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I Final Report {INVESTIGATION CONCERNING THE CAPACITY OF { INHALED KAYLO DUST TO INJURE THE LUNG ! " to the Gwer.s-Illincis Glass Conroany a. Saranaci^GQJNfijPENTIAlf January jO, 1952 ............................. 01 034 00011 CONFIDENTIAL Final Haport INVESTIGATION COECHRNT'-.'G THE CAPACITY OF INHALED ZAILO DUST TO INJURE THE LONG to 0n8-Illinoi3 Glass Company Toledos Ohio by The Saranao Laboratory Saranac Lake, New York January SO, 1952 Submitted by: Arthur J, Vorwald, I4D, Director, The Saranac Laboratory and The Trudeau Foundation In cooperation with* Thomas M, Durkan, M.E. ' Philip C. Pratt, M.D. Edward C.J. Urban, M.S.E. Anthony B. Delahant Donald A. Bailey OIL 034 0002: Final Report ' Owens-Illinois 1-50-52 INVESTIGATION CONCERNING THE CAPACITY OF INHALED KAYLO DUST TO INJURE THE LUNG I. INTRODUCTION In 1943 the Owene-Illinois Glass Company had developed to the pilot plant stage a new Industrial produot, known as Kaylo, which oould be used as a thermal Insulating material and for other applications. According to information supplied by the oompany, this product was prepared by heating under steam pressure a suspension of calcium hydroxide, silica, and asbestos to form a hydrated caloium silicate. Studies at The Saranao Laboratory showed that although the principal component of Kaylo was the hydrated caloium silicate, some unchanged raw materials, including asbestos and freo silica in the form of quarts or diatomaoeous earth, were also present in the final product. Before Kaylo was used commercially on a large scale, it was important to determine whether or not any dust liberated from this product during manufacture, handling or fabrication would be likely to constitute a respiratory hazard to exposed individuals. Therefore, a comprehensive investigation was undertaken in whioh the effect of inhaled Kaylo dust on animals was studied. The investigation was started in February 1945 and was carried on for more than five years. It explored the nature of the pulmonary tissue reaction to inhaled Kaylo dust in normal animals and in animals harboring a tuberculous infeotion. In this final report of the study the principal findings given in previous interim reports will be reviewed briefly and the conclusions derived frem the entire experimental program will be stated. i 01 034 0003 Final Report Owens-Illinois 1-50-62 II. DUSTING MATERIAL USED The material U8sd for generating atmospheric dust in the experimental dust room was finely-divided Kayla obtained from the dust colleotors of the Berlin, New Jersey plant, in which Kaylo is manufactured. The material mas received in separate shipments, as follows: Lot #1 - Received in January 1945. #2 - " " February 1947. ^5 " " January 1949. A. Composition: The results of ohemioal and petrographic analysis of a representative sample of the material as received at The Saranac Laboratory, are given in table 1. It will be noted that the only asbestos mineral revealed by the petrographic examination of this sample Is ohrysotile, Slnoe Kaylo is a commercial product, it is possible that minor alterations in composition may occur from time to time in accordance with ohangos in the raw materials used. For example, the quarts value for lot #1 of Kaylo was less than 1 per cent but the quarts content of lots $2 and #3 was between 2 to 5 per cent. In the final shipment of Kaylo, received in January 1949 and first used in this experiment in September 1949, the asbestos component contained the mineral amosite as well as ohrysotile. The fibers of amosite, in comparison with those of ohrysotile, are less flexible and have a higher iron content. However, since experiments with animals have shown that both amosite end ohrysotile are capable of causing ashestosis, it is unlikely that the substitution of the one mineral for the cthar in ..Kaylo would cause a significant change in tha toxic properties of the final product. 2 01 034 0004 Pinal Report Owens-Illinois 1-50-62 B. X-Ray dlffraotion studies: The X-ray diffraction pattern of samples of two different shipments of Kaylo are shown in fig. 1. The prominent lines present are probably those of a hydrous calcium silicate, which is the principal component of Kaylo. Com parison of patterns a and b discloses that the quarts line is stranger in b than in a, indicating that the Kaylo received in January 1943 has a higher quarts content than has the earlier product, reoeived in January 1945. Faint lines representing oalcite and chrysotile are visible in both patterns. Diffraction lines produced by amosite are also present in pattern b. C. Slse-frequency studies; The size-distribution of Kaylo dust is shorn by the graph in fig. 2. The lines of the graph represent the parent material in the dust machine and two samples of atmos pheric dust collected in the dust room on different days. A magnification . of 1200X was used in making the sise-froqueney counts. It will he noted that about 85 per cent of the pertiolee in the parent material visible at that magnification are smaller than 5 microns in si$ej the corresponding values for the samples of atmospheric dust aro 90 and 95 per cent. This difference In ths two samples of atmospheric dust, which were derived from the same parent material, may have been due to the day-to-day variation in humidity Inside the dust room. III. REVIEW OF PREVIOUS REPORTS During the course of the investigation with Kaylo dust five interim reports were issued. The first report, dated ?