached to the in- side surface of the hopper- ; and to the rotating paddle. This arrangement gave satisfactory results end was used for the remain!: 1 months of the experiment. i7. The composition of the ras? material vshort--fiber asbestos) and of atmos pheric dust 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-suspended material was undoubtedly less than the lp per cent value given in Table 10. In an inter im report, it was stated that the air-horns 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-stilled 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. U8 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 lU5 million. inside the animal i .'he eu'wc::e; 'OJV V. wc'j.c. dust collected tatic apparatus is reported and one after., It frill be noted that a: into the air 50. Reaction in Animals to Inhaled ICO Per Cent Bali-Milled Asbestos Duet. Guinea pigs, rats and mice were used in the inhalation asperiment. Triti the 100 per cent ball-milled asbestos dust. The results are summarized in Table 12. 51. Guinea Pigs. The experiment was started with 100 guinea pigs. As the dust exposure proceeded, there were 39 accidental deaths. 32 of pneumonia in an epidemic. After 23 months of dusting the 16 surviving guinea pigs were transferred to normal air. 52. Rate and Type of Reaction. For the first year of exposure practically the only reaction to the dust was the pre-- eence 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 could be seen. At 2h 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, 7h* lungs cf animals exposed f:!* the full dusting period (23 months) and then living in normal air for 2 months revealed the charges described above and also very slight peribronchiolar fibro sis, After 8 months in normal air the findings were similar but at 12 months 3 of k animals showed grossiy-visible characteristic peribronchiolar fibrosis wiL-1 adenomatoid change. 5b, Lymph fa'ode Involvegient. 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 exposin'e many monocytes filled with yellow granules were present. At 30 month there had been a slight increase in reticulum but no fibrosis, ho further changes occurred in the nodes. Asbestosis bodies were not seen in the nodes of ary of the guinea pigs. 55* Asbestosis Bodies. Minute 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 all were intracellular, although at 20 months 3ome were long enough to project beyond the cell borders. It is important to note that in the later months of exposure there was a dis tinct increase in the number of long fibers (up to 70 microns in length) in the lungs and that after exposure ceased characteristic long asbestosis bodisa were seen. 56. White Rats and Mice. In this experiment k0 rats were exposed for periods up to 20 months and 2k mice for periods up to 12 -2k- was limited to phagocyte: C` lymph nodes. Ic asbcsto asbestos!s end reaction dialed petioles .eiy-scattorsd dust cells isported to the tracheobronchial but in the race there $7* Summary and Interpretation The tissue reactions c-bserved. in this experiment -were much less extensive and slower in development than in the previous investigation y/ith short-fiber asbestos. Since presumably there were fever fibers longer than 3 microns in the made in Section hh of the short--fiber experiment that the reaction 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: 'When the reaction is well-developed at the termination of exposure,the contraction of the fibrous tissue would obscure ary 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. Lvrn IRHALATTCJ? EXPSitliSHT V.ITT! J/Srfl-FSBBF ASBfSfOS FUST Aftar 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 tvs6 given added support. Sine the King's .floats asbestos used in the first inhalation experiment had a rather low content of fibrous ohrysotile and contained considerable serpentine and other impurities, it wae decided to conduct a new 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 Msnwille plant of the Johns-Hanvilie 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 75 per cent, was selected as moat 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 14. 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 oomparing the approx imate analyses of tb long-fiber and short-fiber dust 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 duet. For the first year of the experiment with long-fiber asbestos the average of the light field counts was 32 million; for the second year* L.C million; lor the third ;' "filler 5 end for the fourth year, h3 million. Examination of the ibred not .piss with dark field illumin- ation disclosed that many fine particles less? t one micron in size accompanied the larger particles and dark field, count;- -`.-re -d the average, about $ or 6 times larger than the light field counts. 62. The si2e-frequency of atmospheric samples of the long-fiber asbestos dust and of the ball-sailed dust is shown in Table 16, Both samples were collected with the electrostatic precipitator. It frill 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 inhalation experiment frith long-fiber asbestos. Results of the experiment, summarized in Table 17, are described in greater detail below. 61*. 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 than, 36 more guinea pigs were added to the surviving group in the dust room. * Rate and Type of Reaction. Histological examination revealed grossly visible lesions in the lungs after 8 month3 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 tic on the reaction increased in extent and in the amount of collagen and -27- by the 3hth month, it- had ibnn&d out into the parenchyma, The lesions were rather sharply localised .7 ad the extensions from different, bronchioles showed no tendency to fuse, ere.- in animals exposed for the maximum, period (3 years}.-. Although the intra-puls/nary reaction sometimes reached the pleura, there was no involvement of that m-jibrane. No emphysema was visible at any point. Some thickening of the larger bronchi with a chronic inflammatory infiltration was re vealed, but it proba :ly was no mors than would be produced by a similar exposure to any dust. For t'.o first $ months the phagocytes consisted of monocytes or very small giant cells5 later, giant cell formation was more prominent. After 16 months the gian. cells were large, filled with yellowish-bream, pigment and sometimes vacuolar-d. An occasional animal showed an admixture of polymorpho nuclear `.eTikocyt -s and, in gvinoa pigs exposed for a con/ddex*able period, eosinophiles. The reaction was at first entirely cellular but by 16 months fi~ broiv. tissue formation was definite. However, it never attained a stage of hyalinizat/on suggestive of silicosis. A moderate individual variation occurred among the exposed aiimals, both in the ratj of developing lesions and in the 3tage of development attained at the end cl exposure. Analyses of the lungs (Table 18) disclosed that although the tissut. response was much greater in these guinea pigs than in those exposed to either shoi t-fiber or ball-milled asbestos, the amount of mineral matter in the lung ash was less. 66. Progression. In guinea pigs exposed to the dust for 20 months and th-.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 sine of the focal lesions. None -28- of theso anineIs,illed at Various periods up to 14 months after QXpasure, revealed lesions as large as those in hho group sacrificed at the end of the 20-month exposure period or those, in anixcals which remained in the dust room for more than 20 months. Fourteen men the after duct exposure cessed, the foci in four of the 3ix remaining guinea rugs were so email that they were visible only with a hand lens. Reaction in the group exposed for 27 months and than transferred to a normal atmosphere ms quite 3istilar to the response in the 20-month exposure animals mentioned above, However, small foci were always visible on gross inspection of sectiona of all guinea pigs of the 7-nonth series but in no instance was there evidence of extension of the reaction, 67. Lymph Node Involvarient Reaction in the tracheobronchial lymph nodes was first visible at the third month of ex posure At 13ie 8th month patches of cellular connective tissue began to appear in the medulla and by the 14th 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 sniraals, as a variant, showed heavy sheets 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 sesnc 66. Asbestosis Bodies. Although asbestosis bodies were seen in the lung as early as one month after exposure began, they were rare and hard to find. At 6 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 continued although in some later animals the asbestosis bodies were only moderately numerous. # ^0* c I.1 oijj.