Document 0q0xvOd3QZ3QOmmZZRnXgQOyJ

.. I. y A - /r 1y ,/'b S * 'if j\sf >`i r\S' v 0' TUttdv*, % rff *' y /V*v rrrr^ ff<,vtJrj rl" mrnr rf E*0*A^hMallj Imlui*"4 I'fct Asbestos, Fibrous Glass find Related Minerals' Hearl F. Stanton and Constance Vrench, Laboratory of PatholQftv, National Cancer .InstituteSetV>*;sdaT Maryland ?Q014 ^Received Kovessber 8* 1971; accepted , %atiooal Institutes of `Health, Public Health Service, U.S. t*partaent of Health, Education, and Welfare. 02 215 0731 WZJ31204 SUMMARY Seventeen potential carcinogens (three types of asbestos In seven forms, six types of fiorous glass, two types of silica and two types of metal particles) were applied directly to the pleura of rats on a fibrous glass vehicle. In a 2 year period saosite, chrysocile, and three different specimens of crocidolite yielded equally high incidences of pleural mesotheliomas. Kand-oilled crocidolite ore not exposed to extraneous oils or metallic milling yielded dose-related tumor responses comparable to those of conventionally milled asbestoses. Neither pulveriied fragments of the steel mill nor nickel metal at doses exceeding potential contaminating levels yielded tumors. The intact fibrous glass vehicle did not induce mesotheliomas nor did the absence of the glass vehicle alter the incidence of crocidolite induced tumors. : ij However, when the fibrous glass vehicle and two other types of fibrous^ glass of relatively large diameter^were reduced to smaller fibrous fragments and applied to the pleura a small number of mesotheliomas were observed. Similarly, one of two samples of. non-fibrous silica^ yielded a single mesothelioma.. Two forms of an especially fine fibrous , <** ' ./ glassyjfurther reduced by silling to approach the sise of asbestos particles, induced moderately high incidences of mesotheliomas,^ Notably, the crocidolite that induced a high incidence of me&atheliom&s induced fever mesotheliomas when reduced to a much finer form by excessive milling. These observations suggcst^thaTthe*carcinogenicity of asbestos arid iglaas is related to structural features rather than acquired or intrinsic physicochemical properties. Cld>J sJ.f y r-j <0 J*' Xi s'-:' > f fi- , ii l * 1 Introduction Mesotheliomas of the pleura and antecedent fibrous plaques, comparable to those resulting from asbestos exposure in nan, can be Induced .readily in the rat and hamster by direct intrapleural application of asbestos (1-8). Such experiments not only attest to the carcinogenicity of asbestos but offer an excellent means of investigating those carcinogenic mechanises inherent to the asbestos fiber. Three years ago we began a aeries o? experiment* based on initial Investigations which suggested chat quantitative data could be obtained if treated Tats were observed for two years (7). Open thoracotomy was employed of apply asbestos on gelatin-saturated glass pledgets directly to the pleura. The glass pledgets acted as a scaffold to uniformly distribute and retain asbestos over a large area of the pleura, and in the absence of asbestos acted as an irritant that could serve as a control on the specificity of the carcinogenic response. In themselves, the glass pledgets do not appear to be csrcinogeneic, nor do they appear to increase the incidence of asbestos-induced mesotheliomas. However, we have continued to use the pledgets as a vehicle for the sake of con venience and accuracy. The results of 28 experiments which demonstrate the remarkable carcinogenicity of asbestos and offer some insight into the nature of this process arc reported here* oM1 02 215 0734 \1 2 Methods ; ^ i Twelve hundred Meaning, pen-bred, pathogen-free, female Osborne- Mendel rats, a strain noted for hardiness and tolerance to surgical procedures, were house five to a metal hanging cage and fed conventional laboratory chow and water ad lib. Test materials were Implanted on the pleura when rats were between 11 and id weeks of age, with the exception of two groups treated between 42 and 47 weeks of age. All groups were observed daily for two years, and survivors were killed during the 25th month after treatment. Metalled gross necropsy, findings were recorded on 97Z of the rats and histologic sections were examined from the implant site and all other grossly abnormal tissues. Selected neoplasms were stained with PAS, toluidine blue, smcican&inc, and Hale's stain for acid mucopolysaccharides, before and after digestion, with diastase and hyaluronidase. Concurrently, 256 untreated rats were killed in groups of 50 at 6-month intervals during the 2-1/2 years. These controls, . like several thousand previously studied in this laboratory had no primary -* iotrathoracie neoplasms other chan a rare thymic lymphoma and a distribution of neoplasms outside the chest essentially like that of the experimental groups. 