*ay 13, 1946, revealed that in guinea pigs 01 034 0005 Final Report Owens-Illinoi > 1-30-52 exposed for 14 months to Xaylo dust the tissue reaction consisted only of miorosoopio focal collectio* of dost cells. The assooisted tuberoulous infeotion had not boon altered enough to be of significance. In the second report, issued an October 90, 1947, it was noted that there was no evidence suggesting that Xaylo dust when inhaled into the lungs produces pulmonary fibrosis in rats exposed for 18 months or in guinea pigs exposed for 30 months. These findings seemed to shoe Xaylo dust to be a biologically inert material. In the experiment dealing with the effeot of the dust on a tuberculous infeotion the results were inconclusive. Indicating that the experiment should be repeated. The third report, dated October SO, 1948, presented the final observe^ ticos on the effect of Inhaled Xaylo dust on normal uninfected guinea pigs. Contrary to previous tentative impressions, based upon an exposure of SO months, which suggested Xaylo dust to be inert, the study of animals exposed for 36 months disclosed that the material is not inert but is capable, on prolonged inhalation, of producing asbestos!8 in guinea pigs. However, neither silicosis nor the diffuse fibrosis which is oaused by inhaled di&tomaoeous earth developed in the exposed animals. The fourth report, dated April 30, 1949, reviewed the results of the first 8 mouths of a new experiment in which the effect of inhaled Xaylo dust on a tuberculous infection in guinea pigs was studied. TSo definitely adverse effect of the dust on the infeotion during this 8-months period was noted. The fifth report, dated January 1, 1950, supplemented the information given in the fourth report by extending the observations to infected animals -4- 01 034 0006 Final `.spore Owens-Illinois I-3C-52 that had been exposed to Kaylo dust for periods up to 14 months. The findings at that tima suggested that Kaylo dust had a slightly adverse influence on the course of a tuberculous infection in guinea pigs but that the effect was by no means as pronounced as that of quarts dust. IV. INHALATION EITPEBIHEHT \TIT3 KAYLO DUST As a continuation of the previous interim reports and as a summary for thi3 final report, a review of the entire program of study i3 reported. In the inhalation experiment (Saranac Laboratory No. 1012, 1100, 1104) groups of guinea pigs, rats, end hamsters were placed in a cubical duet roots, 3 feet in dimension, in which an atmospheric suspension of Kaylo dust was maintained. The room was operated for more than five years, from February 1945 to June 1950. About 450 animal3 ware used in the experiment and seme of them lived in the dust room for as long as 3 years. A. Conditions of exposure The atmospheric suspension of Kaylo dust was created by the action of a paddle that rotated inside a hopper, looated in a corner of the dust room, which contained the finely-divided Kaylo product. The dust cloud generated in this manner floated out into the room where it was maintained for 8 hours on five days of the week and for 4 hour3 on Saturdays. At regular intervals during the experiment a few animals were killed and the tissues examined grossly and microscopically to determine the nature and the extent of the dust reaction. The tissue was also analyzed chemically to estimate the amount of Kaylo dust retained in the lungs of animals exposed for definite periods of time. B ^ v-> 01 034 0007 Pinal Report Owena~Illinois 1-30-52 Dust oounts of atmospheric samples collected in the dust room with the midget impinger mere made regularly. The concentration of Kaylo dust to which the uninfected animals were exposed was generally within the range of 100 to 125 million particles per cubio foot of air and the overall average was 115 million. In the experiment with infected animals the concentration was higher, the average being 205 million. B Exposure of uninfected animals Three species - guinea pig, rat, hamster - were employed in this phase of the investigation. A summary of the findings is given in table 2. (l) guinea pigs. At the beginning of the experiment 100 uninfected guinea pigs were placed in the dust room and 6 months later 25 more were added to replace animals that had died in an epidemic of aoute pneumonia,, Since this pneumonia occurred at that time also in animals `exposed to another dust in an adjaoect room, the unfortunate epidemic was considered accidental. Epidemics of this kind, which occurred occasionally in experiments at that time, have since been controlled. Groups of guinea pigs, usually 2 to 4 in number, were killed at 2, 4, 6, 8, 12, 18, 21, 24, 27, 50, 33, and S6 months after the beginning-of their exposure to Kaylo dust so that the course of the pulmonary reaction could be followed. In guinea pigs exposed for periods up to 30 months there was only a , mild tissue response to the inhaled Kaylo dust and the reaction was limited to the accumulation of clumps of dust-containing maorophagos in groups of adjoining air spaces (fig. 3). The intervening alveolar walls were only slightly thickened by accumulated lymphocytes. Occasional multinucleated 0o 01 034 0008 Final Report Owens-Illinois 1-30-52 giant cell* were visible but there was no excessive proliferation of tissue histiocytes or deposition of