* cfiVj .i:oiioiXoo. u'rvG j-oo.^ `i C' . -u. ;>. bOS O.ut-G v> for periods of I!;, 20, 33 and h-2 months. : e/s. eectiveitv.y and wc: e Smaediately sacrificed. Two other cats, after being es >oss<i to dust for 18 months, lived in a normal atmosphere for an additional months'. 70. Rate and Type of Reaction. Exposure for lis months was sufficient to produce cellular accumuiatione 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 the guinea pigs, but fibrosis was much slower in development and had not reached the same degree of maturity. Yi. X-Ray Changes. Roentgenograms of three cat3 were made after exposure periods of 20, 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 fbr 19 months and four for 20 months, were free from pulmonary infec tion and offer a basis for conclusions. -30- 73. Rate and Type of Reaction, All four animals sacrificed at 25 months showed a well-marked peribronchiolar fibrosis* In the 19HBOnth animal, reaction was just beginning* A3besto3is bodies wre practically absent at both 19 and 25 months although two small smooth bodies were found in the 19-month animal after a long search. Thus, these animals exhibited fibrosis without aebestoeie bodies. 74. Mice. Out of 20 white mice used in this experiment, 11 lived, a year or more in duet and died or were killed without showing an. appre ciable degree of pulmonary infection. 75. Rate end Type of Reaption. 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 thoir walls. There was no sug gestion of fibrosis. numerous asbestos!s bodies were observed in animals killed late in the expariment* Thus, these animals exhibited a3bestoeia 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 asbestoB. 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 experiment with short-fiber dust it did not develop In this species, Ths oause of the cellular fibrosis in the lymph nodes of the guinea piga is not clear. It did not occur in other inhalation experiments with, asbestos. LJLXVII INJECTION EXPERIMENTS In order to determine to 'what extent the various fibrous minerals possess the capaoity to produce tissue damage, numerous injection experiments were per formed, In those experiments guinea pigs find 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 tests 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 preferred way of in troducing fibrous dust into the experimental animal. In this method the dust suspension is injected by nssans cf a special needle or catheter deep into the treohea, from which it flows into the lungs. 79. Comparison of Fibrous and Hon-Fibrous Dusts, To demonstrate that the ability of asbestos to proauoe fibrosis resides in its fibrous character, the series of injection experi ments reported in Table 19 were performed. The tests were made with unheated long-fiber ohrysotile and with chryeotile 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 oontrol teste were made with serpentine, i/hicn has the same chemical composition as chrysotile but is nen-fibroue, A review of the findings reveals that only the unheated long-fiber chrysotile produced fibrosis. Fibers subjected to ignition or shortened by ball-milling had lost their capaoity to cause serious tissue damage. Ignition produced important changes in the chrysotile fibers, among them being loss ox" water, so alteration from a flexible to a brittle structure and possibly other changes. 800 Comparison of Various Long-Fiber Dusts. Some very interesting findings are 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 anthophyllitc behaved differently from the other asbestos minerals. Unfortunately, 5 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 4 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 asbestosis. Third, no fibrosis resulted from the injection of glass wool fibers, even though glass wool resembles asbestos in some ways* 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 this structure and the associated flexibility are important factors governing the capacity of a mineral to produce peribronchiolar fibrosis. -33- % 81, Comparison of hong-'-Plber and Shorn-Fiber busts. "ith quarts dust it has been demonstrated that the smaller the particles, the more intenso is the tissue reaction, and that there is little reaction to particles larger than 5 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 aeries of tests with fibrous minerals is reported, '-hen the injected dust consisted, of fibers 20 to 0 microns long, all tha minerals tested (except antho?hyHits, as noted in Section 80) produced a fibrosis; when the material was prepared by firstgrinding the fibrous dust -until the length of fibers was reduced to 20 microns and leas (or, in some oases, 3 microns snd less), none of the injected mineral diets 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 intravenous injection of quarts particles 3 microns and less in diameter will cause a typical tissue reaction with the development of hyalinized fibrotie lesions in extra-pulmonary sites, such as the liver and Spleen. .Asbestos minerals, however, on intravenous injection generally produce only an inert type of reaction, as ie revealed by the results given in the table. The reason for the early deaths in the experiment with chrysotile partioles is not clear; it may have been caused by silicic acid liberated by the finely-ground mineral. -34- Experizaar tc Using Intraoeriion 3a 1 She resol ts of injection experiment:; wit; the indraperitone.il technique are given in Table 2S. It rd.Il be noted tb*t toe long-fiber duets produced a fibrou? I* ction grille dusts? composed of particles t : dcrc&s and less in else erased onlj an inert type of response. These experiments indicate also that the fibrosis initiated by die irritation of asbestos fiber is not restricted to the lungs. as wee former! y assumed, but oar. be produs ,n the peritoneum as ?/sll0 0IH2R EXP EE If Eli 7 ? n?!i A T 0 S Iv! I ?] S R A L S A number of additional experiments -were conducted to throw more light on specific phases of the asbestos!s problem,, 85, Protective Aotlon of Aluminum Compounds. Intratracheal injection of e. suspension of long-fiber chrysotile to which colloidal aluminum hydroxide had been added revealed that the addition of the aluminum compemnd 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 lest injection of the dust suspension the bronchiolitis was beooming fibrous, 88, Formation of Asbestoais Bodies, Tho iron in the coating of the asbestesis 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 pigs - ono kind containing 2 per cent and the other 0,2 per cent Fe^Ggthe asbestos!r, bodies wore equally numerous at both sites of injection. bodies .in a ye xt- solution lung tissue removed at autopsy from an asbestos Tho arbei^tosis bodies could be seen in the guinea. pig* for at least e. year after injection This experiment shows that the asbestos!s body has a rather roDistant coating which is not destroyed by moderate hypochlorite treatment and may be msintr-.inei in vivo for & year or longer. LOTTO IHEOBI OF IRRITANT ACTION Two hypotheses have been proposed to explain the tissue irritation and rss.c~ ti.cn 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 as bestos minerals dissolve in the body fluids and that in this process thoir bases are leached av&y to leave silica in a form capable of irritating tissues. Accord ing to this hypothesis, asbestos!8 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.S0 per cent, caused & typical fibrosis like that produced by the asbestos minerals; (2) free-ailica' particles increase in potency as the particle sise becomes loss, but asbestos fibers shorter than about 10 to 20 microns are relatively innocuous; (3) aluminum B hydroxide neutralises the irritating effect of quartz but not of asbestos; (4) serpentine- has the saw chemical composition as long-fiber chrysotile but it does not produce the same kind of tissue reaction} (5) there is a wid rans<5 in the chemicrl composition of the minerals which do cause asbestosie (see Table 24), In view of this evidence it seems more likely that asbestosis is caused by an unusual mechanical irritation from 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. For example, ignition of chrysotile fibers changed their structure and made them inert while the same fibers, before being heated, produced fibrosis (see Table 19), Further support for the theory of mechanical irritation is that asbestos!s occurs in en organ of high 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 extra-pulmonary organs. LXXXVIII 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, suoh 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 nKingfs floats" dust. It is, perhaps, unfortunate that infection studies were not made in the other inhalation experiments also. (jpuxoili.Ty to -U.U. O - 'rXcjJ.i b oz the beginniiJ|r or exposure to asbestos dust, and also of an infection superimpose u;.on an established ashestosis. mis- described in Section? 