Document LKJJe804Yy3BNEYE4MRYoyJEX

October 50, 1047 OWENS ILLINOIS GLASS COMPANY TOLEDO, OHIO Interim Report on Animal Inhalation Experiments with KAYLO SUMMARY: The first inhalation experiment with this dust was begun in February, 1945. Due to a high inoidenoe of fatal pneumonia, another similar experiment was started in August, 1945* Both of these experiments were designed to test the effect of the dust alone by inhalation in guinea pigs. In August, 1945, also, an experiment was begun to test the effect of inhaled dust on tuberculous infection in guinea pigs* In Ootober, 1945, a group of rtiite rats was placed in the dust room in order to study the effect of Kaylo inhalation in this species. It may be said here that there is no evidence to s uggest that the dust alone when inhaled into the lung produces pulmonary fibrosis in rata in an 18 month period, or in guinea pigs in a SO month period. The results of the experiment in which dust exposure and tuberculosis were combined are inconclusive and further work should be done before a definite statement can be made as to the possible effect of the dust on tuberculous in* feotlon. All these experiments are virtually comploted, although several animals in the original groups are being kept alive for more prolonged exposure. INVESTIGATIONS: Petrographic and x-ray diffraction analyses of the dust supplied and used. Analyses were carried out initially and periodically during the course of the experiment. The oomposite of such analyses reveals tho following: Samples from the supply carton Quarts <L% (approximately) Chrysotlle ana Serpentine 15 - 20%; largely fibrous. The fibers and bundles present are usually several millimeters long and seldom are less than 300 p. Calcite 5% 000244 PLAINT . EXHIBu Page 2 Owens Illinois Qlaaa Comparer October 30, 1947 Interim Report - KAYLO Balance consisted of clay-like massoe which gave a definite x-ray diffrac tion pattern not unlike a hydrous calcium silicate. ^"plea collected from the atmosphere of the chamber. Quarts ljC Chrysotlle and Sarpentine 10 - 15; less fibrous material than in sample above; many fibers are shorter than 20 p. C&loito 10 Balance consisted of clay-like masses which gave a definite x-ray diffrac tion pattern not unlike a hydrous calcium silicate. The analyses reveal several points of interest: The low quarts content (1$) indicates that the amount of crystalline free silica in the dust is prsotioally negligible as a possible oause of silicosis* Although dlatomaoeous earth is one of the materials from which KAYLO is mode, only a trace, probably such less than 1>, is unaltered and appears in the final product in lta original fora* Hoarly all hoe been ensnged by the manufacturing process into another chemical compound, calcium sili cate* Host of the material consisted of claylike masses which gave a definite x-ray pattern similar to that of a hydrous calcium silicate. About 10 to 15# of the atmospheric dust in tho experimental dust room was chrysotlle or serpentine. Both of these minerals produce the seme x-ray" pattern. From petrographic ex amination of tho original material, it appears that chrysotlle, which is a fibrous mineral, is present in much greater amounts than tho nonfibroua aerpontlne. In the atmospheric dust also, the fibers still predominate over t .e nonfibrous serpentina. 000245 Pag* 5* 0--as-Illinois Glass Company October 50, 1947 Tnfc.rtm Report - KAYLO In the original material, the fibers wore p* eaent In bundles often a everal millimeters long; not many were shorter than 500 p. The fibrous portion of tho atmospheric sample, however, consisted mostly of shorter bundles and of fibers ^relatively few in number, which were generally a bout 10 to 40 p in length. B. Techniques of Investigation. Two species of animals were Introduced Into the dust chamber assigned to this in vestigation. They included guinea pigs and white ruts, animals which the Saranac Labora tory nas used routinely in its studies apropos pneumooonloala. Appropriate quantities of the original material supplied by the company were Introduced into the dust generator of the cham ber and pilot runs were made to ascertain the character and concentration of the alr-bome dust produced. Having established the desired concentration, tho animals were then introduced sxid throughout the course of the investigation dust counts were taken periodically. The average concentration of the air-borne material was 122 million part idea per cubic foot of air. This figure varied somewhat from month to month, ranging from a lower limit of 99 million to an upper of 122 million particles. This range Is not unique for this material but has been observed also with most agents which we have employed. It results no doubt from day to day variations in atmospheric conditions which cannot be precisely controlled with our present methods, nevertheless, we havo elected to continue with such methods, believing that to do otherwise would introduce factors unoonsaon to aotual Industrial exposure. The results of the experiments will be presented under the following headings I I. II. III. IV KAYLO inhalation alone in guinea pigs. KAYLO inhalation alone in white rata. KAYLO inhalation and simultaneous tuberculosis Infection in guinea pigs. Conclusions. 