connective tissue, A few asbestosla bodies were aeon (fig, 4). Miorosoopic study of the tracheobronchial lymph nodes, whioh became enlarged but remained soft, revealed clusters of inactive dust bells similar to those in the lungs but without aabestoaia bodies. It is pertinent to point out here that animals exposed to asbestos dust for a similar 30-months period exhibit a severe tissue reaction with the production of fibrosis (fig, 6), For comparison, a lung section of a normal guinea pig is shorn (fig* 6), The 9 animals exposed to Kaylo dust for more than 30 months (3 animals for 33 months and 6 animals for 36 months) showed not only the lesions like those of the 30months animals but also exhibited a true fibrosis of a `type % characteristic of the response of guinea pigs to inhaled asbestos fibers, 'This reaction is a rather diffuse type of fibrosis whioh forms principally about the bronchioles, the small ducts from whioh the terminal air spaces, or alveoli, arise, A comparison of fig, 7 and fig, 5 reveals the similarity of the tissue reaction produced in the lungs by Kaylo duet and by asbestos dust. The lesions in the tracheobronchial lymph nodes showed no change between 30 months and 36 months, (2) This species waa employed because typical nodular fibrosis produced by Inhaled quartz dust develops more rapidly in rats than ' in animals of other species. Forty rats were used and some of them were . exposed to Kaylo dust for as long as 18 months. Pneumonia was not a problem, during the first year of the experiment but among the rats exposed for 13 to 18 months 16 animal3 died with terminal bronchiolitis and lobular - 7- 01 034 0009 Final Report Owens-Illinoi s 1-30-52 pneumonia. Since laboratory rats older than one year, even those not exposed to dust, are oomraonly subject to this type of lesion, the high mortality rate la not considered significant. In order to follow the progression of the tissue reaction to the inhaled Kaylo dust rats were killed in groups of 4 after 6, 9, 12, 15, and 18 months of exposure. It was revealed (fig* 8) that throughout the experiment the reaction to the dust consisted of focal oolleotions, in the alveoli, of macrophages whioh contained particles and spicules of dust. The only change observed as the exposure period became longer was an increase in the number of the alveolar foci* Similar dust-filled macrophages were seen in the traoheobronehial lymph nodes. In no animal was there any tissue reaction typioal of silioosis* The peribronchiolar fibrosis of asbestosis was not discovered, though occasion al aabestoais bodies were observed. In view of other experiments with pure `long-fibre asbestos dust, typioal peribronchiolar fibrosis would not be expected in 18 months exposure to Kaylo dust. (3) Hamsters. Twenty hamsters were plaoed in the dust room and after 3, 9, 12, and 18 months of exposure 2 animals were killed at each period for study; at the 6-months period 3 animals that died from other causes ware examined. Histologic examination revealed that the Kaylo dust accumulated very slowly in the lung3. Ho fibrosis was seen except in one hamster, which had been exposed for 18 months. In that animal there was a trace of peribronchiolar fibrosis so slight that it could be definitely recognized only at high magnification. Asbestosis bodies in small numbers and at the usual sites were present. -8 - 01 034 0010 Pinal Report Owens-Illinois 1-30--52 C. Exposure of tuberculous animals There are three types of experiment which are 'valuable for determining the effect of inhaled duat on the development of tuberculosis in experimental animals. In the simultaneous infection experiment normal animals are infected with attenuated tuberole bacilli by the inhalation method and then are immedi ately transferred to the dust room. Thus, the tuberculous disease and the reaction to the inhaled dust will develop simultaneously. In the reactivation experiment the animals, after being infected with the tubercle bacilli, are allowed to live in a clean atmosphere for several months before being ex posed to dust. During this period the tuberculous disease generally regresses and some healing takes place. Following exposure of the animals to some dusts the lesions may continue to regress or, with other dusts, the tuberoulous process may became active end spread. The predisposition experiment is one `in vhioh animals ars exposed to dust for several months, are then infected, and are immediately returned to the dust room, where their exposure to dust is oantinued. In this way the effect of a previous dust exposure on the early course of a tuberoulous infection can be studied. For investigating the effect of inhaled Kaylo dust on the course of tuberoulous infeotion a particular attenuated strain of human-type tubercle bacilli, known as the strain, was employed. This strain has a low degree of virulence for guinoa pigs and, in the normal animal, produces a self-limited disease which will progress for 2 to 3 months and subsequently heal. In an animal which has inhaled quartz dust or certain other toxio dusts, however, the disease usually progresses continuously and ultimately oausos the death of the animal. Sinoe experience has indicated that tuberole *9 01 034 0011 Final Report Owens-Illinois 1-30-62 bacilli of the R^ strain Rave about the same degree of virulence for the guinea pig as tubercle bacilli of the human strain have for