02 215 0735 In our colonies, the average lifespan of this strain of rats is less than three years; consequently, mortality from disease other than mesothelioma was high during the latter half of the experiment. Periodic epidemics of acute pneumonitis were readily controlled by oral tetracycline therapy; however, the partially compromised function of the treated left lung greatly increased mortality in rats that had contraV. lateral pneumonitis. During the second year of life the mean incidence .* of mammary neoplasms in this strain is approximately 36X. Mammary tumors were surgically removed, care being taken through histologic examination to insure that those of the left thorax did not represent invasion from underlying pleural neoplasms. Neoplasms of the adrenal cortex and genital organs are also common during the second year of life. Because these and other diseases represent a significant mortality factor, notation of significant lesions as well as time of death of each rat is recorded, on the text-figures. 02 215 0736  4 The method of lot reducing the test material to the pleura was described previously (). Thin pledgets of autoclaved coarse fibrous glass measuring approximately 30 X 20 X 2 m were trimmed to weigh 45 agos. The test material at appropriate dosage was suspended in warm 10% gelatin and, during centinous agitation, 1.5 ml aliquots were spread over the surface of each glass pledget. Control nl ** ura treated with either gelatin alone or additional glass. Ail pledgets were allowed to harden at 4C. The treated surface of a pledget was placed directly against the visceral pleura of the left lung through a 3 cm rib-spreading thoracotomy in rats anesthetized with open-drop ether. The incision was closed in layers to Insure against leakage to subcutaneous sites. Post-operative mortality was less than 32. > . >-<B &> ^ -- ^ a *> s%9 A03 Q n SB o o <> ** A i- 1 oN o /- A o M d.'<r A qS O o 3 fn '<<l<5 iojt*a'>-*--* A A AA t1 ii i" O -* si AA a f r-r-ii A 11/. 4' v . A M O -20 >20*40 >40-80 >80-160 >160-320 5 .7 1i ..4l 0 ,3 0 ,6 IS 9 .6 5 .6 4 .2 0 .6 n13 170 4 ,4 34 77 4 .0 U 27 1.8 1.3 7.4 3.9 7.4 24? 13? 26 60 3 .5 163 10 25 0 .4 3? 3.6 6,3 0.3 14 6-7 2,5 0 .2 4 ,6 1.0 3.8 0.3 5 .2 2.2 0 .3 5 . Materials Sevanteen materials differing in composition or structIon were applied to the pleura. Physical and chemical data on the asbestoses have been reported'!* detail <-!) ; 4ata on other materials vara often sparse. In each glass and asbestos sample, particles varied from euhodcfoscoplc to macroscopic dimension in both length and diameter. Assessing this range quantitatively is a problem not yet solved, but to obtain a simple estimate of size distribution,' aliquots of the materials that had been applied to the pledgets were suspended In water (table 1). Similar preparations impressed on formvar-coated copper grids *Ve are indebted to Johns-Kanville Research and Engineering Ctr., Manville, N.J.; Owens-Corning Fiberglas Corp., Toledo, Ohio; Corning Class Works, Corning, H.Y.; Pneumoconiosis Research Unit, Llandough Hospital, Cardiff, Wales; National Bureau of Standards, U.S. Dept, of Commerce, Gaithersburg, M4., dnd the National Bureau of Hines, U.S. Dept, of Interior, College, Park* Md, 02 215 073 6 Corasa Coated fibrous Class Vehicles .The glass pledgets L6*ariB------- *11 experiments were from a single source of flexible, thermal insulation fibrous glass of the type used in building construction. They consisted of aeshworks of long incertvising strands of boreallicate glass costed with a heat cured phenol-formaldehyde resin. They contained in addition to Sifts the oxides noted in table 2. The strends were of diameters --'"7 * 3- to 10-fold that of the asbestoses with a Been diameter of 6. 