000246 Page 4* Owens-Illinois Olaas Company October 30, 1947 Interim Hoport - KAYLO I# K4YL0 Inhalation alone In guinea pigs* The course of thla experiment through the first fourteen months was discussed in a summary written by Doctor Gardner In Hay, 1946* In this report he described the difficulty encountered with acute pneumonia in the guinea pigs and concluded that it was an accidental epldealo* Ills conclusion is supported by the fact that subsequently only four animals have died of pneumonia<, This occurred, however, only after all apparently sick guinea pigs wore disposed of, the apparently well piga isolated, the dust room thoroughly cleaned, aired and disinfected, and all the cages sterilised* A third and new series of guinea plga were intro duced after control of tnc epldealo described* Throughout the course of the study, all animals were exposod continuously to the chamber cloud for eight hours a day, five and ono-half days a week* Thus far, a total of thirty-nine pigs have been killed in order to follow the oourso of the pulmonary reaction resulting from the deposition of inhaled dust in the lung* The period elected for study ranged from two to thirty months* Over this period no definite gross changes occurred in the lungs* The tracheobronchial lymph nodes have enlarged slightly, but re mained soft* Microscopic study of the lungs roveol gradually increasing numbers of dust-containing macrophages* Those inaotive "dust cells" are found characteristically in dumps within groups of adjacent air spaces* The intervening alveolar walls are only slightly thiokoned by accumulated lymphocytes, but there is no undue proliferation of tissue histiocytes and connective tissue* Fibrosis is not present. Occasional aultlnuoloatod giant cells are seen* In addition, there are a very few elongated segmented brown structure which take an iron stain and are clearly asbestosis bodies* Microscopic study of the tracheobronchial lymph nodes reveals dusters of inactive "dust cells" similar to those demonstrated by the lung* There is no apparent over development of connective tissue, and again fi brosis is conspicuous by its absence* Results of the eheaioal analysos carried out on tho lungs of the exposed pigs sacrificed at tho various periods are re corded in tho following table: TABLE I Psge 6 Owene-Ullnola Olaaa Cgnpeny October SO* 1047 Interim Report KAYI>0 CHEMICAL AHALISIS OF GUINEA PIG LONGS Anlaal Binbsr tla* top. Dry Mutter ' Jbdi " ~B1V Hg6 Ash ' --515^--HgC '"'ey 2 6 10 11 16 18 28 54 43 60 45 46 67 68 205 106 49 50 2 mo* 2 mo. 4 mo. 4 no. 6 mo* 6 mo* 8 mo* 8 mo* 10 mo* 10 mo* 12 mo* 12 mo* 18 mo* 18 mo* 21 mo* 21 mo* 24 mo* 24 mo* 4*42 4*94 4*47 4*42 4*13 3*74 4*59 4*54 4*58 4*81 4*80 4*99 4*83 4*90 5*12 5*23 5*02 4*71 0*24 20 22 19 25 20 *31 28 46 *41 53 51 59 86 04 54 66 60 0.07 08 09 08 08 08 06 11 21 14 22 08 26 32 11 08 06 06 0.06 06 09 06 08 10 13 11 14 11 14 16 09 10 19 15 IS 14 557 4 .06 4*87 4*42 5.83 5*56 6.84 5*36 10*12 8.14 10.80 10.25 12*27 17*65 12.64 10.30 12.70 12*52 1.59 1.62 1.99 1.89 2.00 2*19 1.77 2*43 4*30 3.02 4.66 1*60 6*31 6.56 2.03 1*45 1.20 1.72 1.85 1*23 1.99 1*89 2.00 2*68 2.75 2*43 3.04 2*20 2ao 3*17 1*83 2.06 3.68 90 2*54 2*92 TAME I 00024S Page 6. Owens-Illinois Olass Compear October 30, 1947 Interim Report - KAYLO In tIov of the observations recorded above, oertain pertinent ooaaenta appear to be warranted* The microscopic studies of the lungs and traoheobronohial lymph nodes make it obvious that duet was suspended in the atmosphere of the chamber and that portions of that duet were inhaled and Mined aooess into the lungo of the exposed* This is further substantiated by the ohemloal analyses of the lungs whloh show a gradually increasing amount of the various mineral components of the dust used in direct propor tion to the exposure times* In the case of 310q, for example* the pigs sacrificed after ten months exposure revealed a two to three fold increase. The MgO and CaO content also inoraaod but the increments are inconsistent end less manifest. The silica component of the mineral dust is of such low concentration that a significant pulmonary reaction would hardly be expooted in consequence of its presence in the lung. This proved to be the case even in those animals exposed for a period of twenty-four months and revealing an average total of 12.61# 310s of the lung ash* The character of the tissue response at thirty months is comparable and it la anticipated that the 3102 oontant will not be appreciably different. Undoubtedly soow