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American Industrial Hygiene Association Journal Volume 31 MARCH-APRIL, 1970 Number 2 The Pulmonary Response to Fibrous Dusts of Diverse Compositions ................................. 125 Paul Gross, M.D., Robert T. P. deTreville, M.D., Lewis J. Cralley, PhD., William T. Granquist, Ph.D., and Fred L. Pundsack, PhD. Guide for Respirable Mass Sampling .......... ............................................... ...................... ..................... Aerosol Technology Committee, American Industrial Hygiene Association "Respirable" Dust Sampling ....................................................................................................................................... Morton Lippmann, PhD. Dust Control Practices in the Bituminous Coal Mining Industry ....................................... 133 138 160 Kenneth M. Morse 170Evaluation of Tetraalkyl Lead Exposure by Personnel Monitor Surveys ................ Adrian L. Linch, Emil G. Wiest, Ph.D., and Morris D. Carter Vascular Sequelae to Cadmium Fume Exposure .......................................................................................... 180 Mitchell R. Zavon, M.D., and C. Douglas Meadows, M.D. SlIBCELLULAR TRACE MeTAL ALTERATIONS IN RATS EXPOSED TO 50 PPM OF CARBON Monoxide.................................................................................................................................................................................... 183 Stanley C. Mazaleski, Ph.D., Ronald L. Coleman, Ph.D., Robert C. Duncan, Ph.D., and Carl A. Nau, M.D. Why 92 dB A? ................................................................................................................................ 189 V. H. Hill The Effect of Threshold Macular Lesions and Subthreshold Macular Expo sures on Visual Acuity in the Rhesus Monkey ........................................................................ 198 D. N. Farrer, E. S. Graham, W. T. Ham, Jr., W. J. Geeraets, R. C. Williams, H. A. Mueller, S. F. Cleary, and A. M. Clarke Biotransformation of Ethyl Benzene, Styrene, and Alpha-Methylstyrene in Man ................................................................................................................................................................... 206 Zdenek Bardodej and Eva Bardodejova 211Toxic Effects of Allergenic Macromolecular Compounds on Guinea Pigs and Mice Velta Goppers and Harold J. Paulus, Ph.D. Metabolism and Toxicity of Inhaled and Injected niI in the Rat .................................... 213 Randi L. Thomas, M.S., J. K. Scott, M.D., and T. L. Chiffelle, M.D. Activities of the AIHA-ACGIH Respirator Committee During the Past Three Years .......................................................................................................................................................................................... 221 William H. Revoir A Convenient Optimized Method for the Analysis of Selected Solvent Vapors in the Industrial Atmosphere ............................................................................................................................ 225 Lowell D. White, David G. Taylor, Patricia A. Mauer, and Richard E. Kupel Industrial Hygiene Engineering and the Process-Environment System ...................... J. E. Mutchler Fluorine Toxicosis and Industry ............................................................................................................................ 233 240 James L. Shupe, D.V.M. Analytical Guide Series ................................................................................................................................................... 248 Summary Reports .................................................................................................................................................................... 250 Community Air Quality Guide Series .............................................................................................................. 253 1970Abstracts of Technical Papers of the Conference are in the back section of this issue American Industrial Hygiene Association Journal, published bi-monthly by the American Industrial Hygiene Association, Dohrman H. Byers, Editor in chief: Robert G. Keenan, James O. Pierce. II. Allen E. Dooley, Thomas T. Mercer. Bruce A. Hertig, and John W. Clayton, Jr., Associate Editors. Editorial offices, Dept, of Industrial Health, School of Public Health, The University of Michigan. Ann Arbor, Michigan 48104. George D. Clayton, Publications Manager. Business office. 