man, it is possible to obtain some correlation between the influence of various dusts on the Rj tuberculous infeotion in guinea pigs and the effect of those dusts on a tuberculous disease in industrial workers. For infeoting the animals with tubercle bacilli an inhalation teohnio was used. A glass tube was fitted over the animal*s nose and into this tube a suspension containing a known number of bacilli of the strain was sprayed with a DeVilblss atomiser. As the animal breathed, he inhaled the atmospheric suspension of bacilli into the lungs, where they beoame localised. (1) Simultaneous infection:^guinea pigs infected at the time their exposure to Kaylo dustjwafl started. In the first experiment in which animals were infected with tubercle bacilli at the time that their exposure to Kaylo Quart was started (table 3), 25 guinea pigs were used but an epidemic of pneumonia during the first 10 months reduced to 16 the number of animals available for study. Two of these died at 14 months, apparently from progressive tuberculosis, and 2 others died from undetermined causes, but not from tuberculosis. In the remaining T2 animals the course of the tuberculous disease was followed by killing 1 or 2 animals at 1, 6, 9, 12, 15, 18, and 24 months after infection. In 11 of the 12 animals the lesions were isolated, encapsulated, and regressing; in the remaining animal, whioh was killed at 15 months, the lesions were widespread throughout the irg but fibrosis had been produced, whioh indicated a tendency to heal. In an attempt to develop a strain of guinea pig resistant to the Infeotion that had caused the pneumonia, some of the animals were used for breeding piping - 10 o 01 034 0012 Final Report Owens-Illinois 1-30-52 this experiment. The 2 animals that died from progressive tuberculosis (at 14 months) and the single animal, killed at 15 months, that exhibited wido~ spread lesions were all used as breeders during the experiment but 4 other animals in the experiment, -which wore also employed for breeding, failed to show extensive disease. Owing to the element of uncertainty introduced by this factor of breeding, and also because of the high mortality from pneumonia, it was considered impossible to establish, from this experiment, definite conclusions about the effeot of inhaled Kaylo dust upon tuberculosis^ na therefore the experiment was repeated. The simultaneous infection experiment was conducted a second time (table 3) with SO guinea pigs, instead of 25, end animals were killed in pairs for study at 2, 4, 6, 8, 12, 15, and 24 months after infection. Again the incidence of pneumonia was unusually high, 6 of the animals dying from that cause during the first 8 months of exposture aid 2 more during the following 16 months. Of the remaining 22 guinea pigs 4 animals revealed, at 15, 23, end 24 months after infection, a spreading tuberculous disease, of liriTHmal extent (fig* 9), which was confirmed by microscopic examination. Three other animals si so had a spreading tuberculosis but in them the disease had extended to the spleen and other organs. These animals were considered ex ceptional and in the past have been classified as "sports".' The term "sport" is given to a guinea pig in which the inhalation infection with the attenuated bacilli is not confined principally to the lungs and tracheobronchial lymph nodes but extends also to other organs and produoes in them tuberculous changes of sufficient extent to be recognised in the gross. Since "sports" represent a departure from the normal pattern of tissue reaction to attenuated 11 * 01 034 0013 Final Report Owen 9-Illinois 1-80-62 tubercle bacilli, -whether the infeotian ia combined with dust exposure or not, those animals must bo excluded in assaying the effeot of an inhaled dust upon a tuberculous infection. It is believed that "sports" are animals whose native resistance to the attenuated organism is unusually low. Support for this belief is given by experience with quarts dust, & definitely hazardous material, which reactivates m inhalation infection produced by attenuated bacilli but ordinarily is not associated with tuberculous extension to organs other than the lungs and tracheobronchial lymph nodes. The control non-dusted group (table 8) of guinea pigs for this experiment was infected at the same time as the experimental group of animals subsequent ly dusted. The same culture of organisms was used for all animals. The tuberculous infection followed its usual course in the control group and resolved in all of the animals except one, a "sport", in which the disease spread. Although a progressive tuberculous disease was noted in 4 guinea pigs of the seoond experiment, the degree of extension of the disease la them was slight. The remaining 25 animals, 8 of which died of interourreat pneumonia, showed no spread and the infection followed the natural course of regression and healing as in the non-dusted control animals. It ia believed, therefore, that the inhaled Kaylo dust had no sigiificantly adverse effeot on the tuberculosis. ( 2) Reactivation infection: guinea pigs, after being infectadlived in^clean air for several months before being exposed to Esylo dust. In the reactivation experiment (table 4) 26 guinea pigs wore infected with the attenuated bacilli and allowed to live in a clean atmosphere. Animals - 12 - 01 034 0014 Final Report Ovena-Illinois 1-50-52 of this group were