of the dust cells constituting the mild cellular reaction observed stay be in consequence of the serpen tine components of the dust deposited in the lung* This, howovor, cannot be verified since serpentlno is a relatively benign duet and falls to produce reaction other than mere dust laden macro phages. This interpretation la in aocord with Doctor Gardner's observation that the silioate serpentine la a biologically inert substance producing at best a mild resotlon consisting of small widely scattered lobal collections of dust laden phagocytes and an occasional giant cell without other collulor reaction and without fibrosis* The presence of a rare curious body (asbestosis body) indi cates that some of the air-borne fibrous component of the chrysotile gained access to the lung. However, the absenoo of cellular reaction or fibrosis about the terminal bronchioles would Indicate that the Inhaled quantity of such fibers was of extremely low intensity and insufficient to produce permanent damage. Thla view is supported by the petrographic end x-ray diffraction studlee which disclosed that a major amount of the chrysotlle component of the dust consisted of- structures too largo for dispersion into the breathing atmosphere and for easy access into the lung. In oonsequonoo of the experimental studies with guinea pigs to determine the biological activity of EATL3, it may be tentatively co eluded that KAYLO alone falls to produce significant pulmonary damage when inhalod into the lung. 00024& Pago 7. Owens-Illinois Ginas Cocoanr October 30, 1947 Tnt^jplm Report - KAYLO II. KAYLO Inhalation alone In white ratal This spooled was used because too rat la know to develop fibrosis from quarts Inhalation noro rapidly than other anliaals. Forty rata were used In tola experiment and, aa Indies ted by Ocxstor Gardner In his report In Hay, 1946, no difficulty alto early development of pneumonia vaa experienced. In fact, during toe first thirteen months of the experiment no spontaneous deaths occurred. Prom thirteen to eighteen months, about 40 of the animals died with terminal bronchiolitis and lobular pneumonia. This is of no significance, however, because it is veil known that laboratory rats beyond the age of one year arc subject to this typo of lesion. The results of exposure to KAYLO were followed by killing four rats at o&oh of the following periods: six, nine, tsolve, fifteen and eighteen months. The duration of toe experiment was eighteen months and all animals have been killed. The reaction to the dust throughout the experiment has consisted of focal collections in the pulmonary alveoli of macrophages containing particles and splculos of dust. As toe duration of exposure increased, the only observed change was an lnorease in the number of such foci. Similar dust filled macrophages were seen in the traoheobronohlal lymph nodes. In no animal was there any tissue r action of the sort seen in allloosia or asbestosis. Chemical analyses of too lungs of these rats show that the Slop reached the average level of 16.6 of the lung ash within nine months and subsequently diminished. This is difficult to explain, since in the same period too percentage of Slop in guinea pigs' lungs exposed simultaneously (see Table I) was increasing. The table of these results follows: TABLE II. 00025C Pag 8* Qgana-Illlnols Glass Company Octobsr 30 # 1947 Interin Report - KAYLO XAYLO IKHALATIOH SILICA DCTER&IXtATIOKS OP UJBQ TISSUE IB BATS OHLY Animal Ho* Group Aah In Dry SiOp in t),ry TIssuo Tiaou _______ * ... SiOo in Ash * 1012-202 IV 4*8 0*668 14*0 1012-201 IV 4G 0*735 16*1 Duration of Exposure 6 months 6 month 1012-205 1012-206 IV IV 4*6 3*8 0*86 0*528 18*3 13*9 9 month 9 months 1012-209 1012-210 IV IV 4*2 4*4 0*55 0*655 13*2 15*0 12 months 12 months 1012-216 1012-223 IV IV 4*2 4*2 0*443 0*562 10*6 13*3 15 aontha 15 months 1012-226 1012-234 IV IV 3*1 3*9 0*43 0*44 13.7 11*3 18 months 13 months TABLE II 000251 Pago 9* Owens-Illinois Glass Company October 30, 1947 Interin Report - KAYLO In commenting upon Part II of the investigation using white rats as the experimental animals, reference is made to tho 'comments recorded under Part I, which apply* III* KAYLO inhalation and s^ "*], taneous tuberculous infpotion in flulnoa plrca* This experiment was carried out by the use of only one bf tho throe methods which have been found valuable in determining tho effect of dust on the course of tuberculosis* In this case tho animals were infected with tubercle bacilli by inhalation and im mediately placed in the dust room* Thus, we are testing the offeet of tho early accumulation of dust on the early course of tubercu losis* The other two methods available consist in exposing the animals to either the dust or the infection alone for sovoral months, subsequently exposing them to the' othor agent* We may then atudy the effect of accumulated dust on the early oourao of