1' unc. to of :.y,.r. re port .. It will be noted that asbestos, wl^en classified according: to the si~er-; of a dust on tuberculous infection, would be placed belc-v an active dust quarts but above inert dusts, such as calcic. and gypauru In animals infected with attenuated tubercle bacilli, quartz vrill cause the infect.io-::.s process to progress until the animal dies of tuberculosis. Inert dusts mil have no effect or the infection and the lesions Till usually heal and the disease disa-ipear , Asbestos dust is in a different category, bhen the fibrous dust was being in haled during the evolution of the infection, thare was & 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 cf exposure to asbestos dust, 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 piextra.. Such tubercles healed in a few months and there was nothing to suggest any influence on the course of the disease. 91. Susceptibility to Non-Tfiberculous Infection. There was no pointed ex periment concerning uh 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 59 per cent,. This incidental evidence suggests the possibility of an effect of asbestos dust on non** tuberculous infection. Nevertheless, since ouch 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 asbestod dust does not exert a significant effect on the susceptibility to non"-tuberculous pulmonary infection* . -S8- XCII SDKJiAHT Owing to the vest amount of data included in thia report it ssoms most convenient to state &3 precisely as possible the various points which emerged from the investigations and, when necessary, follow each v/ith a brief resume of the evidence* A. Various foras of asbestos fibers produce a peribronchiolar fibrosis of the lungs of guinea pigs, rats, cats and rabbits but not of mice and dogs. Both inhalation, and injection experiments provide ample support for this statement. Figures 5 and 6 show the reaction to two dif ferent kinds of asbestos mineral in the guinea pig ling. Similar but less extensive fibrosis occurred also in rats, cats and rabbits (Table 1), Mice and dogs failed to respond. This variation in re sponse of different speoies to identical duet exposures is still to be accounted for.. B. The mode of action of the asbestos fiber is primarily mechanical rather than chemical in nature. 3*** The evidence for this ia given in Section LXXXVII. Figures 1, 2, 3, 4 and 7 illustrate the important points. The fibrous fila mented structure of asbestos plays an essential part in the irri tating action, since the solid fibers of glass wool do not produce fibrosis (see Figure 8). C0 Long asbestos fibers are essential in the production of peribronchial fibrosiB short fibers are incapable of producing this reaction, Experimental evidence discloses that fibrosis develops follow ing the injection of asbestos fibers between 20 and 50 microns in length, but not following injection of particles less than 20 microns, (Tables 21 and 23). This shows that the capacity to pro duce fibrosis develops 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* Typical experimental asbestotic fibrosis was produced by the inhalation of an atmospheric suspension containing -n average of 138 million particles per cubic foot of air of which only 0*6 per cent consisted of fibers longer than 10 micron So In the inhalation experiment with 100 per cent ball-milled asbestos dust containing 0*6 per cent of fibers longer then 10 microns (Section 45 to 57 inclusive) a typical fibrosis was ob tained (see Table 12),, The evidence presented shows at least that an atmospherio concentration of asbestos dust containing 0,8 mil lion (0.6^x138,000,000) fibers greater than 10 micron* per cubic foot of air is capable of producing experimental asbestosis. The actual lower limit of concentration of long fibers necessary to produce asbestosis cannot be established on the evidence available. E* The duration of exposure required to develop the pulmonary reaction to in haled asbestos dust is - inversely proportional to the concentration of long fibers in the atmosphere, viz. as the concentration is increased, the reaction develops in ehorter time. Evidence for this statement is presented in the inhalation experiment with Long-fiber Asbestos Dust (Sections 68 to 76 inclusive). Study of the size-frequency of the long-fiber dust disoloses that it contained S.7 per cent of fibers greater than 10 microns in length (Table 16). The lungs of animals exposed to this dust revealed that the pulmonary reaotion developed in approx imately one .half the exposure time required for its development in animals subjected to the ball-milled dust,whibh contained only 0o6 per oent of long fibers. -41 F. Established experimental asbestosis ceusssto progress on discontinuing exposure to the dust. Evidence shows in fact that on discontinuing exposure there was an appreciable clearing of the mature pulmonary lesions due to contraction of the fibretic reaction. In contrast, an immature tissue response consisting primarily of cells with little or no fibrosis continued to progress. It is assumed that following at tainment of fibrctic maturity, the same process of contraction would ensuw (Section 57)c G. The formation of asbestosis bodies represents s. coating of the fibers by blood and tissue elements which results in loss of ability of the fiber to produce fibrosis. Intratracheal injection of asbestesis bodies failed to produce the -typical asbestotic tissue reaction in experimental animals (Section 86). The cessation of progressive reaction to inhaled asbestos dust soon after exposure terminates may be due to the formation of asbestosis bodies. H. Aluminum hydroxide failed to neutralise the fibrosing aotion of the long fiber asbestos (Section 85). I. Inhalation of asbestos dust did not alter significantly the final outcome of experimental tuberculosis in two series of guinea pigs exposed. This interpretation is in distinct contrast to the stimulating effect of inhaled quarts upon a tuberculous process in the lung. It must remain tentative, however, since it is based upon evidence limited to two series of guinea pigs exposed to only one kind of asbestos, namely "King's floats" (Section 19-20, 90). Reference to Table 3 shows that when infeotion was coincident with onset of dust exposure, there was temporary progression of the infectious prooe3s with subsequent healing; when infection was initiated after 25-3/4 months of dust exposure the course of the tuberculosis was not (Continued on Page 42a) -42- appreciably .O.t*;r c>. this letter fording is in marked contrast with our usual experie: ro rnrolming quarts dusts or mixed dusts containing quarts wherein th- adverse influence of quarts upon a tuberculous infection is most strikingly manifested v/here infection is initiated after a period of rust exposure, vis. superimposed upon a background of established silicosis. As indicated above, application of this more sensitive test tc asbestos dust failed to demonstrate that such dust had an adverse influence upon a tuberculous infection. Thus, there is strong evidence in support of the interpretation. However, it is recemended that further investigation be undertaken to establish this point. /O ---- ~/j6G&6a* ^ y TB ~ jftfeok - /%>$ " 4*br fit*1' 'fftf.ru.MSl-ii' - (3k\iiF#z> - ftyliaJl&A V J Table I FACTION TO ROFG-F7BT3 CERYSOTIUi XH L'OKGS OF 3,mST A 01 SP3CIES OF AH3JAU.I Species Lc.m Guinea Pig Rabbit Pfx4unite House unite Hat Dog Kodo of Exposure Inhalation Inhalation and injection n nu ii n u Inhalation Inhalation and injection Injection Fibrosis i U* 2* -iJj, 0 V 0 Aabootoai Q1,.