transferred to the dust room at 2 months (10 animals), 4 months (8 animals), and 6 months (8 animals) after infection. In order to follow the course of the tissue reaotion a pair of animals of the first sub-group (2 months in clean air) vas killed after only 2 months of dust exposure and, in addition, animals from all groups mere killed in pairs after 4, 8, and 12 months of exposure to the dust. Examination of the tissue of the dusted animals failed to reveal a significant reactivation of the tuberculous disease by the inhaled Kaylo dust. Only one animal shoved evidence of spreading pulmonary tuberculosis, minimal in extent (fig. 10). That animal vas 1 of 5 that had lived in clean air for 2 months after in fection and then had been exposed to the dust for 12 months. In the control group (table 4) the tuberculous disease behaved in the V. normal manner and healed, vith the exception that one animal (a "sport") developed a spreading disease and died from tuberculosis 6 months after ' infection, (5) Predisposition infsctiont guinea pigs vere infected 5 months after their exposure to Kaylo dust vas started, in experiment of this type Is a severe test because a dust that is only very slightly toxic may produce tissue changes ufoich, though minor ln~ character, may be sufficient to alter profoundly the development of a fresh tuberculous infection. In this experiment (table 5), which vas designed to study the effect of Kaylo dust accumulated in the lung upon a newly-developing tuberculous disease, 30 guinea pigs vere exposed to the dust for 3 months and then were infected vith attenuated tuberols baoilli. The dust exposure wbb immediately resumed and carried on for another 21 months. -- 13 -- 01 034 0015 Final Report Owens-Illinois 1-80-62 During the eourae of this experiment 5 animals were killed at the end of the preliminary S months dusting period. They showed only an occasional clump of dust phagocytes within the alveolar spaces. Of the dusted and infected animals, 7 died from intercurrent pneumonia, which eliminates them for purposes of analysis; 1 animal died at 14 months after iafeotion from spreading tuberculosis; and tho remaining 17 animals were killed in pairs at 2, 4, 6, 8, 10, 12, and 16 months and 8 were killed at 21 months after infection. Of the latter group of 17 animals, 1 showed many spreading tubercles, 10 revealed scattered spreading tubercles and 6 inhibited healed tubercles. These findings indicate that a tuberoulous infection Is guinea pigs that originates several months after & prolonged exposure of the animals to Xaylo dust was initiated may be unfavorably influenced by the inhaled dust. This lnfluenoe, however, is minimal in degree. The tuberculous infection in the control nimals (table 5) in general regressed and healed. Of the control animals there was, as usual, 1 "sport animal", killed 10 months after infection, which revealed a few soattered isolated tubercles that were of the spreading type. D, Analysis of tissue of exposed animals Chemical analysis of the lungs of uninfected guinea pigs that had inhaled Kaylo dust for periods up to 86 months yielded the data reported in table 6. It will be noted that as the period of exposure became longer p the ash values gradually increased, thus shewing that mineral matter was - accumulating in the lungs. There was a pronounced increase in the silica component up to about 30 months and then a alight decrease. The reason for - 14 - 01 034 0016 Final Report Owens-Illinois 1-30-62 this condition, vhioh has been observed in experiments with other dusts, is not entirely clear but it seems to be due in part to dilution of the Inhaled dust with the inorganic components mobilized as a result of the increased tissue reaction whioh developed, as already mentioned, in the guinea pigs between SO and 36 months of exposure to Kaylo dust. Since only 2 animals were analyzed for each exposure period, individual values occasionally were out of line bub the general trend was clearly indicated. V. REVIEW OF EXPERIMENTAL FINDINGS The findings derived from the various inhalation experiments will be reviewed briefly and some points of significance mentioned. A. Findings for normal animals inhaling Kaylo dust The results of the inhalation experiment prove that Kaylo dust, when .inhaled into the lungs of guinea pigs for a prolonged period (30 to 36 months), is capable of producing the peribronchiolar fibrosis oharecteristic of the disease asbestosis. No fibrosis was formed by the inhaled dust in % rats or in hamsters, with the exception that fibrosis was barely recognizable in a single hamster, exposed for 18 months. In animals exposed for 30 months or less the lesions showed no fibrosis but certain aepeota of the lesiona, although mashed by the reaction to the inert components in Kaylo dust, mero compatible with an early stage in the development of asbestosis, a disease in which there is a predilection of the fibers to localize in the terminal .bronchioles. This behavior is unlike that of partioulate material, which is initially deposited chiefly in the alveoli rather than in the lumens of - 16 = 01 034 0017 Final Report Owens-Illinois 1-30-52 the bronchioles. Since the major portion of Kaylo dust is particulate, a great deal of it lodged in the alveoli and the reaction overshadowed the developing peribronchiolar response to the asbestos fibers of Kaylo. It was noted that in the region of the