tuberculosis, or the affect of dust on healing tuberculous lesions* In tho experiment which, has been completed twenty-five animals were used* Of these, five died within tan months of tho pneumonia which has been a problem throughout all the experiments with this dust* Four others were killed b eoause they were in poor condition, and it was hoped to out down the incidence of pneumonia by removing them from the room. Theae four also showed some evidence of pneumonia* Of the remaining sixteen animals, eleven were killed in order to follow the course of the infection* One was killed at one month and two were killed at six, nine, twelve, fifteen and eighteen months* Two died at fourteen months, apparently from progressive tuberculosis; those will bo discussed further below* Two mlmals died, one at thirteen and one at seventeen months, of undetermined causes, but not from tuberculosis* Of the animals killed to follow the course of tho infection, the lesions were Isolated, encapsulated and regressing in all exoopt one of the two killed at fifteen months* In this animal, lesions were widespread throughout tne lung, but were associated with the production of fibrosis. Indicating a tendenoy to heal* The interpretation of the results of this experiment is com plicated by the accessory faotor that some of the animals were used for breeding during the experiment* This was done in an attempt to develop a strain of animals resistant to tne infection which was producing the frequent pneumonias* A review of tho records shows that each of tho two animals which died of progressing tuberculous infection had been used as brooders* In addition, tho animal whloh was killed at fifteen months and showed widespread lesions had also been used for breeding* 000252 Pago 10 Owens-Illinois Olaaa Company Ootober 30, 1947 latorlm Report - KAYLO Four other animals used Tor breeding during the same period did not ehow the extensive disease seen in the animals Just described. Thus, it acesa hazardous to a ttempt to attribute the spread- ing of the tuborouloua infection entirely either to the effect of the dust or to that of breeding. , IV, Conclusions * 1. It aeeaa clear from the results preaantod in parts I and II above that KAYLO acta as an inert duat when inhaled by guinea pigs and rats; that is* it is phagocytisod by macro phages , but does not injure theta or produce fibrosis or anything suggestive of either silicosis or aabeatosls. This indicates that the dust will probably be harmloss on inhalation in moderate amounts over prolonged periods by normal human beings. 8. The results observod in the experiment concerning the in* fluenoe of tho dust on tuboroulous infection are equivo cal. Although It is our feeling that this dust exerts little or no adverse influenoo on tuberculosis, the possibility that it playsd a part in spreading the infection cannot be excluded. 5* In view of this possibility, further studies concerning this aspect of the problem should be oarried out. It is imperative that the original experiment with infection and simultaneous dust exposuro be repeated in its entirety. One further study with a small series of guinea pigs is now in progress. It is designed to reveal the influence of the dust on partially healed tuberculosis, however, it is now felt that thle experiment should be oarried out on a larger scale by increasing the number of animals. Should this expanded investigation be pursued, it might bo identified as follows* a. Repeat experiment to determine tho biological activity of KAYLO when inhaled Into the lung harboring a tuberculous infection, which otherwise would regress an.* heal; vis. * Does KAYLO stimulate tho disease? b. Repeat experiment to determine the blologloal activity of KAYLO when inhaled into the lung harboring a regressing or quioscont tuberculous focus; via.: Does KAYLO re activate a focus of infection? c. Introduce a now experiment to determine the biological activity of KAYLO inhaled into the lung upon infeotlon; vis. * Does KAYLO predispose the lung to tuboroulous lnfec- 00025' Page II* Owens-Illinois Glass Company October 30, 1947 Interim Report - KAYLQ 4* la addition, it appears important to explore the biological activity of hydrous oaloium silicate either by inhalation or at least pilot-vise by injection methods should it be impossible to generate a Sufficient cloud of atmoaphorlo dust because of tho character of the material* / 5* These recoomended investigations can be carried ^ out simultaneously and completed in a period of eighteen months to two years* It is anticipated that these studies would remove the doubt raised by the results of the original experiments herein reported and would enable us to conclude finally that KAYLO is inert not only in regard to the production of pulmonary injury, but also in relation to associated tuberculosis* AJV:L8 CC \ ^ 1 Arthur J* Vorvald, IUD* Director of Research The Edward