5 y and a few fibers la both the < l V and > 25 u range* The counts indicated in table 1 are skewed by the predominant large fibers that were complexly intertwined and exceeded lengths of 320 jj. The great length of the fibers made it impractical Co assess the cumber of whole fibers per unit weight with accuracy. PICC Standard Reference Samples of Asbestos (Crocidolite, ^aositer Chrysotlie A); The' International Onion Against Cancer on the recoatfoendation of a working group is 1964 established Standard Reference Asbestos Samples (SRA3) of the major types of asbestos prepared from as pure a parent material as possible (9,10). Three of these samples, crocidolite, aaosite end chrysotlle A are Included in the studies. The IHCC-SKAS crocidolite served as a baseline for comparative studies with the other materials. The samples were received from the Pneumoconiosis Unit, Llandough Hospital, fenarth, Vales in polythene plastic bags and were stored in glass containers. All three samples have been characterised both physically and chemically ib previous reports (11, 12), Our own measurements on the samples coincide with the reported range; however, different parameters of oeasureaent were used in table 1. 02 215 0740 7 South African CrocldoHte (Wagner Preparat1onl-.TMs * .... ... sample, obtained from t>r. J. C. Vagner, 'Is fine milled croc Idol I te from a single source of Northwest Cape Blue asbestos. U was originally used to determine carcinogenicity In the rat by Vagner et aU and Harlngton, et aU(j_, j Jb J>)-d later by our laboratory (2)* Physical and chemical properties have been documented and coincide with the measurements In table I (i2mi)- UfCC-SRAS CrocldoHte (partially pulverized!--To treat asbestos In a manner similar to that of some glass samples, the UICC-SRAS croc I Oolite was partially pulverized In a Spex model $000 stainless-steel ball mill (Spex Industries, inc., Hetuchen, N. J.), operating at 3200 rpm for 1-1/2 minutes, using \$ mgm loads In 2 ral chambers. The resultant differences in particle size and reduction to particles, that by optical standards appeared non-flbrous (l.e.v dimensional ratios of are Indicated In table 1. X-ray diffraction analysis of samples of this cfocidollte after comparable pulverizatlon was carried otrt -isy^both the'Nat? one I Bureau of Standards and by the Johns- hanvllle Research and Engineering Center, The results of these analyses Indicated a persistence In the 'diffraction lines characteristic of CrocldoHte, although areas beneath selected peaks were reduced. Electron microscopic examination Indicated that the non-flbrous particles by optical standards were essentially clumps of submlcroscopic fibrils (fig. 8), consequently the changes In the X-ray diffraction pattern most probably represented changes In fiber size rather than loss In structure. 02 215 0741 02 215 0742 Virgin Crocidol Ue tre. Hand Hilled;--This material was obtained as a single hand cobbed ore specimen measuring approximately 8 X 8 X 10 cm through the courtesy of Mr. U N. Kuyper, of Cape* Asbestos South Africa (Pty.) Ltd. Much of the stratified fibrous vein was separated from extraneous minerals by hand^ then, using the two halves of the remaining fibrous surface as a grinding media, the fibers were ground by hand to a fibrous quality that resembled that of the UICC-SftAS crocIdol Ite. Virgin CrocidolUe Ore. Bal 1 Hilled;--Other portions of the stratlfled fibrous vein from the above ore sample were subjected to brief fragmentation In the Spex mill until, again, they were reduced to a quality comparable to the UICC-SRAS crocldoMte. $pex Stainless-Steel Hill Fragments:.--A stainless-steel chamber of the type used In our milling process was reduced to minute fragments with a carbide steel lathe, and these fragments a were further reduced In size by prolonged. JO^oi note pulverization In the mill. Host particles ranged In size from 2.5 to 40 n. this nickel-chrcroe steel was'not arva\y2ed for trace elements. Metallic nickel fragments.--The nickel was purified precipitate from disintegration of nickel carbonyl, composed of 2 to 100 p aggregates of particles that rarely exceeded I p In diameter. By electron microscopy, these particles possessed sharply 9* angulated spine-like projections over their surface. The sample originally obtained from the International Nickel Company, Toronto, by Dr. tf. C. Huepef has been reported to yield local sarcomas in the rat at a cumulative Intrapleural dose of 300 mgms (}. Other Classes.