** ' *v " 0 0 Very small atypical asbeatosis bodiesc 9 -43- VsdXo A OF K2FG:S sSTOE Psfcrc- Chrysettle ?ergf>nfei;v> r.`5.gno0tt<i 0? ft -r-s.ti.'i rslc Offers 10GO 10 10 10 :. 100 ^Values gi^sn aro on the jEuscer of psrtlcioo., ex&vrst uhryactilc, :. filer tb'in 10 "loro:.!;:: for :Fry>- 2i'~il fibers the rieei-~;r. dir.r.iiic-r, ttos 100 rf. rrer.r* . *. . z Mature of Experiment i.'UO'O exposure continuous through out life. Dust exposure followed by a prolonged residence in. normal air. Tuberculous infecticn* at start of dust exposure. Controls to infection; no dust exposure. Tuberculous infection* after 25=3/4 months of dust exposure^ then residence in normal air Controls to infeetionj no duct exposure. 72& or Oi aiunio j ,i?:AlTr:Ur! I Duct j X..XV5C o | ..v.oxjjnum | Survival j After Dust { Exposure Results go rigs rabbits 18 w;-> 25 iz- 25 fp piS? 1 rabbit 1 rabbit 40 go pige I 33 months >.j Bioni t,, 0 ` i _.l. r Typical, peribz-onchiolar .f5.brcsie Foreign body fcrouchitiSo Little or no react!on0 j >5 I:;OTi'ui'lft j | 8=3/4 /h ^ 18=2/3 Sz nonthz | i i i I i | 1 k i s !i 35 months JIonprogressiTe fi brosis. `-1 r-.- Honprogrossive fi bre si Do *T<'. ou N J Absorption of for 34=1/3 ) eign body reaction. 0 months Temporary progression of infection followed by hoaling with fibreelSc 23 S pigs 12 go pigs 0 month 25=3/4 i 1 l { i 14 i f l l | Mealing by resolution (one exception).. Ko appreciable increase in susesptibility to tuberculouo in fectionc Healing 'rith fibrosis. 12 go pigs 0 month | 10=1/2 ** I j i ! Healing by resolution* 1 i j * With low-*virulent i:-. strain of tubercle bacillus. After infe-ctioro -45- .'V i.:>-.)v Lb ' <>J C r\Q ;>'5V "10O So 03 0,K 0,09 G..35 30.9 6 0. 10 o,,20 0 98 .: gnltion) 10.,09 loss locoii .! too .".ntor/.ai cc^tcinr. prec-crv.!.r.-'vtco of filvrouc chr/sr~ tile i';"d platy ( oca-fibrcv.;;; sg-rpe.-utlnoo Icoospor>r ~iuorr?.r are dolcuivcr* ohrc33;tet :r.r.~;r. trS/ho a little tronolitc "'c cctinolitoc Ecoa* vaiforrr. nl.'n dis-tributios* Fibrous bvr.t l-.;." vary fro-a. o.tcut 00 jaicro"'r vo 1 zniororje Koro fibore. :ln Si2,5sC flop.to^ Table g SIZS-F-HEQUiT^CY OP AWOSPSERIC DUST ISSIDE CAGES FOR BJHALATION EKPERIMSNT V5ITH SHQKT-FIBER ASBESTOS Size microns <1 1-2 2-3 3-5 5-10 > 10 North -wall per cent 50.1 24.5 12.4 7.4 3.9 le7 100,0 Eaet frail per cent 60.8 1808 10.4 6.1 207 1.2 100.0 Soulii wall per cent 70.3 14c0 5.5 4.2 3.4 2.6 100.0 -47- Table 6 SUMtfAEY ILATIOh Ti jiYIi SSOHT-PIBSa ASBESTOS (OHP^SOTVLH) ; --------------------------------- Maximum f ViTiX'". 'XT,v~; Survival *i ....... ......... . ; \t Mature of Humber of Dust After Dust Experiment Animals Exposure Exposure EcruIts j H6: g P^-gs 33 month 0 months Eat of reaction, about the earn as in oxperlBjBct with Sing's floats asbestcs but extent of involvement very much less Bee Bote BolcWo ** i j ! Dust exposure con tinuous throughout lifo 73 rats 18 cats 32 53-2/3* 0 0 Only slight reac tion; no anbeptosis bodies,.- Keacti on 0nonti ally tnat to c.n. *> ert durst c 7 rabbits 46-3/4* 0 Jio fibrosis seen grossly; microscopic evidence of elvod.sr wall thickening after 40 months exposures ! 13: g Pigs i 20 months 13-2/3 months Dust exposure fol*= lowed by a Pro" longed residence in normal air. 2 cate 1 rabbit 31-1/2 62* 24 6 Progression after removed from dust doubtful: neither cloarly established nor definitoly excluded Same as for continuous exposure Similar to continuous exposure; evidence of slight regression.. .......... . * Exposure after S3 months was to 100 per cent, ball ---ml. lied asbestos ** Reaction probably due to long fibers in the ungroun.d material which was mixed with the ground asbestos dust to produce a satisfactory dust cloud Refer to text* page li>. c Table ? Dust Exposure Continuous During Life ANALYSES OP LUNGS OF GUINEA PIGS AFTER PROLONGED INHALATION OF SHORT-FIBER ASBESTOS (CHRYSOTILE) r } ITime from boginninf months 12 months | months 15 J 20 Exposure to dust Period in normal air 12 15 00 20 0 Amount of ash (per cent of dried lung) per oent 5,02 4058 5016 per cent 5,00 4,76 4,95 per cent 5,86 6,43 Total SiOg (per cent of dried lung) 0,51 0, 46 0,54 0,49 0,43 0o55 0,85 0,90 Total SiOg (per cent of ash) Tissue Reaction# 10,23 10,08 10c 84 + 9,96 9,00 10,60 + L. # ' 14,46 14,07 2+ Dust Exposure Followed by Prolonged Residence in Normal Air Exposure to dust Period in normal air Amount of ash (per oent of dried lung) Total SiO(per cent of dried lung) months *= per cent months - per cent = - <= months - per can1 Total SiO_ (per cent of ash) m Tissue Reaction# -- - *Th symbols merely represent the relative degree of reaction, ranging from!' (questionable) to 4 + (the maximum for this , experiment)r The relationships apply only within this table* and cannot be compared with symbols in other tGbles0 / Tabl_7 LOITGS OF GUINEA PIGS LONGED INHALATION OF ASBESTOS (GHEYSOTILE) it months 12 e to dust in normal air 12 0 of ash ent of dried lung) per cent 5.02 4058 5016 i2 ent of dried lung) 0,51 0 .46 0o54 iOg ont of ash) Reaction* 10o25 10.08 10c 54 + Time from bceinning of dust exposin' months months months months 15 20 24 30 15 20 24 30 0 000 per cent per cent per cent per cent 5,00 40 76 4.95 5.66 6,43 5.42 6.50 5.35 6.55 0.49 0<>43 0.53 0o86 0,90 0,78 0,78 0,96 1.27 9.9S 9,00 10o60 }i * s re i'jA 14,46 14,07 2+ 14,48 14,20 3* 17.89 ,19.46 u+ months 33-3/4 33=3/4 0 per cent 6.06 6,55 0,75 0,96 12,37 15.11 u+ a to dust in normal air of ash ant of dried lung) i? ent of dried lung) i0_ mv of &Bh) Reaotion* months = - per cent at months - per cent = c= ca? months - months 20 4 months 20 10 months 20 13=3/4 per cent < per cent 5.16 5,11 per cent 6oll 3.98 per cent 4.77 5.18 4.77 . 0,48 0.62 0.25 0o36 0,34 0.26 0.22 _ 9.30 10.21 5.31 7,16 8.51 5.00 4.60 - 2* 3+ 2+ rely represent the relative degree of reaction* (quest!enable) to 4 * (th maximum for this fhe relationships apply only within this tablq^d compared with symbols in other tables, 1 Table 8 ANALYSES OP LUNGS OP WHITE RATS THAT HAD INHALED SHORT-FIBER ASBESTOS (CHHYSOTIIE) Duration of Exposure * 0 months 2 months 4 months 6 months 8 months 10 months per cent per cent per cent per cent per cent per cent Amount of ash 3*9 3,5 3.3 3.3 3.9 4.9 (per cent of dried lung) 4,3 3,6 3,6 3.4 3.6 4,6 2.9 2,9 3.4 3.7 3.5 5,3 3,6 3,2 4.4 4,7 3,3 3.7 3,9 3,4 Total Si02 (per cent of dried lung) 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,08 0,13 0,09 0,05 0,08 0,11 0,07 0.07 0.05 0,04 0.08 0.18 0,15 0.17 0.16 0,18 0.16 0.15 0.13 Total SiOg (per cent of ash) 0,0 2,1 2,3 2,1 2.2 3,8 0,0 3,5 3,0 1.5 5.5 3,5 OcO 3,2 2.2 1.1 3.4 2,8 0,0 1,5 4.0 2,8 0,0 4,2 0,0 0,0 * Normal controls (no dust exposure) 50- OOr^FAEISCI: 07 O'O r-5:< I:"us 00:01: Oil: :`0;I ...c.uW'S 00 ::..:J. ..-.o _:j v.-..'<.vl;- (L-ings oa3.y without included lyvv-b. ?iodec). -- r~-- V Q ( i: lc?) of Exposure Z months 4 7'c 6 months 8 months 10 months gar cent per cent per cent per cent Amount of Aah 8fcortfiber asbestos 1r,;* <j jr.' 3h 3.G 4,9 (per cent of Quarts dried lung) Chert Gyp sum^querts '6 i> 4.5 7.1 4.S 7.8 CoS 2oS OV/ OC\ *c-.*' <o/` e,,9 5.4 9.0 14.1 3.6 4.1 Total SiOp (per cent of dried lung) Short-fiber asbestos Quarts Chert Gyp su.Ti~qu.Grta 0.0S Go 51 0o25 0.G3 0o0iJ 0o5S Vr.