bronchioles of animals which were exposed for less than 30 months there was evidenoe of more dust oell activity than is usual in the reaction to an inert du3t. Little significance was attached to this observation at first, however, because there was no indication of fibrosis. The early reaction to Kaylo dust is somewhat'different from that to asbo8tos dust, since the particulate coznposonts modify the type of lesion. In silicosis modification of the lesions is a well-recognised condition which f j.llews the inhalation of a mixed dust containing quarts and oert&in inert materiel, such as iron oxide. The rate of pneumonia among the animals exposed to Kaylo dust was ' unusually high. Since the pneumonia rate has been elevated also in several inhalation experiments conducted with asbostos dust, it is possible that the asbestos component of Kaylo may have an unfavorable influence on the incidence of pneumonia. Verification of this possibility must await further study. While the experimental investigation proved that Kaylo dust is oapablo of producing asosatoeis, the results showed that the dust -trill not cause silicosis in animals of the throe species tested. In none of the animals, even those exposed for the full 3-year period, was there any focus of the hyaline nodular fibrosis characteristic of silicosis, A study of the exposed animals disclosed also that Kaylo dust did not produce the diffuse fibrosis which follows the inhalation of diatoaaceous earth. In regard to the hydrated -- 16 01 034 0018 Pinal Report OwenIllinois 1*30-52 *t oaloium silioate which is the major constituent of Kaylo it nay be said that although there is no speoifio information about the effeot of that oonpound on pulmonary tissue, there is much clinical and experimental evidence to suggest that it is biologically inert. Furthermore, in the inhalation experiment with Kaylo dust there was no deleterious effeot noted which could be attributed to the oaloium silioate component of the inhaled material. This observation is supported by our studies concerning similar oaloium silicates in oemcnt. B. Findings for tuberculous animals inhaling Kaylo duat The inhalation experiments in which tuberculous guinea pigs were exposed to Kaylo dust indicate that when the animals were infected with attenuated tubercle bacilli at the time exposure to the dust was started, or were infected prior to that time, the inhaled Kaylo dust had little or no unfavorable effect of significance on the tuberculous infection. In most of the exposed animals the tuberculous disease healed in a normal manner and in the few animals that exhibited a spreading type of lesion the aggravation of the disease by the dust was minimal. Bhen the animals inhaled Kaylo dust for several months before being infected, the subsequent accumulation of the dusts in the lungs seemed to have a slightly unfavorable influence on the usual course of the tuberculous disease. V!. SUMMARY A. Kaylo dust, alien inhaled for a prolonged period, is oapablo of produoing - 17 - 01 034 0019 Final Report Owens-Illinois 1-30-52 in tbs lungs of guinea pigs, but not of rats, tho peribronchiolar fibrosis typical of asbestosis, B. The inhalation of Kaylo dust will not cause in guinea pigs, rats, or hamsters either silioosis or the diffuse fibrosis which sometimes follows the Inhalation of diatomaeeous earth, C, The inhalation of Kaylo dust does not significantly influence the usual course of tuberculosis in guinea pigs infected with attenuated tubercle baoilli, provided the infection is given to the animals before they are first exposed to the dust. In guinea pigs that have inhaled Kaylo dust for several months before being infected, a continuation of the exposure appears to have a slightly unfavorable effect on the disease. -- 18 -- 01 034 0020 Final Report Owens-Illinois 1-30-52 Table 1 ANALYSIS OF KAYLO (Analysis of lot #2, received in January 1947) t Chemical analysis Si09 Fe0 AI0O3 Ti02 JSnO CaO HgO Ha20 X20 Loss helow 105C tt above 105C 43.803C 1.69 1.97 0.12 0.09 24,94 7.10 0.17 0.15 4.52 15.19 99.74 Petrographic and X-ray diffraction analysis The material consisted principally of clay-like masses ahich produced a definite X-ray diffraction pattern, presumably that of a hydrous calcium silicate. Quarts particles up to 30 miorons in sise were present, but they were not numerous and by weight probably made up less than 1 per cent of the sample. About 5 per cent of the material was oalcite and approximately 16 to 20 per cent was chryaotile or serpentine, which was largely fibrous. The fibers and bundles present were usually several millimeters in length and were seldom shorter than 0.3 millimeter. 01 034 0021 Final Report Owens-Illinois 1-30-52 Table 2 SUMMARY OF INHALATION EXPERIMENT WITH KAYLO DUST Exposure of Uninfected Animals Nature of Experiment Animals used f 125 6. Pigs umnx oc w^a animals exposed continuously to Kaylo dust until death. 40 rats 20 hamsters duration of exposure to Kaylo dust Results months 36 Peribronchiolar fibrosis, like that of asbestosia, produoed after 36 months of exposure. At 30 months of exposure reaction was limited to accumulation of clumps of dust-containing macrophages, without fibrosis. A fear aebestosis bodies were seen. Tracheobronchial lymph nodes became enlarged but remained soft and without fibrosis. 