--The coarse fibrous glass that served as a vehicle, and four other types of fibrous glasses, were tested w+\ i\ - *t*ky<t .^4-y' for carcinogenicity after reduction to fiber lengths^roughly comparable to those of asbestos by brief mUHng In the Spex mill. Table I and figures 3-16 indicate the distribution of fiber sizes and particularly the quantity of such materials that ware reduced to non-flbroas particles by the milling, .While It was difficult to satisfy ourselves that sufficient materiel could be examined electron microscopically to make valid quantitative assessments of the finest fibers It was apparent that fibers less than 0,5 U in diameter were present A** In all partially pulverized glasses and were particularly ^7,. r abundant In the AAA fibrous glass. Table 2 Indicates the . mineral content of these glasses exclusive of the major silicate component and other features prior to milling are Indicated below. Fibrous pyrex brand glass wool.Is a commercial product used In coarse filtration procedures. It consisted of long silky fibers, '5-to l2-p In^di^meter coatedWit'h a binder of undetermined composition. Old glass wool is an obsolete loose connerclal fiber, popular several decades ago as house Insulation- The fibrous glass was notably Irritating for the skin and consisted of abundant short fibers ranging widely frcm 1 to 35 p In diameter * with many hook-ended fibers. It had the typical glass wool mineral oxide content indicated In table 2. * 02 215 0744 11 AAA fibrous glass was an especially fine fibrous glass that was tested because It tended to approach the fiber diameters of asbestos. The mean diameter of whole fibers was 0*22 \i with a range of .06 to 3 H- Two forms of this glass were tested after partial pulverization to reduce fiber length: uncoated glass designated AAA-u and Identical glass coated with a urea-formaldehyde resin designated AAA-c. The mineral oxide content and size range of the two after partial pulverization were essentially Identical (tables 1 and 2). . Silicon Dioxide.-- Two forms c' --- eel ally fine particulate $I02 (silica soot and Cab-C-Sil) were tasted. Both samples were prepared by flame hydrolysis of silica tetrachloride and were more than 99-5% pure. A considerable difference In particle size was apparent between the two types of silica at the optical level {table I, figs. 12 and 15), However, of more significance at the submlcroscoplc level, these particles- were composed of agglutinated clumps .of. minute spheres that ranged In size from 5*)5 nra for silica soot and 50-150 nm for Cab-O-SU. . 02 215 0745 Result 12 Analysis tff-luaor ydaldi- The rsulie of 20 experiments, each employing y> rets, are shown la taxt~figures 1-7. The text-figures ere arranged ia identical format for comparing data, end ere sufficiently detailed to perait alternative method* of calculation* Since the earliest mesothelioma van observed 54 week# after treatment, ve arbitrarily considered only those rats that survived the first year after treatanat as the effective number. The over-ell incidence of oesothelloass is this effective nucbsr is recorded In the heading of each text-figure* In general, deaths from other causes during the second year were sufficiently coopamble to make direct tumor incidence comparison* vUid. Vithin each texUfignre the space below the headline represent* rats dead during the fir*t year. **bove this line the solid area represents rata dead with mesothelioma, and the outlined area^rat* dead from other causes. Humbert at the base of each text-figure tabulate this information by week. The extent of pleural fibrosis at the implant site was acae&eed in all rats that died during the second year on a seals and the predominant value is indicated at the lover right of each text-figure. The xajor lesions noted in each rat at death are indicated by an alphabetic code vlthln the text-figure, Each alphabetic character represents an organ eystea^vlth upper case letters indicating neoplasms and lower ease lettore representing other leeIona, primarily of inflammation or degeneration* 02 215 0746 A - adrenal, 8 - bone, C - cutaneous tissues, D muscle, E - pancreas, F - peritoneum, G - digestive tract, H - liver, I - EENT, J - nervous system, l - respiratory tract, H - mammary glands, 0 - genitalia, F - pituitary, Q - thyews, R - lymphoreticular system, S - spleen, T - thyroicf, U - urinary system, V -- vascular system, V - salivary glands, X - no lesions, Y generalized disease, 2 - lost or unaccounted* 02 215 0747 Text-figure 1 a-d compare the incidence of mesotheliomas after a maximum dose level of 40 agas for four different types of asbestos. The earliest neoplasm in the entire aeries of experiaents vas detected la. the a&QSlt* group* during the 54th week. Two further tisors were detected in this group before the 68th week, suggesting an earlier onset of neoplasms with aoosite than with ehrysotile or the crocidolites--a phenomenon also apparent in Wagner's experiments <,^7). However, during all comparable periods of time in the last three-quarters of the second year, the cumulative incidence of deaths with mesothelioma from *v the four types of asbestos vas not significantly different, and the final incidences of pleural neoplasms resulting from the three UICC asbestoses were remarkable similar (15/25, 15/26, 14/23). The final 15/20 incidence for the Wagner erocidolite does not exceed this range sufficiently to indicate a statistically significant difference in tumor response. It vas considered that this high incidence-range might represent a maximum tumor response end that, for comparative purposes, 40 mgms might repre sent an excessive dose, hut this seems unlikely since half this dose ef erocidolite in a subsequent experiment showed an appreciable reduction in tumor incidence. The extent of fibrosis at the Implant site vas uniformly high in all four groups and served as a basis for comparisons to follow. One rare neoplasm, a generalized lymphosarcoma not involving the pleural site, was encountered in the ehrysotile group. a4 02 215 0748 Text-figures* 2a-d Illustrate the response vith DICC-SRAS croeidolite Xn graded doses fxoa 20 to 1 mgta. Vith the data from text-figure 1c on the 40 mgs dose, It was clear that a staple dose response relationship existed both for the apparent ttae of tumor onset and final incidence of pleural' neoplasms. From these data, a dose of approximately 23 agios U>"* vas calculated by graphic probit analysis to yield a two-year 502 tumor # incidence if deaths from other causes remained constant (J8). Fibro sis less extensive than that at 40 mgms vas present in ell rats at doses of 10 and 20 mgms, and appreciably lesser degrees of fibrosis occurred at lower doses. In these groups, four relatively rare neoplasms were noted: an osteogenic earcoma of the skull* two hemangiomas of the splden, and a peritoneal lipoma. These experiments, along vith the controls In text-figure 3, served as a standard of reference for the experiments to follow. 02 215 0749 u 16 Tert-figuree* 3e-e illustrate 3 controls for the previous and subsequent experiments in which the pledgets of large fibered glass laden with gelatin alone were employed. In the three experiments, none of Che 90 rats had pleural neoplasms. However, only 58 rats survived long enough to*serve as valid treated controls; thus incidences of mesotheliomas 9 less than 3/30 rats in- comparable experiments could conceivably result from the vehicle alone (19). The distribution of ail neoplasms in these groups was essentially like that in several hundred untreated controls, unusual neoplasms remaining as isolated examples. Although initial inflammatory reactions to the pledgets appeared to be as vigorous as to pledgets saturated with asbestos, the subsequent histological changes 'indicated far less fibTous proliferation than with 1 mgra of croeidolite. In rats killed at the end of these experiments only minute macroscopic residues of glass and scar tissue could be detected. 02 215 0750 17 Text-figure 3d represents the filiale converse control of the 3 experiments above in determining the potential effect that the glass pledget sight have on neoplasms Induced with asbestos. A median dose of ID agoi tJXCC-SRAS crocidolIte was suspended la physiologic saline and delivered to the left pleura through an open thoracotomy without the glass vehicle. Ve expected less tumors because asbestos* injected In this manner tended to pool in the costophrenlc angle and elicited less fibrosis than when applied with the glass vehicle. However, comparing this result with that of text-figure 2b, it is evident that, although Che neoplasms tended to be lethal scoevhat later in the glass vehicle group, the final incidence in the two experiments (9/21 vs. 11/27) ^indicated that the glass neither enhanced nor suppressed the induction of mesotheliomas by asbestos.