$ U-7 mr rvJ- <3 ovs 9 ( 0*05 ScS4 5.45 0.11 Go 15 1,44 2.40 Vo vT ^ 0.15 4.40 6,09 0.28 Total SiOg (per cent of ash) Short"fiber asbestos Quarts Chert Gypsuaqtiarta 2.6 11.7 3oS 2,6 2,5 11*4 c3 6 2*0 lo6 41.5 34.4 3.4 3.8 23.4 2Gc>o 3.1 3c2 56.6 43.2 6o? . ... .. Table 10 COMPOSITION OP 100 PER CEtTT BALL-MILXJBD ASBESTOS DUST Chrysotile Serpentine Magnetite Quarts Bruclte Hematite Other Minerals Short-fibar Asbestos Used in Dusting Machines per cent 17 55 10 2 5 0 11 100 Atmospheric Dust Collected with Preoipitator per oent 15 60 10 2 S 0 10 100 -52- Table H SIZE-FREQUENCY OP ATMOSPHERIC DOST COLLECTED INSIDE CAGES FOR INHALATION EXPERIMENT WITH 100 PER CENT BALL-HILLED ASBESTOS DUST Sample taken Sanple taken before wire brushes after wire brushes installed installed Distribution of ( Particles* Clumps ( Particles and clumps and Fibers ( Fibers per oent 98.7 1,3 100.0 per cent 98c 6 1.4 lOdoO Site-frequency of Particles (< 3 microns ( 3-10 (> 10 8S01 11,9 2.0 95c0 5.0 0.0 TOOTS Site-frequency of Fibers ( < 3 microns ( 3-10 ( > 10 22 44 34 TO 0 58 42 100 -53- Table 12 8UMAEZ OF 'jm&LATim EXPERIMENTS TsITE 100 PER CERT BALL-MILLS) ASBESTOS BEST Nature of Experiment Duet exposure continuous throughout life Humber of Animals 84 go pigs 40 rats 24 mice Maximum Dust Exposure *.. --------------Maximum Survival After Dust Exposure Results 24 months 0 months 20 0 12 0 Ho appreciable pulmonary reaction. No suggestion of aabestosis. Ho suggestion of asbeetosis. Dust exposure followed by a prolonged residence in normal air 16 g. pigs 28 jaonths 12 months Fibrosis typical of as bestos!s was present 12 months after exposure ceased in an amount suf ficient to be visible grossly; smaller foci could be seen microsco pically at 2 months and 8 months. -54- ANALYSES OF LUNGS OF GUINEA PIGS EXPOSED TO DUST IN INHALATION EXPERIMENT WITH 100 PER CENT BALL-2SILLED ASBESTOS DUST Exposure to dust Period in normal air Dust Exposure Continuous During Life Amount of ash (per cent of dried lung) > Total SiOg (per cent of dried lung) Total SiO,, (per cent of ash) Tissue Reaction-^ months 1 months 2 T1 1- 2 00 3 0 per cent 4.85 4,30 4.35 per cent 4.66 4.60 5.05 per c< 56( 5:0' 5, ?< 0.21 Qc 0.24 0.23 0.34 0.61 0,7< Oc 31 05< 4.28 7.05 5.60 4.90 7.40 12.01 nr 0o Dust Exposure Followed by Prolonged Residence in Normal Air Exposure to dust Period in normal air Amount of ash (per cent of dried lung) Total SiOg (per cent of dried lung) Total SiOg (per oent of ash) Tissue Reaction1? Time from beginning of exoosure months months 30 36 montl 40 28 28 28 28 12 per cent per oent 7.25 5.25 8.67 5.96 per o< 6.3< 5.1' 1.57 2.19 0.68 0.87 0.8' 0.& 21.63 25,24 A 12.99 14.55 13,0; 12.4: 2+ *The symbols merely represent the relative degree of react!onc ranging from 0 to (questionable) to 2''* (the maximum observed in this experi ment. The relationships apply only within this table and cannot b compared with symbols in other tables, ^ -55' 3ST0S DUST months 1 10 months 2 2 0 Time from beginning of auat exposure months months months months months 3 5 8 12 16 3 5 8 12 16 0 00 00 mon'the 20 20 0 months 24 24 0 per cent 4.85 4:30 4-o 35 per cent 4066 4o60 5,05 0,21 0:30 0,24 0C23 0,34 0o61 per cent 5:60 5.07 6:74 per cent 5.10 5., 08 . `5-0 02 per cent per cent 4.35 5,65 6.24 per cent 5,40 5,01 per cent 5.66 5,20 per cent Go 50 5.56 0,70 , 0:32 0.56 0.38 0oo8 0:39 0,52 1.25 1.45 1.02 loll 1,28 1,28 1,85 3.0 2Z 4:28 7,05 5c, 60 0 4o90 7.40 12o01 12.47 , JUfTTV" 6 58 B^^9.77 00 7.51 7.47 7.72 0 11.86 0 22.16 23.28 0 18,96 21.95 + 22.60 24c 61 29.05 21c 70 Time from beginning of duat exDosure months 30 months 36 months 40 28 28 28 28 12 per cent 7 c 25 8,67 per cent 5.25 5.96 1,57 2,19 0.68 0.87 per oent 6.38 5.17 0.84 0.64 ` 21.63 25:24 <r 120S9 14.55 13.08 12,41 2+ 3 of reaction,, ranging jserred in this orpori table and cannot be j j j Too !.o lU 01? asbsstqs dust (as recoiled) Chemical Si02 FegOg Al'20,3 SSnO CaO MgO HagO KgO C02 ttgO S840?S 5$i>2 0*78 0*08 0*31 40*18 0,,0S 0*06 0o57 14*00 99 76 Petrographic The material contains about 75 per cent of fibrous asbestos. probably chrysotil, with some serpentine and small mounts of calcite, magnetite and chloritic or micaceous minerals. Only a trace of quarts was seen* There were shreds of non^separated fibers 10 to 50 microns long and 5 to 15 microns vd.de -56- '-..I.-* -I f>* COMPARISON OF APPROXIMATE ANALYSES OP LOHG-FIBER AND SHORT-FIBER ASBESTOS DUST Chrysotile Serpentine Magnetite Quartz Bruoite Hematite Other Long--Fiber Original Atmospheric Material Dust per cent par cent 75 SO 16 20 53 tr 3* 24 0 10* 3 100 100 Short-Fiber Original Atmospheric Material Dust** per cent per cent 17 15 55 60 10 10 22 53 00 11 10 100 100 Contamination from other dusts<, Atmospheric san$>le was 100 per cent ball--milled dust. 57- Table 16 COMPARISON OF SI2S-PESQUMCT OF ATMOSPHERIC LONG-FIBBa AND 100 PER CENT BALL-MILLED ASBESTOS DUST COLLECTED INSIDE CAGES Grains Fibers Clumps ( < 3 microns 3-10 ( > 10 ( < 10 ( ( > 1G Long-Fiber per cent 66.4 1.1 0.0 25.8 6.7 1.0 100.0 Ball-misd per cent 80.6 4.8 0.0 0.8 0.6 3.2 100.0 58- SBI8SAKJT OF INHALATION EXPERIFiSHT L0NG-PI3BR ASBESTOS Nature of Experiment limber of Animals Maximum Duet Exposure Ifeximun* Survive 1 After Duet Exposure Results Dust exposure continuous throughout life 117 g* pigs 4 cats 20 rate 36 months 42 25 20 mice 25 0 months 0 0 0 Botinito fibrosis in 26 monthso Slowly developing fibre'is first seen at 24 months. Marked peribronchiolar fi brosis first seal at 24 months. Limited reactions no fibro= siSc * Dust exposure followed by a prolonged residence in normal air 12 g0 pigs 20 month8 14 months 9 g. pigs 27 months 9 2 cats 18 . 24 Clearing of inflammatory reaction and definite contraction of fibrosis. Clearing of inflammatory reaction and slight con traction of fibrosis. Similar to continuous ex posure groupj suggestion of progression in one of the two animals* Table Ifl ANALYSES OF LUNGS OF GUINEA PIGS EXPOSED TO DUST IN INHALATION EXPERIMENT WITH LCKG-FIBER ASBESTOS I Exposure to dust Period in normal air Dust Expos ura Continuous During Life Amount of ash (per cent of dried lung) Total Si02 (per cent of dried lung) Total SiOg (per cent of ash) Tissue Reaction* Exposure to dust Period in normal air Dust Exposure Followed by Prolonged Residence in Normal Mr Amount of ash (per cent of dried lung) Total SiOg (per cent of dried lung) Total SiOg (per cent of ash) months 1 months 2 months months 3| 5 * 1 235 0000 per cent per cent per cent per cent pe: 4.35 4.33 4.37 0o04 0o09 0*04 4.45 4.48 4.35 0C05 0.05 0.06 4.38 ; 4.38 i 4.51 ; i 0.05 | 0.05 ! 0.06 4*77 4,63 5.08 O.OS 0 12 0-09 t i 4 ( ( C 1.10 2.11 0.93 0 months 20 0 1.23 1.18 1.46 + months 20 4 1.20 1.13 1.48 1.76 2o67 lc77 + EaeSffilsfpr TimeflRS months 20 10 months 20 14 i4 3 *1 of me per cent per cent per cenl per cent p* 3c 54 3c 60 So58 2*>92 2.81 4.18 4.30 5,01 5.04 P t i; 0.43 0*49 0. 52 0.21 0.27 0.24 0.22 0.21 0.18 C c 12.22 13.63 14.59 7.23 9o50 5c 75 5.07 409 3.68 i: ii Tissue Reaction* 2+ t 2+ 2+ - *The symbols merely represent the relative degree of reaction The relationships apply only within this table and cannot be ranging frees -60- ;hs j months S 5 0 months 6 8 0 maothe 12 12 0 months 16 16 0 months 20 20 0 months 24 24 0 months 27 27 0 months 30 30 0 months 34 34 0 TOOT 2 tS ent per cent per cent per cent per sent per coat per cent per cent per cent per cent por 8: 8! i ! I 5! >5 ! '6 4*77 4,63 5 a08 0,08 0,12 0,09 4,72 4,92 4,34 0,10 0,09 0,07 4,87 So02 So 16 0,25 0o20 0o31 2,98 2,83 3,16 0,38 Qo35 0o34 3.54 3,60 S.,63 0o43 0,49 0o52 3.42 3,52 Go 36 0o29 .40 3,74 0e39 0o49 3,63 3,03 3,88 0,37 0,34 0o24 5,85 6,70 0o60 0o84 4o 2, 0<3 Oo 12,220 Iff 76 2,21 6,20 12,70 10,18 11,51 10,56 8,60 9o 1,903 2,67 4,09 12,26 13,65 8,29 13,15 11,22 12,47 12o 8 1077 1,58 5,99 10,91 14,59 6,10 u+ h*___ ______ * 2* 2* 3^ 3* I*4 ha months months months months month# 20 27 27 27 27 14 0 3 7 9 ant per cent per cent per cent per cent per oent 8 5,01 3,40 : 3,56 So 19 0 5,04 3,74 2,54 3,18 3,21 2,75 4 0.21 0,39 0,31 0,25 2 0,18 0,49 0.18 0,28 0,29 0,25 5 4,19 11,51 8,59 7,99 8,86 7 3c 58 13,15 8,94 8072 8o51 3 2* 2* 2* . ranging from 0 to (questionable) to 4 . (the zsMociaaaa for this experiment). with symbols in other tables. Table 19 COLTARISON OF REACTION TO CHHYSOTIL ASTD SERPENTINE ISJ JSC TED IN TRATRACRE/iLLY Dosage Each animal was injected intratracheally with 0.5 co of a 5 per cent suspension of the duet- Two weeks later another similar injection was given. Total amount of dust injected 50 mg. Animals Deed Six groups of 9 guinea pigs each (one group for eaoh type of dust). Sacrificing Periods One or two animals in each group at 1, 2, 3, 8-1/2 and 12 months after injection. Preparation of Dust Chrysotila (ball-milled) unhoated: Chrysotile (hall-milled) ignited : Chrysotila (fibrous) unheated Chrysotile (fibrous) ignited : : Serpentine (ball-milled) unheated: Serpentine (ball-milled) ignited : Ball-milled for 1176 hours, dried and roground in agate mortar0 Ball-milled chrysotile heated for 2 