25711 Southfield Road, Southfield, Michigan 48075. The subscription price is $18 per year in U.S.A., and $19 elsewhere. Single copies, when available, may be purchased from the business office at $4 per copy. Copyright 1969, by the American Industrial Hygiene Association. The Association reserves the right to edit all advertisements and to refuse advertising copy when it does not meet the high standards adopted by the Association. Library of Congress Catalogue No. 57-3191. Second class postage paid at St. Paul, Minnesota Printed for the American Industrial Hygiene Association by THE BRUCE PUBLISHING COMPANY, SAINT PAUL, MINNESOTA 55114| J PLAINTIFF'S fXHlBIT A-3 IM-S33 The Pulmonary Response to Fibrous Dusts of Diverse Compositions PAUL GROSS, M.D., ROBERT T. P. deTREVILLE, M.D.,* LEWIS J. CRALLEY, PH.D.,t WILLIAM T. GRANQUIST, PH.D.4 and FRED L. PUNDSACK, PHD. Industrial Hygiene Research Unit, Department of Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 @ Fibrous quartz, chrysotile asbestos, and tremolite talc dust, all of respirable particle size, injected intratracheally, produced polypoid proliferative inflammations with in smaller air-conducting tubes as well as more peripherally. With time, the inflam matory tissue became converted into collagenous scars which often caused permanent deformities of bronchi and bronchioles. After intratracheal injection of a fibrous dust such as synthetic chrysotile, ceramic aluminum silicate, silicon carbide, glass, or brucite, the main pulmonary response was a macrophage reaction with minimal stromal participation. In addition, within 4 days after the injection, there were foci of poly poid proliferative inflammation but limited to the more peripheral respiratory bron chiole and alveolar ducts. Because these polypoid lesions did not collagenize and did not destroy the anatomic integrity of the air spaces, and because the lesions were reversible, the dusts calling forth this type of response must be classed as biologically "inert." Furthermore, the polypoid lesions are believed to be artifactual in the sense that their production is determined by the method of introducing the dust into the lungs, since snch lesions are not seen in animals inhaling high concentrations of the same dusts. Introduction to many in the past, was that the patho WITH THE INCREASING production genicity of asbestos, and, therefore, of all and use of fibrous materials, both the fibrous dusts, was related to the fibrous shape naturally occurring and those industrially proof the particles. According to this theory, duced, the dust created by their fragmenta when the fibers are inhaled, their sharp ends tion is becoming more prevalent. We know traumatize the cells they contact, and fibrosis that one type of naturally occurring fibrous results from the multiple traumata. dust, namely asbestos, is biologically active Although some years ago we had investi and is capable of causing extensive and fatal gated the pulmonary response to one indus scarring of the lungs, and some kinds of this trially produced fibrous dust, namely ceramic mineral have been associated with the pro aluminum silicate fibers, and found it to be duction of cancer. Inasmuch as it is not known exactly what it is about the asbestos dust particle that is responsible for its pathogenicity, the simplest explanation, which seemed to be attractive biologically "inert"1 in recent years the needle like character of fibrous dust has again been implicated as the pathogenic factor. This has occurred in connection with fibrous glass dust, the pathogenic potential of which has been questioned in spite of the fact that nonfibrous Industrial Hygiene Foundation, 5231 Centre Ave., Pitts* burgh, Pennsylvania 15232. tBureau of Occupational Safety and Health, Department of Health, Education, and Welfare, 1014 Broadway, Cin cinnati, Ohio 45202. tMellon Institute, 4400 Fifth Ave.. Pittsburgh. Pennsylvania 15213. . SResearch and Development, Johns-Manville Research and Engineering Center, Manville, New Jersey 06835. glass dust has been found to be biologically "inert."2 This paper is concerned with a study of the pathogenic potential of fibrous dusts not here tofore documented and with the pathologic 125 126 March-April, 1970 Table I Tabular Protocol of Rats Injected Intratrachcally with Dusts - ------- . ------------- ____------- Dust No. Fiber of Diameter (n) Rats Dose Mortality* -- Mirro<|uartzb 1.2 62 25 ms 26% in 6 months 45% in 15 months Natural chrysolilce Talc (tremolitc)'1 0.05-0.2 0.2 -t 0.1-0.2 55 10.5 mg + 14 nig 90% in 18 months 100% in 24 months jl) 25 mg 40% in 6 months 44% in 6 months Synthetic chrysotile' 0.O2 0.1 M 53 14 ing + 45 mg 22% in 12 months 53% in 24 months Oramic aluminum silicate* (ilavsff 2.0 1 nn 10.5 mg 75 10.5 mg 72% in 18 months 85% in 18 monLhs 67% in 12 months 100% in 18 months Bruritr 2-3 15 10.5 ltifi 47% in 12 months 73% in 24 months Silicon carbide1* 22 3.5 mg 18 rats sacrificed at in tervals (no deaths in 6 months) Amorphous magnesium silicate' ID 75 mg 10% in 6 months Range of mortality in mouths of different groups. nickel, 35 rats; medium nickel, 31 rats; and low nickel, 27 rats. 