L* Trudeau Foundation 000254 The Saranac Laboratory OF THE EDWARD L. TRUDEAU FOUNDATION Saranac Lake, N.Y. Arthur J. Vorwald. M.D. OmCCTOR October 30, 1947 OVEIS ILLINOIS OLABS COMPAST TOLEDO, OHIO Interim Report on Animal Inhalatl T;Experiments with KAYL SUMMARY: The first inhalation e begun in February, 1 fatal pneumonia, another alml August, 1945* Both of thes the effect of the dust alo August, 1945, also, an exper of inhaled dust on tuberculous October, 1945, a group of ^xite in order to study thr^effect^ of ent with thus dust was to a high incidence of ent was started in s were designed to test tion in guinea pigs. In begun to test the effect tion in guinea pigs. In placed in the dust room elation in this species* It may be sale re is no evidence to suggest that the duet alon< ed into the lung produces pulmonary fibrosis in rats li SO month period* period, or in guinea pigs in a the experiment in which dust exposure and tuberc further wqrk^&hquld bV^done combined are inconclusive and fore a definite statement can be made as to/^htfpbssibleeffect of the dust bn tuberculous in fection* /Ail these experiments are virtually completed, although several animals in thft original groups are being kept alive for sure prol\ mVESTIQATlOH: A. Petrographic and x-ray diffraction analyses of the dust supplied and used* Analyses were carried out initially and periodicallyduring the course of the experiment* The composite of such analyses reveals the following: Samples from the supply carton Quartz <l (approximately) Chrysotlle and Serpentine 15 - 20%; largely fibrous* The fibers and bundles present are usually several millimeters long and seldom are less than 300 p Calcite 5% 00025* Pag 2 Ovens Illlnola Qlaaa Company October 50, 1947 Interim Report - KAYLO Balance eons la ted of clay-like aaaaea vhich gave a definite x-ray diffrac tion pattern not unlike a hydrous calcium silicate* fl*plea collected from the atmoaphere of ike chamber* Quarts 1% Chryaotlle and Serpentine 10 - 16*; leas fibrous material than in sample above; many fibers are shorter than 20 p. Calolte 10* Balance consisted of clay-like masses vhloh gave a definite x-ray diffrac tion pattern not unlike a hydrous calcium silicate. The analyses reveal several points of interest: The low quarts content (1%) indicates that the amount of orystalllne free silica In the dust la practioally negligible as a possible cause of silicosis* Although diatomaceous earth Is one of the materials from which KAYLO is made, only a trace, probably much less than 1*, is unaltered and appears in the final product in its original form* Hearly all has been changed by the manufacturing process into another chemical compound, calcium sili cate* Most of the material consisted of claylike masses which gave a definite x-ray pattern similar to that of a hydrous calcium silicate* About 10 to 15* of the atmospheric dust in the experimental dust room was ohryaotlle or serpentine* Both of these minerals produce the same x-ray pattern* From petrographic ex amination of the original material, it appears that chryaotlle, which is a fibrous mineral, is present in much greater amounts than'the nonfibrous serpentine. In the atmospheric dust also, the fibers still predominate over the nonfibrous serpentine* 00.0:2 5 fc Fag* 3. Owens-Illinois Glass Company October 30, 1947 Interim Report - KAYLO In the original material, the fibers were pr eaent in bundles often a everal mini ma tars long; not many were shorter than 500 p The fibrous portion of the atmoapheria sample, however, consisted mostly of shorter bundles and of fibers, relatively few in number, which were generally a bout 10 to 40 |i in length* B. Techniques of Investigation. Two species of animals were introduced into the dust chamber assigned to this in vestigation. They inoluded guinea pigs and white rata, animals which the Saranac Labora tory has used routinely in its studies apropos pneumoconiosis* Appropriate quantities of' the original material supplied by the company were introduced into the dust generator of the cham ber and pilot runs were made to ascertain the character and concentration of the air-borne dust produced* Having established the desired concentration, the animals were then Introduced and throughout the course of the investigation dust counts were taken periodically* The average concentration of the air-borne material was 122 million particles per cubic foot of air* This figure varied somewhat from month to month, ranging from a lower limit of 99 million to an upper of 122 million particles* This range is not unique for this material but has been observed also with most agents which we have employed* It results no doubt from day to day variations in atmospheric conditions which cannot be precisely controlled with our present methods* levertheless, we have elected to continue with such methods, believing that to do otherwise would introduce factors uncommon to actual Industrial exposure* The results of the experiments will be presented under the following headings: I. II. III* IV. KAYLO inhalation alone in guinea pigs* KAYLO inhalation alone in white rats* KAYLO inhalation and simultaneous tuberculosis infection in guinea pigs. Conclusions. 