hours at about 700 C., then ground in agate mortar 2 or 3 minutes. Ground in agate mortar to pass 200 mesh. 200-mesh material heated for 2 hours at about 700 C. Ko further grinding. Ball-milled for 1488 hours, dried and reground in agate mortar. Ball-milied serpentine heated for 2 hours at about 700 C.. then ground in agate mortar 2 or 3 minutes. Mineral Siae of Dust Particles Chrysotile 3 microns and (ball-milled) less unheated Chrysotile 3 microns and (ball-milled) less ignited Results Grinding destroyed capacity to cause fibrosis. At 1 month considerable inflammatory edema and cellular proliferation and localization of dust particles about bronchiolesj at 2 months, only a very slight proliferative reaction; at 6, 8-1/2 and 12 months, widely-scattered Bmall mononuclear phagocytes. At 12 months, a few microscopic patches of thin alveolar wall thickening with come adenomatoid change in portion of air spaces abutting on thickened bronchi. Do asbestosis bodies seen. Reaction limited to large foreign-body giant cells without production of fibrous tissue-. (continued on page 62 ) Sable IS continued from pago 61. ** Size of ] Hin.fs.ral Dust Particles j Results Chrysotile (fibrous) unheated 20-50 microns approx - distinct fibrosis. Reaction localized to connective tissue about terminal bronchioles; little within those tubes Contraction caused adenomatoid appearance of air spaces given off directly from terminal bronchioles. Reaction area became smaller with progress of time; no now regions invo3.vcd No chronic pleurisy even at points abutting intrapulmon- ary change. At 1 month considerable inflsmmatory edema and foci of cellular prolifera tion; at 2 months well-marked cellular proli feration and fibrosis occurring focally about respiratory bronchioles This reaction de veloped before asbestosis bodies had formed and was as advanced as that produced by 2 years inhalation of asbestos dust. At 6 months., re action less extensive than at 2 months, appar ently due to contraction of fibrous tissue; asbestosis bodies were abundant At 8-l/Z months, reaction still less extensive, confined to the immediate vicinity of the small terminal bronchioles, where the soar tissue was quite dense and was becoming hyaline in character. Sometimes it even obliterated the bronchiole. Asbestosis bodies had boooms scarce At 12 months, the well-developed peri- and intrabrcnohial adenomatoid area3 of fibrosis had produced considerable distortion More peripherally were patches of pneumonitis with eosinophilic infiltrationB some of which was being transferred into fibrous tissue0 These seemed to be precursors of the localized, diffuse patches of thin alveolar wall fibrosis seen elsewhere | 1 Chrysotile (fibrous) ignited 20-50 microns approxe Reaction limited to large foreign-body giant cells without proliferation. Heating the fibera, which made them brittle, .. destroyed their capacity to produce significant reaction Serpentine (ball-milled) onheated 3 microns and loss Dust relatively inactive. At 1 and 2 months, simple phagocytosis without proliferation; at 6 months, no change except possibly lymphoid cell infiltration; at 8-l/2 months, a slight chronic pneumonitis; at 12 months, only a lit tle pneumonitis without suggestion of fibrosis. Serpentine (ball-milied) ignited 3 microns and less Dust relatively inactive. Reaction essentially the same as for unheated serpentine. Rith ig nited serpentine, less tendency for dust to be carried to bronchial nodes0 --_____________________________ ___________________________ r* n Table 20 \*/ w -tU- * V*. v INJECTED JJTTilAfKACHEALLY Dosage See Table 3 Animals Used Prom 6 to 9 guinea pigs for each dust. Sacrificing Periods Usually at 1, 4, 8 and 12 months after injection* Sise of Dust Particles Separated so that most fibers are from 20 to 50 microns long. Mineral ChryBotile (Thetford) Chrysotile (Arizona; low iron oontent; 0eZfc Fe^g) Amosita Results A distinct fibrosis* Additional information given opposite Chrysotile (fibrous) unheated, in Table 19. Reaction virtually identical with that to Thetford Chryso tile* Both fibrosis and asbestosis bodies produoed with an asbestos containing very little iron. Fibrosis ocourred as plugs within terminal bronchioles and as finer deposits at periphery. Fibrosis developed before asbestosis bodies seen and was in cellular state well-formed at one month. With age, fibrous tissue contracted and occupied smaller area but was more dense. Adenomatoid changes similar to those with Thetford Chrysotile. Pleurisy limited to immediate vicinity of early reaction about areas of massive local isation. Asbestosis bodies formed but were few in number. At 1 month after injection, minute fool of mononuclear pro liferation about bronchioles and in areas of ateleotasis; at 1-1/2 months, heavy peribronohiolar patohes of fibrosis often with papillary projections partially closing lumen of branohiole; adenomatoid appearance marked; connective tis sue reaction showed heavy collagen but no hyalinization; at 2 months, minute foci of well-matured fibrosis about bronch ioles; at 6 months, mature asbestosis fibrosis with evidence of contraction; considerable ohrcnic pneumonitis with in filtration of lymphocytes and eosinophiles. At 9 months, small intrabronchiolar fibrous plugs Tilth foci of more deli cate fibrosis at periphery. Sypiool fibrous endo- and peribronchiolitis with formation of atypical asbestosis bodies. Haustration of bodies began before 4th month after injection,, well-developed by 8th month. Bodies persist after 12th month. Reaction at 1 month heavy endo- and peribronchiolitis already showing (Continued on Page 64) Ctbxv '0 coat!nueu fram Peg 6S j Mineral j ............... j Amosite (coat'd) 1* 1 f ! < i ti i" | Crocidolite { (Bolivia) | Crocidolito (So Africa) Anthophyllite Tremolite i i | i Revolts fibrous changes; atelectasis and fibrosis with some ne crosis at sit of massive localisation of dust. At 4 months*, heavy*, widely-scattered endo and peribronchio litis, now fibrous., with marked deformity of bronchioles and with an ademonatoid appearance . At 6 and 10-1/2 months, reaction in lung essentially the same as at 4 months* At 12 months, foci of fibrous endo- and peri bronchiolitis still large with more dense scar tissue and more deformity of bronchial tubes but no extension into, or atelectasis of* peripheral parenchyma. Advanced fibrous endo- and peribronchiolitisc Beaded asbestos!s bodies noted at 8 months. At 1 months oarly fibrous endo- and peribronchiolitis; many giant cells and some lymphocytic reaction,, At 4 months*, small arses of endobronchiolit-is scattered throughout the lung; cellular fibrosiSo At 8 and 12 months,, areas of bronchiolitis smaller because of contraction of dense scar tissue; at 12 months*, marked lymphocytic infiltration and adenoma toid appearanceo | j ] j j j Typical advanced fibrous endo- and peribronchiolitis pro duced by 0o5?& suspension (1 cc total dose); most animals would not tolerate usual 5% suspension,, Fibrosis trell- developed before asbestos!s bodies seen. At 4 months*, welldeveloped fibrous bronchiolitis with lymphocytes and giant cells and adenomatoid change. A,t 8 months, typical bronchiolitis not quite as extensive or as heavily fibrous as with a 5% suspension*, otherwise the sains,, Many deeply stained fibers with a good proportion of haustrated esbostosis bodieso At 12 months*, heavy fibrous bronchiolitis*, more peri- than endobronchiolitis*, with lymphocytes and giant cells; very marked adenomatoid appearance. | j \ j Lymphocytic infiltration and giant cells but no fibrosiB. A very few atypical asbestos!s bodies. At 1 month, many scattered foci of intrabronchiolar dust without, massive localisation; lymphocytic infiltration of walls and a few giant cellso At 8 and 12 months, little evidence of dust; a few bronchioles and bronchi with giant cells in adjacent alveoli and with lymphocytic infiltration of walls. Fibrosis about bronchioles. At 1 month, area of dust local ization with collapse of alveoli and infiltration with acute inflammatory oells, macrophages and giant cells. Within the area ware a few foci of fibrous tissue and numerous areas of hypertrophy of alveolar epithelium. Many bronchioles pocked with fibers. At 4 months,, general appearance of lesion unchanged; pleura slightly thickened