'Ball-milled dust given to 40 rats and hammer-milled dust to 15 rats. Talc with high and low natural nickel content (given to 25 rats each). *One hatch prepared at Mellon Institute, the other at Joluts-Manvillc Research and Engi neering Center. 'Named Fiberfrax, obtained from Carborundum Company. sGroups of 15 rat* given different kinds of fibrous glass: 1, etched glass; 2, uncoated; 1, coated with starch binder; 1, coated with resin. Silicon carbide whiskers from Carborundum Company. Prepared by reacting sodium silicate with MgCb and washing precipitate. effects of these, as well as of previously in vestigated dusts that have not been reported. This study is part of a more basic investiga tion being conducted in cooperation with the U.S. Public Health Service (Grant No. 1 R01 UI-00849-01 ) and industry, the purpose of which is to determine the locus of pathogeniritv of asbestos dust. Method and Materials A tabular summary of the types of dust studied, the number of rats employed, and the dose of dust administered is given in Table I. Included under any one type of dust may be two or more materials from different sources of slightly different compositions but grouped together because, for the purpose of this study, no significant difference was noted. For instance, microquartz prepared at the Johns-Manville Research and Engineering Center consisted of resintcrcd, acid-leached glass fibers with an originally high alkali con tent. The average diameter of the fiber was 1.2 ju. Two batches had been prepared: one with a metallic nickel content of 0.11% and the other, of 3.1%. The natural chrysotile was also of two kinds. One had been ball-milled and then hammermilled. In the latter process, besides being reduced to subnhcronic dimensions, it also acquired an increased nickel content from the nickel-steel alloy of the hammers. The other was comminuted by ball milling only. The talc dust was of the tremolitc variety, and there were two kinds. One had a high natural nickel content and contained fibers with an average diameter of 0.2 /*; the other had a low nickel content and contained fibers with an average diameter of 0.1 Two batches of synthetic chrysotile were employed. One, prepared at the Mellon In stitute. Pittsburgh, Pennsylvania, had a purity of about 90%. The impurities consisted large ly of brucite (Mg(OH)2). The diameter of the tubular crystals averaged 0.02 /i and their length varied from 0.08 to 0.17 g.. The other batch was synthesized at the Johns-Manville Research and Engineering Center, Manville, New Jersey. Its purity was 99.4%. The aver age diameter of the crystals was 0.03 to 0.04 American Industrial Hygiene Association Journal 127 with a solution of sodium silicate. The result ing precipitate was washed with abundant water, and a standard suspension was pre pared. All dusts were suspended in water, the con centrations depending on the amount sus pended in 1 ml of water which could be in jected without killing the rats. Most sus pensions contained 3.5 mg of dust per milli liter. Several suspensions contained 25 mg of dust per milliliter. A total of 424 rats was injected intra tracheally with these dusts. In some groups the total dose was administered by as many as four injections. The injections were made under light ether anesthesia with the aid of an illuminated laryngeal speculum which al lowed the introduction of a spinal-type needle between the vocal chords under direct ob servation. Figurp. 