^ ' 02 215 0751 18 Text-figures Aa-c Illustrate the results of applying croeldol Itedlfferent from the. UICC-SRAS croeldol Ite In that It was from a single specimen of northwest cape crocldollteore that was silled In our laboratory using only the ore Itself as a milling media without exposure to extrinsic oil or seta I After hand milling to a consistency like that of the UICC-SRAS crocldollte, serial doses were applied. The Incidence of mesotheliomas at levels of AO and 20 mgms was virtually Identical to that of the UICC-SRAS crocldollte, and at the I mgm level no significant difference In response was evident (2/2$ vs A/30) (p>0.5)* A graphic prob ft analysis of dose response over this range Indicated that a 25 mgra dose would yield a two year 50% tusor . Incidence compared with the 23 mgm 50% tumor dose for UICC-SRAS . crocldollte (18). The extent of pleural fibrosis correlated with dose and closely .matched that of comparable doses of the UICC-SRAS crocldollte and again no exceptional deviation in the incidence of neoplasms outside the thorsx was noted* The control experiment, text-figure Ad, illustrates the result of Implanting AQ mgms of the same crocldollte ore after milling In a stainless steel ball mill. Here again the 15/23 Incidence of pleural neoplasms was virtually Identical with both the IA/23 incidence from UICC-SRAS crocldollte (text-fig. 1c) and more Importantly with the 18/2? Incidence from the crocldollte ore milled by hand (text-fig. Aa). Both Implant site fibrosis and extra-thoracic tumor Incidence followed that of the two previous crocldollte tests. These Indications that mill contamination was not a factor In asbestos carcinogenesis were further supported by the . -. experiments Illustrated In text-flqure 5* 02 21 K 0752 19 The experiments In text-figures $a-b explored the carclffegenlc potential of nickel chrome steel, the prime metallic contaminant- In the sill ling process and nickel, the most carcinogenic constituent of the mill. Pulverized particles of nickel-chrome steel frc*n a mill similar to that which was used to process the crocldolUe ore yielded no mesotheliomas at a dose equal to that of the hlghest.-level of asbestos used. Pure nickel metal at similar high levels was excessively toxic; all rats were dead with hemorrhagic pneumonitis within 60 days. Consequently, a I mgm dose, still far In excess of potential nickel contamination from the mill, was employed. Toxicity of the nickel even at this level was high In the first year, but no pleural neoplasms developed In the group of 18 rats which had a survival rate during the second year comparable to that of the asbestos treated groups. -The experiments In text-figures 5c-d are relevant . to testing the carcinogenic potential of silicon, t.he predominant constituent of both asbestos and glass. Silicon dioxide in the \ r *' ~ a form of smooth surfaced spheres of submcroscopic site was usttd'In both experiments. The result of these two experiments was a single mesothelioma developing in the early part of the second year. All four of these experiments with non-flbrous materials resulted In a minlmvea of pleural fibrosis, although the degree of fibrosis seemed greater with the steel fragments than with the other caaterlals, in the nickel treated group two relatively rare tumors, a pituitary adenoma and a carcinoma of the kidney, were noted. 02 215 0753 a 20 The subsequent experiments tabulated in text-figures $ and 7 concern the role that the physical characteristics of asbestos might play In carcinogenesis. Text-figure $ Illustrates our first attempts to examine non-asbestaferafibers of a size similar to asbestos. The Urge fibered glass that we had used as a vehicle was reduced to short fibers by brief milling In a stainless-steel ball mill and Implanted In two groups of rats. The first group was young rats as In the other experiments, but the second group was hO-week-old rats that were used to test age-related susceptibility. Two further experiments (textfigs. 