over heavy lo calizations of dust. An occasional segmented asbestosis body seen. At 8 months, many foci of fibers in bronchioles and alveolar ducts with cellular reaction as before; also,. j (Continued on Pag 65) SB Table 20 continued from Pago 64 Mineral Result Tremolite (cont!d) ecm fool eliowod distinct collagen deposition* At 12 and 18 months* reaction as before with fibrosis about bron chioles more apparent because of contraction and decrease of inflammation* Giant cells prominent* Pleura markedly involved. Brucite Typical fibrous endo- and peribronobiolitis like reaction to asbestos mineral30 At 1 month., extensive endo- and peri bronchiolitis with giant cells; dees fibrous loops within bronchioles and cellular fibrosis about them; adenomatoid change present-. At 2 months* heavy intrabronchiolar and peribronchiolar fibrosis producing marked deformity with distortion of tubes and obliteration of surrounding air spaces; fibrosis pale without hyalinisation but with few nuclei; no necrosis. Typical asbestosis bodies seen. At 4 and 8 months, little change; fibrous tissue contracting* At 10<=l/2 months* dense fibrous bronchiolitis with asbestosis bodies* Ho pleurisy* Ho extension to stirrounding lung* Glass Wool Ho fibrosis within a year* At 1 month, no reaction inside bronchioles; in peripheral air spaces clumps of giant cells packed with fine spicules of glass with lymphocytic infil tration of adjacent walls; no asbestosis bodies* At 2 months* reaction less intense than at 1 month; f&lr-siaed clumps of elongated giant phagocytes containing spicules and particles of glass; no endobronchitis* At 4 end 8 months* reaction still diminishing. At 12 months* focal areas of pneumonitis with no fibrosis or endobronchitis; moderate number of smooth iron-staining fibers* ________________________ Table 21 rwsTjAPTprsTT np PPODUOED BY losg-fteee and short-pibsr dusts EJECTED 233TEATRACHSALLY uosago Animal a VIgod Sacrificing Periode ) SEE TABLE 19 Mineral. Uhrysotile (Thetford) I Siso of 1 | Dust Particles Results Longfiber 20=50 microns A distinct fibrosis. Refer to Chrysotile (fibrous) unhoated in Table 19. Short=fiber 3 raioran3 end lees Ho fibrosis. Refer to Chrysotile (ball-milied) unheated in Table 190 Amosite Long=flber 20=50 microns Short-fiber 20 microns end less Typical endo- and peribronchiolitis. Refer to Table 20. Reaction limited to phagocytosis with lymphocytio infiltration of adjacent walls. Short fi= bers packed inside swollen phagocytes; longer ones free# some coated to form typical asbestosis bodies. At 1 month after injection, alveo li contained good=sized giant cells; most pha gocytes were within air spaces and had not mi grated to walls. At 4 months,, free extracel lular fibers had worked themselves into inter stitial tissue where there was extensive proli feration of lymphoid cells and monocytes but no fibrosis. At 8 months, foreign body reaction with some pneumonitis, no bronchiolitis. Typical asbestosls bodies present. Crocidolite (Bolivia) Long-fiber 20=50 microns Short-fiber 20 microns and less (Continued on Page 67) Advanced fibrous endo- and peribonchiolitis. Refer to Table 20. No fibrosis. At 1 month, air spaces compressed and largely filled with giant cells packed with dust needles. Walla heavily infiltrated with monocytes and lymphoid cells. At 4 months, a i moderate degree of cellular infiltration of walls; small giant cells packed with dust spicules. Table 22 SUMMARY OF INJECTION EXPERIMENTS BY INTRAVENOUS TECHNIQUE Total amount of dust : 1,0 gram, divided into 20 equal doses (each dose tto.g 5 cc of i Mineral Size of Dust Particles Animals Used ! Maximum Survival After Last Injection ! Chryeotil (Thotford) 3 microns and less (ball-milled IS2 hours) 6 rabbits The rabbit the 6 died after 27 1 Reaction 1 lungs. 3?o for fatali thrombi. Amosite Crooidolit Anthophyllite Tremolite (soda-iron) Tremolite (soda) 3 microns and less (ground in agate mortar) 5 rabbits 3 microns and less (ground in agate mortar) 4 rabbits 3 microns and less (ball-milled 1400 hours) 4 rabbits 3 miorons and less (ball-milled 140 hours) 3 microns and less (ball-milled 48 hours) 4 rabbits 4 rabbits 17 months 12 months 24 months 19 months 24 months Advanced p shortening sacrificed the 6 mont (us focal necr EM'' n isHr ^oao^iri w rap at 3, 4, a sir? no ohange etroyed th rather tha: Reaction e Only eugge the liver, nuclear an animal. T in the llv No fibers Reaction e liforation No evidepc An inert f 12 and 24 I tto,g 5 cc of a i per oont suspension) which were given twicQ a week for 10 weeks. Results The rabbits c&d not tolerate intravenous injections of finely-ground chrysotilo and 5 of the 6 died after 1 to 5 injections of even diluted suspensions; the other animal died after 27 injo&ticne of one-quarter strength, suspension (69 days after first injection). Reaction limited to few large giant phagocytes of inactive type in liver, spleen and lungs. J?o thrombi of dust cells seen in pulmonary capillaries. Ho definite explanation, for fatalities discovered but material my have been retained in heart and caused local thrombi. Advanced pulmonary Infection killed 3 animals at 9e 11 and 17 months after last injection, shortening intended duration of experiment and complicating picture. However, rabbits sacrificed earlier (3 and 6 months) showed only inert phagocytosis with no progression in the 6 months animal. The last two (11 and 17 months) were probably the some although - focal necrosis of the liver and amyloid of the spleen made interpretation difficult. m ------------------------------------------ ---- _ _ r. _ . _ , _. . . . _ _ ------------- . ------------------------- - ------------ -. | . - . _ . _ r._ ,__ -- ;# Reaction was that to an inert substsnoe with no change in 12 months. (Other observations r: at 3. 4, and 6 months). Simple phagocytosis of particles. Ho tendency to agglomerate and t:' no change in adjacent tissues. Grinding the dust to sizes of 3 microns and under de stroyed the fibrous structure of this mineral and injected material resembled plates rather than fibers. Reaction essentially that of an inert mineral. Observations made at 3. 6, 12 and 24 months. Only suggestion of irritating properties manifested in spleen and lymph nodes, but not the liver, ofl$he 24 month rabbit. In this animal there had been proliferation of mono nuclear and gmt cells that was not present in either spleen or lymph nod of 12 month animal. The absence of associated fibroblastic reaction in these organs and of any change in the liver condition justifies the classification of enthophyllite as an inert silicate. Ho fibers wera retained in lung to demonstrate whether asbaatosis bodies would develop. Reaction essentially that of an inert mineral. Last animal sacrificed shewed a little pro liferation and lymphocytic infiltration in liver, not seen earlier (at 3, 6 and 12 months). l?o evidence of any activity in lesions in other organs. iba inert foreign-body reaction with no change in 24 months. Observations made at S, 6, 12 and 24 months*. Table 21 ecnfcinued from Psg 65. Mineral Crooidolite (Bolivia) cont'd | Sigo of i Dust Particles Results At 6 and 8*l/2 months, masses of giant colls. containing mineral particles, in small bronchi but not in respiratory bronchioles.; smaller ones widely scattered in terminal air spaces. Hum orous asbosto3is bodies. Ho reaction in connective tissue. Bb endobronchial proliferation. At 12 months, many scattered small monocytes packed with dust. Ho endobronchitis. Ho peri pheral fibrosis. In lymph node, a slight reticulosis: no fibrosisr j Anthophyllit Long-fiber 20v50 microns Short-fiber S microns sad less Tremolito Long-fiber 20~5Q microns Short-fiber 20 microns and less Lymphocytic infiltration and giant colls but no definite fibroaiSc. Refer to Table 20. Bo fibrosis and practical^ no aebestosis bodies At 1 month, focal collections of dust-fillod monocytes and a few giant colls; at 4 months, some adematoid epithelial reaction; at 8 months, simple pneumonitis with phagocytosis of shortfibers; at 12 months, isolated and sharply lo calised collections of dust cells inside air spaces shout terminal arterioles. Reaction in walls limited to lymphoid cell infiltration. Ho fibrosis. In lymph node, reaction limited to slight prominence of reticulum. Fibrosis about bronchioles. Refer to Table 20. - Simple foreign^body reaction. No acute inflamstation. Ho accumulation of dust in or about terminal bronchioles. Ho endobronchitis. At 1 month, scattered small giant cells and consi derable infiltration of adjacent email giant oells and considerable infiltration of adjacent walls with monocytes and lymphoid cells. At 4 months, little change except more cellular infiltration of connective tissue. At 8 months, lymphoid infiltration and thickening of walla about soma but not all terminal bronchioles. Bruoite Long-fiber 20-5Q microns Short-fiber (made by crushing long fibers with rubber policeman) Typical fibrous endo- and peribronchiolitis-like reaction to asbestos minerals. Refer to Table 20 c Inert type of reaction. At 1 month after injec tion, small monocytes widely scattered through air spaces; focus of atelectasis with lymphoid infiltration of compressed air-space walls. Ho endobronchial reaction as with chrysotile. At 2 months, reaction similar to that at 1 month: typioal aBbeatosis bodies seen. At 12 months. Table 23 ! o`2-C.