1. Tubular crystals of synthetic chrysotilc prepared at Mellon Institute. Pittsburgh. Pennsyl vania. fi with a length of 1 p or less, although a few fibers were up to 5 p in length. Both lots gave x-ray diffraction patterns typical of c.hrysotile (Figures 1 and 2). Five different varieties of fibrous glass were injected into rats. These averaged about 1 p in diameter. One was etched, two were un coated, one was coated with a textile-type of binder (mostly starch), and the last was coated with a phenol-formaldehyde resin type of binder ( used largely for insulation). The ceramic aluminum silicate fibers (Fiberfrax from Carborundum Company) had an average diameter of 2.0 p. Two batches were used: one had been hammer-milled to in crease its nickel content, and the other was briefly comminuted in a glass tissue grinder. The silicon carbide whiskers, also obtained from the Carborundum Company, had a fiber diameter ranging between 0.5 and 3 p and a length ranging between 100 and 750 p. Amorphous magnesium silicate was used as a nonfibrous control dust. It was prepared by leading a solution of magnesium chloride Figure 2. Crystals of synthetic chrysotile pre pared at the Johns-Manville Research and Engi neering Center, Manviile, New Jersey. Note that these crystals are longer and needle-like; also the magnification is approximately one-tenth of that in Figure 1. 128 In order to study the early pulmonary re sponse to the various types of dust, four rats were killed from each group four days after the first intratracheal dust injection. The rest were allowed to live out their lives. The lungs of all animals were distended with 4% formaldehyde solution under a head of 10 to 12 cm of water. Paraffin sections of the lungs were stained routinely with hema toxylin and eosin. Pertinent fields were pho tographed, and after impregnation with silver the same fields were rephotographed in order to study the relationship between cells and stroma. In order to study the relationship of the dust to the lesions, some sections, cleared unstained, were examined or photographed under dark-field illumination; other sections were subjected to microincineration and were similarly examined or photographed under March-A pul, 1970 Figure 4. Acid-insoluble ash pattern superim posed on the same field as in Figure 3. It shows three dense deposits of chrysotile dust in what prob ably was originally the lumen of the bronchiole. The "snow" in the background is artifactual. Microincincration, 150X. Ficure 3. Contracted, densely collagenous scar in the lung of rat injected intratrar.healiy with 3.5 mg of very finely comminuted chrysotile 23 months previously. The scar probably represents an obliter ated bronchiole as judged by the size of associated blood vessels on its right border. Hematoxylin and eosin, 150X. dark-field illumination. Results Four days after the intratracheal injection, the lungs burdened with asbestos, talc, and microquartz showed a proliferative inflamma tion involving widely scattered smaller bronchi and bronchioles. This was characterized by polypoid processes of avascular fibroblastic tissue rich in argyrophilic fibers which en meshed large amounts of the injected dust. These polypoid structures originated from one or several widely separated ulcers in the mu cosa and distorted the bronchial lumen, con verting it into disconnected circumferential channels that tended to encircle the central stromal plug. These channels quickly became invested with normal-appearing ciliated col umnar epithelium. Within a few months, the American Industrial Hygiene Association Journal 129 latter, considerable shrinkage of the lesions occurred months later when the initially argyrophilic stroma became converted into dense collagen. The main pulmonary response to the dusts of synthetic chrysotile, ceramic aluminum sili cate fibers, fibrous glass, brucite, and silicon carbide whiskers was the mobilization of macrophages which, filled with dust, occupied alveoli evaginating off respiratory bronchioles and alveolar ducts along with much extra cellular dust. The walls of these alveoli were thickened by a combination of surface cell enlargement and arborescence of the septal argyrophilic stroma. Perhaps the most interest ing feature of the pulmonary response was the development of fibroblastic tissue proc- Figure 5. A polypoid mass of inflammatory tissue occupies the lumen of a respiratory bronchiole. Scattered macrophages are seen in many alveoli. Rat injected intratracheallv with 3-5 mg of ceramic aluminum silicate and killed four days later. Hema toxylin and eosin, 150X. argyrophilic fibers were replaced by dense collagenous tissue. The lungs injected with asbestos dust had, in addition to the proliferative inflammation in the smaller bronchi and bronchioles, similar lesions in the respiratory bronchioles, and al veolar ducts. These more peripheral polypoid structures originated from one or more of the evaginating alveoli. Although the former did not become covered with epithelium, as happened in the terminal bronchioles and larger passages, they did become converted into dense collagen and, as