18 Focal collections of dust- containing macrophages in the alveoli and in the tracheobronchial lymph nodes. Increase in number of focal collections was the only change observed as exposure period became longer. No silicosis or asbestosis. 18 Dust accunulatod very slowly. No fibrosis except in one An-iw^l whioh showed very slight fibrous reaction after 18 monthB of exposure. Asbestosis bodies in small numbers | and at usual site3, 01 034 0022 Final Report Owens-Illinois 1-30-52 Table 3 : 1 SUMMARY OF INHALATION EXPERIMENT WITH KAYLO DUST Exposure of Infected Animals (Simultaneous infection phase) Nature of experiment Animals used Animals infected with tuberole bacilli'* at beginning of exposure to Kaylo dust 25 go pigs Controls to 25 * infection; no E. Pigs ' exposure to dust Animals infected with tubercle bacilli* at beginning of exposure to Xaylo dust. (Repetition of previous experi ment) Controls to infection; no exposure to dust 30 g. pigs . 34 E pigs Mn-Hmira duration of exposure to .Kaylo dust ( months 24 0 24 Results Results inconclusive; adverse influence of Kaylo dust on a tuberculous infection not defi nitely established. 'Progressive disease in 3 animals that had been used as breeders. Infection followed usual course; no spreading disease. Eo pneumonia. Little or no significantly adverse effect of inhaled Kaylo dust on a tuberoulous infeotion was demonstrated. Confirmed spreads, all of slight degree, in 4 of 27 exposed animals; the other 3 exposed animals were "sports". 0 Infection followed psuql course; no spreading disease except in 1 "sport". *The guinea pigs were infected with tubercle bacilli of the low-virulent Rj strain. 01 034 0023 Table 4 Final Report Owens-Illinois 1-30-52 SUMMARY OF INHALATION EXPERIMENT WITH KAYLO OTST Exposure of Infooted Animals (Reactivation phase) Nature of experiment Animals used Animals infected with tubercle bacilli* and then left in olean air for the following periods before being exposed to dust: 2 months 10 g. Pigs Maximum duration of exposure to Xaylo dust months 12 4 months 6 months Controls to infection; no exposure to dust 8 8 34 12 12 0 Results So definite evidence of reactivation of the tuberculous infection except in 1 animal, exposed to Kaylo dust for 12 months, that had a slight spread of the disease. So definite evidence of reactivation. No definite evidence of reactivation. Infection followed it3 usual course and resolved in all animals except one, a "sport", in which the disease spread to other organs and caused the death of the animal 6 months after infection. *The guinea pigs were infected with tubercle bacilli of the low-virulent Rj strain. 01 034 0024 Final Report Owen8-111inois 1-30-52 Table 6 SUMMARY OF INHALATION EXPERIMENT WITH KAYLO DUST Exposure of Animals That Were Infected 3 Months After Their Exposure to Kaylo Dust Was Started Nature of experiment Animals exposed to Kaylo dust for 3 months, then infeoted with tubercle bacilli*, and the exposure to dust continued Animals used 30 g. Pigs MmriimTrn duration of exposure to Kaylo dust Results months 3 (before infect.) plus Spreading disease in 1 animal killed 2 months after infection and in 2 animals dying 14 months after infection. 21 (after infeot.) Controls to infection; no exposure to dust 25 g. Pig 0 Infeotion followed its usual course and resolved, with the exception that 1 animal, killed 10 months after infection, showed scattered isolated spreading tubercles. --------- - --.. - .i .. i The guinea pigs were infected with tubercle bacilli of the low-virulent strain. 01 034 0025 K* o ** ** p Hi P_ fra O 0 O B <* a 1 o CD P H- o o to ti, O *i P Q. O H- O o Hd*- PO aP c+ P a og d- D> O O- to tr d- to e.a W Si o /co - 09 0 H** oo ., . iff ... *d <M9 9 * o0(+ o(L a0" O* oCl Og d- o * tdo- MM t 05 Ol to o mm to co 63 Ol 'act o en 05 -O oo oo CO ~o oo oo 05 ca oo to to O Aa CO iH A to to MM CD <0 to to MM CO to to Aa A CO to -J oo oo A tO oo oo CO to oo M PO to to %9 O*-t> 6H- to tO Hi O to o CO M 03 --Oo to to * AO CO O to tO 09 < Ol Ol 05 05 C3 AC-1 03 A t *o <C M *o o oo co a> o MO H CD o o MO O so o o Hi H M 03 O O to to O Cl >o o ra 09 CO M 09 Ha iMA.CO a dO- 09 Ol A CO V* CO c<ac+a sO`t A MO CO 09 5 O OA CD AM to 90 o ' - > >A?> it~o> ' o - M MM MA o to ' M 03 CD Ol m as a4P MA . a o> O CT1 05 at 1 ai at a. 63 to MO o tO M O CO 05 05 Hi MOO - P0 CO cn O MM -5 CO a CO Cl -a O o to a to oo 03 tO tO C5 o *o MM Oi A OO H> O O to oo 03 Ol M 08 OO as ci A tO CCOO AQD CO as O 03 o p d- tO 0 oea m oa ' ao Ol 03 MM # ro to O tO 03 Oto 03 CD to CO _ * to 5A MM O CO 6* Ol OA Hi H to to * . tOol -o3 OO OM CO M OO oo co a o o H> H Ol to O O Ol A >P tfy o o ' jO j M M os as. A 01 A Ol Cl to Hi 09 to '- , ' & ov MMd to a s Vi M to toft Ol 03 - O ^3 CO A tO CO A Cl M -9 CD OHl cn to Cl 03 oo t MM Ol to oo H H tp oo A A tO ifw Ol -<3 J--* CD 03 to oo - to to * HI CO to to CO Cl Ca 05 to H CO Cl O! 05 Oo oO oi or o Hi W 03 M M MM to O o oi O) Os 63 tO s 09 w 9I to -O Cl -o to to to to a oi M H* m -a - o HI oO M Ol M Hi M Od tO COCDtoO) w H ro H CO CO rt* H* ui ^ ua cn h* to oi Q o o M to -O M o o H> W W 09 o o Cl to to a o o | o o o o Hi 63 HH -0 (C H* O) M M M Cl A A S CO CR Ol Ol CD Ol Ol Ol 09 d 09 Vi A 03 C61J ANALYSES OF LUNGS OF UNINFECTED GUINEA PIGS EXPOSED TO DUST IN INHALATION EXPERIMENT WITH KAYLO DUST *-* w M m*-* *oo BO t*1t 8t taot 01 034 0026 Pinal Report CwanaIilinoi3 1-30-52 Pig. 1 X-RAT DIFFRACTION PATTERN OF RATLO . Kaylo duet received in January 1946, , Kaylo dust received in January 1949, Both