*nJVi /Ji'/.'JL '1`^S.in tjiii IS BY ISTSAPERITGHEAL TECHNIQUE Dosage Each animal* received a single injection intraperitoneally of 2 co of a 10 per e< Mineral Size of Dust Particles Animals Used Maximum Survival After Injection Chrysotile (Thetford) 3 microns and less (ball "milled 216 hours) 15 g, pigs 36 months Ho fibre nucleate dissolve) fat or a: made at : Chrysotile (Thetford) Amosite Crocidolite Anthophyllite Anthophyllite (originally labeled talc) Tresnolite ( soda-iron) Long fiber (through 100 mesh) 3 microns and less (ground in agate mortar) 3 microns and less; also some long spicules (ground in agate mortar) 3 microns and less (ball=roilled 1400 hours) Mostly 3 microns and less; some fibers 30 microns or more long (ground in agate mortar') 3 microns and less (ball-milled 15 hours) 4 g. pigs 2 months 9 g, pigs ------------------------12 months 7 g, pigs 12 months 5 g,, pigs 25 months 5 g. pigs 12 months 5 g pigs 12 months Definite developed --r tions on! pfilCSSotion Mi-limit p^Jv4SK<j.Hjf:srfI. one a Dust foci amount of that in g: orons, or lightly i j Essential; ; monocytes was also i cytes and Reaction, ocyteSp we experiment connective that it ob Inert type tion about 12 months. Each animal receiving long-fiber chrysotile was injeoted with 2 cc of a 0.5 per cent dust suspension.. (Continued on Pago 70 a 10 per cant dust suspension,. Total amount of dust injected - 0,2 gram. Basults No fibrosis nor asbeatosia bodies, Bust particles Ingested by phagocytes., chiefly multinucleated variety, Six months after injection fibrous elements of du3t appear to have dissolved leaving only the insoluble magnetite,, a contaminant, No reaction in surrounding fat or areolar tissue, No transporting of dust to regional lymph nodes. Observations made at intervals from 1 to 36 months after injection, Definite fibrous reaction produced* delicate and non'hyaline-. Atypical asbestos! s bodies developed but all were unusually small, No evidence of extra-long fibers seen. Observa tions only at 2 months, Infection Interfered with interpretation. Bust reaction appeared to be of inert type [. Cad limited to phagocytosis with a moderate tendency to lymphocytic infiltration. vations at 1, 4, 8 and 12 months. Obser Dust foci consisted only of large mononuclear end giant phagocytes surrounded by a minimum amount of cellular connective tissue. The injeoted dust contained not only fine material that in grinding had been mashed into irregular plates but also many long spicules 10 mi- orons, or more* in length. No asbestosis bodies seen although the longer spicules appear slightly swollen and greenish. Observations at 1* 4, 8 and 12 months. Essentially inert foreign body reaction. In early animals (I* 4 and 8 months) focus of monooytes and small giant oells and a little central neorosis, In the 4 month animal there was also slight peripheral fibrosis. At 12 and 25 months,, non-progressive mass of mono cytes and giant cellsj no fibrosis. Reaction, which consisted of very large giant oells surrounded by a variable number of lymph ocytes, wa$ much heavier to these unintentionally long fibers than to the fine dust in the experiment above. There was more or less proliferation of fibroblasts producing cellular connective tissue visible in areas where the quantity of foreign particles was not so groat that it obscured the reaction. Observations at 1* 4, 8 and 12 months. Inert type *>f response never progressing beyond the stage of very slight lymphocytic reac tion about masses of dust-filled phagocytes. No fibrosis0 Observations at X,, 4, 8 and 12 months. Table 25 on.tinv.rd from Pag 69 mineral Tremoltt (coda) Anthophyllit } (originally ) labeled talc) ) Pyrophyllit ) ) (fibrous) ) ) Pyrophyllite ) (crystalline) ) > Si89 of Dust Particles 3 miorons end lose (b8dl*aslllcd 48 hours) Animals Hoed 5 pigs 15 g* pigs 100 caiorons and less SSaximm Survival ||| gS fA.': After Injection la mBC ; IS months Inert non 4, 8, 12 12 months Distinct queat reg about Ion anthophyl 4 months ; all thro necrosis < I Inert non-progi'ossive foreign-body type of reaction,, Ho fibrosis0 Observations at 1^, 4, 8, 12 ard 16 months* Distinct early fibrosis produced by anthophyllite and fibrous pyrophyllite with subse quent regression; crystalline pyrophyllite inert throughout* At 1 month, giant cells about long thick splinters; at 4 months, definite fibrosis replacing giant cells of anthophyllite and fibrous pyrophyllite reaction; at 8 months, fibrosis which started at 4 months had decreased, especially vdth fibrous pyrophyllite* At 12 monthse reaction to all three dusts consisted of foreign-body giant colls with lyraphooyteo but without necrosis or fibrosis* Ho asbestosis bodies* Table 24 AH&LTSES OP FIBROUS WHERALS 1 i 1"11 Antho- Croci~ Amosite ihaphibolo phyllito Bruoito Ghrysotiles dolito Tresiollte per cant per cant par coat per cant per cent per cent per cent Si02 Fe23 45.23 4o06 55,04 3,05 59o80 0o57 0,90 6c 78 38,56 2c 35 54,99 15,27 56,20 7,21 | 9 FeO A123 G&O 33,83 lo09 2,01 lo69 12c22 -- 0.32 0,44 9,36 0o46 0e04 Oo v>4 OoOS 4,29 1,01 0,80 -0,56 444 MgO N&gO Eg Ignition (<I05C 6028 0,33 0o20 0,66 23o29 043 Oe 12 0,32 33 c 37 0,52 0,15 0,27 53099 0o91 0,15 0,53 38,95 0.29 0,08 4.30 12,25 6.92 0,67 0.02 20,52 6,78 0,99 0,34 Loss (>10SC 5,28 3,60 4,08 26,66 14,99 2,56 2,78 99o97 99,77 99o52 99,79 99,89 99,68 99,82 *71 . * Fig. 1 - King's Floats Inhalation, Guinea Pig, 30 l&mths* The lesion is more pronounced than most of those seen in this experiment. The tiro larger central spaces are bronchioles, and they are surrounded by scar tissue, forming the typical peribronchiolar fibrosis* The so-called "adenomatoid" change is prominent, especially between the two bronchioles. 2001 IK I-? mr~ Fig. 2 - Short-Fiber Asbestos Inhalation, Guinea Pig, 30 Months. A bronchiole is present in the left center of the field. In the central portion, to the right of the bronchiole, is a small zone of cellular fibrosis. The scattered dark structures are phagocytic cells filled with particles of dust. 2001 -72- *4* \ - * *" V' / \ i* Fig. 3 -- 100 Pea* Cent Ball-Milled Asbestos Inhalation, Guinea Pig, 30 Months. This field, shows a rather large bronchiole, virtually without reaction. A few adjacent alveoli contain phagocytes, packed with particles of the dust. 20GX Fig. 1* * Long-Fiber Asbestos Inhalation, Guinea Pig, 30 Months. In the right center is a bronchiole surrounded by cellular fibrosis At the left is an area in which alveolar walls have been involved by extension of the process. 2001 -73-  Fig. $ - Intratracheal Chrysotile, Guinea Pig, 2 Months. There is obvious peribronchiolar fibrosis. 200X Fig. 6 - Intratracheal Crocidolite, Guinea Pig, h Months. There is obvious peribronchiolar fibrosis of about the same degree as in Figures 5 and 7. 200X -74- ( Fig. 7 - Intratracheal Brucite, Guinea Pig, h Months. Note the similarity of the lesion to that in Figures and 6. 200X Fig. 8 - Intratracheal Glass iool# Guinea Fig, U Months. Two bronchioles are present in this field, one seen in cross section and the other in longitudanal section. The asso SciSantecdtupreuslmaornearvyiratrutearllioylenoirsmaplr,easelthnot ubgehnefaibtherthseoJf igttleasrs. w.oaoel be visible if examined with higher magnification. 2001 75-