a result, under went considerable shrinkage (Figures 3 and 4). The lungs injected with talc showed num erous foci of proliferative inflammation in respiratory bronchioles and alveolar ducts similar to those encountered in lungs injected with asbestos (Figures 3 and 4) ; and, like the Figurk 6. Polypoid masses of inflammatory tissue occupy the lumen of a respiratory bronchiole (mid dle letl) and the lumen of an alveolar duct (lower right). I'lie inflammatory tissue is loose ami cel- lulai. Transparent libers and a giant cell are seen in the polyp in the lower right portion of the field. Rat injected intralracheally with 3.5 mg of glass fibers and killed four days later. Hematoxylin and eosin, 150X. 130 March-April, 1970 delicate. Along with the disappearance of the intraluminal polypoid inflammatory tissue and the reduction in macrophages, the amount of dust in the sections appeared to undergo a parallel reduction. The lungs of rats injected with amorphous magnesium silicate also showed occasional proliferative polypoid fibroblastic inflamma tion in respiratory bronchioles and alveolar ducts; like the lesions associated with synthetic chrysotile injections, they were no longer found some months later. In the main, the pulmonary response was a macrophage re action with minimal stromal reaction. Giant cells were also prominent. Figure 7. A terminal bronchiole containing a polypoid mass of inflammatory tissue enclosing numerous opaque fibers. It is of interest that the inflammatory tissue is already (96 hours) covered by bronchiolar epithelium. Numerous leukocytes are present. Rat injected intratracheally with fl.3 mg of silicon carbide whiskers and killed four days later. Hematoxylin and cosin. flOOX. esses from one or several of the evaginating alveoli of respiratory bronchioles and alveolar ducts. This inflammatory tissue, consisting of argvrophilic stroma, extended in a polypoid manner into the lurnen of the parent struc ture (Figures 5, 6 and 7). Well-developed by the fourth postinjec.tion day, these lesions were less numerous by the fourteenth day and could not be found six months and longer after the injection. Collagenization of these lesions was not observed at any time. Evidence of the dust injections was still present in the form of dust-laden macrophages scattered throughout the section, but these were less numerous, loose, and usually separated from one another, and the walls of the air spaces in which they were found now were thin and Comments According to the commonly accepted defini tion of a fiber--a particle whose length is three times its diameter or longer--synthetic chrysotile certainly is fibrous. In one batch (Mellon Institute), the individual particles when viewed under an electron microscope are tubular crystals, the diameter of which in relation to their length is such that by no stretch of the imagination can they be con sidered needlelike (Figure 1). Nevertheless, this material, injected intratracheally, has pro duced proliferative inflammatory lesions sim ilar to those produced by injected brucite. Furthermore, identical lesions have been seen in an occasional animal injected with amor phous magnesium silicate. It appears, there fore, that the proliferative inflammation noted four days after synthetic chrysotile injections may be ascribed to the high local concentra tions of magnesium silicate associated with the intratracheal injections. In view of the proved biologic inertness of ceramic aluminum silicate,1 silicon carbide,3 '' and glass,- it is difficult to explain the pro duction of the proliferative inflammation ob served following intratracheal injection of the needlelike particles on any other basis than that of mechanical trauma. It would seem that the injection under pressure from the syringe causes the fluid to emerge from the needle with high velocity. Also, the fibrous particles, tending to align themselves parallel to the stream, would thereby tend to impinge point first on the mucosa of branching con- American Industrial Hygiene Association Journal 131 inhaled, even in high concentrations. Ex amples of this contradiction are chrysotile as bestos and fibrous glass. Animals have been exposed to high concentrations of chrysotile asbestos dust in inhalation chambers for more than a year without such polypoid-prolifera tive lesions having been observed.5' We have under study at the present time rats and hamsters that have inhaled coated and un coated fibrous glass in concentrations approx imating 100 mg/m3 for over one year, without detecting any such proliferative lesions7 (Fig