00^25'. Fage 4. Owena-Illlnola Glass Company Ootobar 30, 1947 Interim Report - KAYLO I. KAYLO inhalation alone in guinea pigs: The course of this experiment through the first fourteen months was dlaousaed In a summary written by Dootor Gardner In toy# 1946* In thle report he described the difficulty encountered with acute pneumonia in the guinea pigs and concluded that It was an accidental epidemic. Bis conclusion is supported by the fact that subsequently only four animals hare died of pneumonia. This occurred, however, only after all apparently sick guinea pigs were disposed of, the apparently well pigs isolated, the dust room thoroughly cleaned, aired and disinfected, and all the cages sterilised. A third and new series of guinea pigs were intro duced after control of the epidemic described. Throughout the course of the study, all animals were exposed continuously to the chamber cloud for eight hours a day, five anc*< one-half days a week. Thus far, a total of thirty-nine pigs have been killed in order to follow the course of the pulmonary reaction resulting from the deposition of Inhaled dust in the lung. The period elected for study ranged from two to thirty months. Over this period no definite gross ohanges occurred in the lungs. The tracheobronchial lymph nodes have enlarged slightly, but re mained soft. Microscopic study of the lungs reveal gradually increasing numbers of dust-containing macrophages These inactive "dust cells" are found charaoteristioally in clumps within groups of adjacent air spaces. The intervening alveolar walls are only slightly thickened by accumulated lymphocytes, but there is no undue proliferation of tissue histiocytes and connective tissue. Fibrosis is not present. Occasional multinucleated giant cells are seen. In addition, there are a very few elongated segmented brown structure which take an iron stain and are clearly aabestosis bodies. Microscopic study of the tracheobronchial lymph nodes reveals clusters of Inactive "dust cells" similar to those demonstrated by the lung. There is no apparent over development of connective tissue, and again fi brosis is conspicuous by its absence. Results of the chemical analyses carried out on the lungs of the exposed pigs sacrificed at the various periods are re corded in the following table: TABUS I. 00025C Page 5. Owena-Illlnola Place Company Ootober 50, 1947 Interim Report - KAYLO CHEMICAL AHALYSIS OP GUINEA PIG LUNGS Animal Number Time Exp, 2 6 10 11 16 18 26 54 45 56 45 46 67 68 105 108 49 50 2 mo 2 mo* 4 mo 4 mo 6 mo. 6 mo. 8 mo* 8 mo * 10 mo * 10 mo* 12 mo 12 mo* 18 mo. 18 mo* 21 mo* 21 mo* 24 mo* 24 mo* Dry Matter -J.Z5E % SIO2 MgO CaO 4*42 4*94 4.47 4.42 4.15 5.74 4*59 4*54 4*58 4.81 4*86 4.99 4*85 4.90 5*12 5*25 5.02 4.71 0.24 .20 0.07 .08 .22 .09 .12 08 .25 08 20 08 .31 08 28 .11 46 *21 41 .14 55 *22 .51 08 59 *26 *86 52 64 .54 . .11 08 66 06 60 08 0.06 .06 .09 .08 .08 .10 15 .11 .14 .11 14 .16 *09 10 .19 .16 15 14 <J102 Ash MftO oao 5.57 4.06 4.87 4'.42 5.83 5.36 6.84 5.56 10.12 8.14 10.80 10.25 12.27 17.65 12.54 10.30 12.70 12.52 1.59 1.62 1.99 1.89 2.00 2.19 1.77 2.43 4^30 3.02 4.65 1.60 5.51 6.56 2.03 1*45 1.20 1.72 1.85 1.23 1.99 1.89 2.00 2.68 2.75 2.45 5*04 2.20 2.80 5.17 1*85 2.Q6 5.68 2.90 2.54 2.92 TABLE I 00025S. Page 6. Owens-Illinois Glass Company October 30, 1947 TntAria Report - KAYLO In view of the observations recorded above, certain pertinent eoamenta appear to be warranted* The ad.oroacoplc atudlea of the lungs and tracheobronchial lymph nodes make It obvious that dust was suspended In the atmosphere of the chamber and that portions of that dust were Inhaled and gained access Into the lungs of the exposed* Th<* Is further substantiated by the chemical analyses of the lungs which show a gradually Increasing amount of the various mineral consonants of the dust used In direct propor tion to the exposure times* In the case of SiOg, for example, the pigs sacrificed after ten months exposure revealed a two to three fold Increase* The MgO and CaO content also increased but the Increments are Inconsistent and less manifest* The silica component of the mineral dust is of such low concentration that a significant pulmonary reaction would hardly be expected In consequence of Its presence in the lung* This proved to be the case even In those animals exposed for a period of twenty-four months and revealing an average total of 12*6l 3102 the lung ash* The character of the tissue response at thirty months is cosq>arable and it is anticipated that the 3102 content will not be appreciably different* Undoubtedly same of the dust cells