patterns contain lines which represent a moderate amount of chrysotile and of caloite end a small amount of quarts. Faint lines representing what 1b probably the hydrous oalcium. silicate component of Kaylo are also present in both patterns, Zh addition, the original film of pattern b contains a very iraak line corresponding to axsosite but the line is too faint to appear in the reproduced pattern. The quartz content indicated by pattern a is less than 1 per cent and by pattern b, about 4 per cent 01 034 0027 i Cwsna-^Illinois 1~-jO-52 Fig. 5 Fig. 3, Luag tissue of a guinea pig exposed for 30 months to Kaylo dust by inhalation and then killed. A cross section of a bronchiole is shown just above end slightly to the left of center of the figure. The other spaces are alveoli. Note the dark masses within same of the smaller spaces, especially to the right of the bronchiole. These masaos are macrophages and multi* nucleated giant oells. Note the thinness of the wall of the bronohiole; it is virtually normal, as- is apparent on comparison with the tissue section of a normal guinea pig (fig. 5). An asbesto3is body is present in this field and is revealed in fig. 4 at higher magnification. (200 X) Fig. 4. High magnification of the tissue section shorn in fig. 3. This higher magnification of part of the field of fig. 3 reveals a -typical form of asbestosis body, the dark curved elongated structure to the right of oenter. (1200 X) Fig. 5. Lung tissue of a guinea pig exposed for 30 months to King's floats asbestos dust by inhalation and then killed. The bronchiole in this photo~ micrograph is just afcove and to the left of center. Note the thickening of its walls. Strands of fibrous tissue are apparent within the wall of this bronohiole as well as in the adjacent alveolar walls. (200 X) 01 034 0029 Final Report Owens-Illinois i-30-52 Fig. 8 Fig. 6- Lung tissue of a normal guinea pig. This field shows two bronchioles, one in cross section at the right of center and the other in longitudinal section The small associated blood Tesesl is visible above the bronchiole in longitudi nal section. Compare the thickness of the wall of these bronchioles with the thickness of wall of the bronchioles in figs. 3, 5, and 7. Note also the empty alveoli and the thin alveolar walls in the normal animal. ( 200 X) Fig. 7. Lung tissue of a guinea pig exposed for 36 months to Kaylo dust by inhalation and then killed. In this photomicrograph a bronchiole is just above and to the right of center. Note the thickness of the tissue between the lumen of the bronchiole and the lumen of the adjacent alveoli in com parison with the thin bronchiolar walls in figs. 3 and 6. Note also that the greatly thickened bronchiolar walls of the animal exposed to asbestos dust for 30 months {fig. 5) are similar to those of this animal, exposed to Kaylo dust for 36 months. Fine strands of fibrous tissue are visible near the bronohiole in this photomicrograph but they are more apparent, because they are less dense, in the area at the right of the field vhere fibrosis has extended to adjacent alveolar walls. (200 X) Fig. 8. Lung tissue of a rat exposed for 18 months to Xaylo duet by inhalation and then killed. The photomicrograph reveals simple accumulation of cells in alveoli. The cytoplasm of the cells is packed with particles of dust. Note that the alveolar septa, even those of spaces containing numerous dust cells, are thin and delicate. (200 X) V.I--- (El 034 0030 final ieoor" \ A.. Fig7~il Fig, 9. Lung tisau of a guinea pig infected with attenuated tubercle bacilli and immediately exposed to ilaylo duct by inhalation for 24 months, and then killed. The figure is a slightly magnified photograph of the routine histologic preparation of the lungs, including both lungs from apex to base. The cluster of dark fool of irregular outline in the lower center (median lobe) and at the right (left caudal lobe) are foci of active spreading tuberculosis. The widely distributed minute dark foci are merely lymphoid cell dusters and are unrelated to the infoction. The extent of the tuberculous activity is seen to be exceeding ly small. (1.5 X} Fig,, 10. Lung tissue of a guinea pig infected with attenuated tubercle bacilli and left in clean air for 2 months, then exposed for 12 montha to Kaylo dust by inhalation, and killed. The dark zone, irregular in outline, in the left upper quadrant (right cephalic lobe) is an area of active spreading tuberculosis. A smaller but similar foous is visible at the lower margin of the Deft cephalic lobe. Again, as in fig. 9, the extent of the tuberculous process is very small. (1.6 X) ^ig. 11. Lung tissue of a guinea pig exposed to Xaylo dust by inhalation for 3 months, then infected vnth attenuated tubercle bacilli and exposed to the dust for an additional 16 months, and killed. The numerous poorly-defined dark zones, irregular in outline, are all sites of active, spreading tuberculous disease. The tuberculous involvement exhibited in this photograph is the most extensive noted in any animal killed during the experiment. The involvenaent is relatively slight in comparison with the progression which occurs in animals exposed to quartz dust under similar conditions; in fact, all of the animals exposed to quartz dust would have died within 12 months after infection because of almost complete loss of functioning pulmonary tissue. (1.6 X) Ofl 034 0,031 01 034 0032j