ures 8, 9 and 10). We are, therefore, forced to conclude that fibrous dust, when injected intratracheally un der pressure, may produce mechanical trauma resulting in inflammatory foci which, how ever, resolve and disappear with time. These lesions must be considered artifactual. Figure 8. This field is typical of findings in the lungs of rats that had inhaled fibrous glass dust (100 mg/m:t) for 232 days, 6 hours per day. It is noted that there is no fibrosis. The alveolar walls are thin and delicate but small clusters of darkly staining alveolar macrophages are present in alveoli clustered about some alveolar ducts. Hematoxylin and eosin, 150X. ducting tubes, and the possibility of multiple small traumata, amounting to abrasions, be comes a probability. Nevertheless, we are faced with apparent contradictions. When we first investigated the biologic potential of ceramic aluminum silicate fibers,1 we did not observe the prolifer ative inflammatory lesions described above. The reason for this failure lies in the fact that these lesions disappear with time, and w-e had not examined the lungs during the first two weeks after the intratracheal injec tion. Another highly significant contradiction lies in the fact that such polypoid intraluminal proliferative lesions as are found following the intratracheal injection of certain fibrous dusts are not encountered when the same dusts are decolorization and silver impregnation showing min imal stromal reaction which is limited to the re gions where macrophages are clustered and con sists of arborescent reticulin fibers. Gordon and Sweet, 150X. 132 March-April, 1970 and bronchioles caused by permanent scars and short-lived reversible lesions. Viewed from another angle, the prolifera tive lesions produced by all the fibrous dusts investigated except those of quartz, asbestos, and talc have the following characteristics: 1. Significant collagenization in the react ing lung tissue is absent. 2. The anatomic integrity of the air spaces is maintained in spite of the presence of dust therein. 3. The lesions are reversible. These features are those of biologicallly "in ert" dusts" and justify classifying synthetic chrysotile, fibrous glass, brucite, silicon carbide whiskers, and ceramic aluminum silicate in this category in spite of the polypoid prolifer ative inflammation produced when these dusts are injected intratracheally. The proliferative inflammation is considered to be artifactual, dependent on the injection technique. Figure 10. This is the acid-insoluble ash pattern superimposed on the same photograph as in Figure 8. The large amount of fibrous glass dust demon strable and the insignificant tissue reaction to its presence point to the biologic "inertness" of this fibrous dust. The "snow" in the background is artifactual. Microincineration, 150X. The difference between the proliferative lesions produced by the intratracheal injection of fibrous quartz, asbestos, and talc on the one hand, and those produced by similar in jections of synthetic chrysotile, silicon carbide whiskers, fibrous ceramic aluminum silicate, fibrous glass, and brucite on the other hand, is the difference between the deformed bronchi References 1. Gross. 1\. M. L. Wrstrick, H. H. Schrf.nx and J. M. * McNernky: The Effects of a Synthetic Ceramic Fiber Dust upon the Lungs of Rats. A.M.A. Arch. Jnd. Health 13: 161 (1956). 2. Gross, Ij., M. L. Wrstrick, and J. M. McNerney: Glass Dust, A Studv of Its Biologic Effects. A.M.A. Arch. Jnd. Health 21: 10 (I960). 3. Gardner, L. U.: Studies on the Relation of Mineral Dusts to Tuberculosis. III. The Relatively Early Lesions in Experimental Pneumoconiosis Produced by Carborun dum inhalation and Their Influence on Pulmonary Tu berculosis. Am. Rev. Tuberc. 7: 344 (1923). 4. Gr<?ss, P., M. L. VVestrjck and J. M. McNerney: Ex perimental Tuberculopneumoconiosis. A.M.A. Arch. Jnd. Health 19: 320 (1959). 5. W.aonkr, J. C.: Asbcstosis in Experimental Animals. Brit. J. Ind. Med. 20: 1 (1963). 6. Gross, P., R. T. P. dcTreville, . B. Tolxer, M. Kaschak. and M. A. Babyak: Experimental Asbcstosis, The Development of Lung Cancer in Rats with Pulmo nary Deposits of Chrysotile Asbestos Dust. Arch. Environ. Health 15: 343 (1967). 7. Gross, P., R. T. P. deTreville, E. B. Tolkek, M. Kaschak and M. A. Babyak: Fibrous Glass Dust, The Pulmonary Response to Long-Term Inhalation of High Concentrations. To be published. 8. Gross, P., and C. A. Nau: Lignite and the Derived Steam-Activated Carbon. The Pulmonary Response to Their Dusts. Arch. Environ. Health 14 : 450 (1967). Received June 12. 1969