constituting the mild cellular reaction observed may be In consequence of the serpen tine components of the dust deposited in the lung* This, however, cannot be verified since serpentine is a relatively benign dust and fails to produce reaction other than mere dust laden macro phages* This interpretation la in accord with Doctor aardner*a observation that the silicate serpentine is a biologically inert substance producing at best a mild reaotion consisting of small widely scattered local collections of dust laden phagooytes and an occasional giant cell without other cellular reaction and without fibrosis* The presence of a rare curious body (asbestosis body) indi cates that some of the air-borne fibrous component of the chryaotile gained access to the lung* However, the absence of cellular reaotion or fibrosis about the terminal bronchioles would indicate that the Inhaled quantity of such fibers was of extremely low intensity and insufficient to produce permanent damage* This view is supported by the petrographic and x-ray diffraction studies which disclosed that a major amount of the chrysotile component of the dust consisted of structures too large for dispersion into the breathing atmosphere and for easy access into the lung. In consequence of the experimental studies with guinea pigs to determine the biological activity of KAYLO, it may be tentatively con cluded that KAYLO alone fails to produce significant pulmonary damage when inhaled into the lung. 00026G Page 7. Owens-Illinois Glass Compairy October 50, 1947 Tntarlm Report - KAYLO II. KAYLO inhalation alone In white rats! This species was used because the rat is known to develop fibrosis from quarts inhalation more rapidly than other animals. Forty rats were used in this experiment and, as indicated by Doo tor Gardner in his report in Hay, 1946, no difficulty with early development of pneumonia was experienced* In fact, during the first thirteen months of the experiment no spontaneous deaths occurred. From thirteen to eighteen months, about 40% of the animals died with terminal bronchiolitis and lobular pneumonia. This is of no significance, however, because it is well known that laboratory, rats beyond the age of one year are subject to this type of lesion. The results of exposure to KAYLO were followed by killing four rats a+ each of the following periods! six, nine, twelve, fifteen and eighteen months. The duration of the experiment was eighteen months and all animals have been killed. The reaction to t he dust throughout the experiment has consisted of focal collections in the pulmonary alveoli of macrophages containing particles and spicules of dust. As -the duration of exposure increased, the only observed change was an increase in the number of such fool. Similar dust filled macrophages were seen in the tracheobronchial lymph nodes. In no animal was there any tissue r eaction of the sort seen in silicosis or asbestosls. Chemical analyses of the lungs of these rats show that the SiOg reached the average level of 16.6^ of the lung ash within nine months and subsequently diminished. This Is difficult to explain, since in the same period the percentage of SIO2 in Sinea pigs' lungs exposed simultaneously (sea Table I) was creasing. The table of these results follows! TABLE II. 000261 Pag* 8* Oyene--Illinois Glass Company October 30, 1947 Interim Report - KAYLO KAILO IBHALATIOI SILICA DETERMUIATIOHS OP LUBG TISSUE IS RATS OHLI Animal So* Group Aah. in Dry SlOp in Dry Tissue Tissue ............ . * Si02 in : Ash 1012-202 IV 4*8 0.668 14.0 1012-201 IV 4*6 0.755 16.1 Duration of Exposure 6 months 6 months 1012-205 1012-206 IV IV 4.6 3.8 0.86 0.528 18.3 15.9 9 months 9 months 1012-209 1012-210 1012-215 1012-225 IV IV IV IV 4*2 4.4 4.2 4.2 0.55 0.655 0.445 0.562 15*2 15.0 10.5 13.3 12 months 12 months 15 months 15 months 1018-225 1012-234 IV IV 3.1 5.9 0.43 0.44 13.7 11.3 18 months 18 months TABUS II 00026i Page 9. Owens-Illinois Glass Company October 30, 1947 Interim Report - KAYLO In commenting upon Part II of the investigation using white rata as the experimental animals, reference is made to the comments recorded under Part I, which apply* IH* &AYL0 inhalation and simultaneous tuberculous infection in guinea pigs* . This experiment was carried out by the use of only one of the three methods which have been found valuable in determining the effect of dust on the course of tuberculosis* In this case the animals were infeoted with tubercle bacilli by inhalation and im mediately placed in the dust room* Thus, we are testing the effect of the early accumulation of dust on the early course of tubercu losis* The other two methods available consist in exposing the animals to either the dust or the lnfeotlon alone for several months, subsequently exposing them to the other agent* We may then study the effect of a ccumulated dust on t he early course of tuberculosis, or the effect of dust on healing tuberculous lesions. In the experiment which has been Completed twenty-five animals were used* Of these, five died within ten months of the pneumonia which has been a problem throughout all the experiments with this dust* Pour others were killedbecause they were in poor condition, and it was hoped to out down the incidence of pneumonia by removing them from the room* These four also-showed some evidence of pneumonia. Of the remaining sixteen animals, eleven were killed in order to follow the course of the Infection* One was killed at one month and two were killed at six, nine, twelve, fifteen and eighteen months* Two died at fourteen months, apparently from progressive tuberculonis; these will be discussed further below* Two aiimals died, one at thirteen and one at seventeen months, of undetermined causes, but not from tuberculosis. Of the animals killed to follow the course of the infection, the lesions were isolated, encapsulated and regressing in all except one of the two killed at fifteen months* In this animal, lesions were widespread throughout the lung, but were associated with the production of fibrosis, indicating a tendency to heal. The interpretation of-the results of this experiment is com plicated by the accessory factor that some of the animals were used for breeding during the experiment* This was done in an attempt to develop a strain of animals resistant to the infection which was producing the frequent pneumonias* A review of the records shows that each of the two animals which died of progressing tuberculous infection had been used as breeders. In addition, the animal which was killed at fifteen months and showed widespread lesions had also been used for breeding. 00026S Page 10. Owens-Illinois Glass Company October 30, 1947 Interim Report - KAYLO Four other animals used for breeding during the same period did not show the extensive disease seen in the animals Just described. Thus, it seens hazardous to attempt to attribute the spread ing of the tuberculous infection entirely either to the effect of the dust or to that of breeding, IV, Conclusions: 1, It seams clear from the results presented in parts I and II above that KAYLO acts as an inert dust when inhaled by guinea pigs and rats; that is, it is phagocytised by macro phages, but does not injure them or produce fibrosis or anything suggestive of either silicosis or asbestosis. This indicates that the dust will probably be harmless on inhalation in moderate amounts . over prolonged periods by normal human beings, 2, The results observed in the experiment concerning the in fluence of the dust on tuberculous infection are equivo cal, Although it is our feeling that this dust exerts little or no adverse influence on tuberculosis, the possibility that it played a part in spreading the infection cannot be exoluded, 3, In view of this possibility, further studies concerning this aspect of the problem should be carried out. It is imperative that the original experiment vith infection and simultaneous dust exposure be repeated in its entirety. One further study with a small series of guinea pigs is now in progress. It is designed to reveal the influence of the dust on partially healed tuberculosis. However, it is now felt that this experiment should be carried out on a larger scale by increasing the number of animals. Should this expanded investigation be pursued, it might be identified as follows: a. Repeat, experiment to determine the biological activity of KAYLO when Inhaled into the lung harboring a tuberculous infection, which otherwise would regress and heal; vix,: Does KAYLO stimulate the disease? b. Repeat experiment to determine the biological activity of KAYLO when inhaled into the lung harboring a regressing or quiescent tuberculous focus; viz,: Does KAYLO re activate a focus of infection? c. Introduce a new experiment to determine the biological activity of KAYLO Inhaled into the lung upon infection; viz,: Does KAYLO predispose the lung to tuberculous infec tion? 000264 Page 11. Owens-Illlnola Glass Company October 30, 1947 Interim Report - KAYLO 4. In addition. It eppeera important to explore the blologloel activity of hydrous calcium silicate either by Inhalation or at least pilot-wise by ;lnjeotlon methods should It be impossible to generate a sufficient cloud of atmospheric dust because of the character of the material. 5. These recommended investigations can be carried out simultaneously and consisted in a period of eighteen months to two years. It is anticipated that these studies would remove the doubt raised by the results of the original experiments herein reported and would enable us to conclude finally that KAYLO is inert not only in regard to the production of pulmonary injury, but also in relation to associated tuberculosis. AJV:LB Arthur J. Vorwald, M.D. Director of Research The Edward L. Trudeau Foundation 000261;