6c-d) were done on fibrous glasses of slightly smaller diameters, the highly Irritating old glass wool and pyrex fibrous glass. The results of all four experiments were essentially alike In that only single pleural neoplasms occurred in each of the four experiments, the degree of pleural fibrosis was minimal, and unusual neoplasms occurred only rarely. Although no tumors had been noted with the Intact fibrous glass vehicle, the occurrence of four mesotheilnas In these experiments clearly Indicated that fibrous glass could be carcinogenic If reduced to short fibrous lengths* Kxever, the 1cm Incidence of'mesotheHcmas, whether the four experiments were considered separately or collectively (W, was not sufficient to conclude that the partially pulverized glasses had a higher potential for tuaor induction than the Intact glass. 02 215 0754 -.^L ^ 21 Text-figures 7a-b Illustrate two further experiments carried out In the same way after partial pulverization, but, <-*u-' Vi-At) lJ * employing fibrous glasses of much finer diameter than the K ^ vehicle glass* As noted In the nwterlals section and table 1, these two samples were Identical except for e coating of ureaformaldehyde resin on the latter. The range In fiber diameters -> ;-*vrrs.w 'after pulverization closely approached that of medium sized asbestos fibers. The result of each experiment was a small but significant number of mesotheliomas, 3/26 and 5/28. These Incidences If taken together were sufficient to Indicate a statistically significant Increase In Incidence over both the Intact coarse glass vehicle {8/54 vs 0/58) or the combined groups of partially pulverized glass of more coarse dimension (8/5A vs A/gi). The extent of pleural fibrosis was also greater In these groups than the groups treated with coarse fibrous glasses. 02 215 0755 9 Text-figures 7c-d represent a final, set of experiments designed as controls on the experiments with partially pulverized glass. Groups of both young and old rets were Implanted with UJCC-S&AS crocidollte that had been treated In the same manner as the partially pulverized glass. That Is, the UICC-milled crocidollte was further pulverized In our ball mill for l-1/2 minutes. Gross effects of the additional sluing were not apparent; however, subsequent microscopic examination Indicated appreciable reduction In particle size {table I). The result of both of these experiments was a reduction In the number of pleural neoplasms and the extent of pleural fibrosis as compared with the four groups treated with crocidollte milled conventionally. Since this difference seemed of particular Importance a series of statistical calculations compering the differences were done. Although data from the experiment with hQ-week-old rats suggested a reduced Incidence, no rats of comparable age had been treated with conventionally milled crocidollte. To compensate for this difference and the associated Increased mortality frcro extraneous causes, a emulative mortality rate from mesotheliomas corrected for extraneous deaths was plotted for the two groups of rats treated with partially pulverized UICC-SJWS crocidollte - (text-figs. 7c-d) and for the four more conventionally milled crocldolites (text-figs. 1c, Id, Ua, and 4d) by the method of 02 215 0756 Pilgrim and Oowd (20). Text-figure 8 clearly Indicates the consistent mesothelioma Incidence from the conventionally milled croc I dolltes, and the consistent reduction In mesothelioma Incidence throughout the course of the two experiments with croc idolise subject to prolonged milling. A chi square test of comparison of the overall Incidence of mesotheliomas between the single group of young rats treated with partially pulverized crocldollte (text-fig. ?c) and each of the four groups of comparable age treated with conventionally milled croeidollte (text-figs. 1c, Id, Aa, and Ad) Indicates the probability of a valid difference to be 95% when calculated for two of the four groups and more than 99% for the other two groups, or for the four groups combined. 02 215 24 Considerations of Fiber Size In Relation to Tumor Yield.--The results of the experiments In text-figure 7 pointing to differences In tunor response relevant to particle size or structure motivated an assessment of the size distribution of fibers of the 17 implanted materials. The wide range in dimensions In any one of the 17 materials made a precise tabulation of the site of Individual particles a formldible task. Consequently, the assignment of 1000 consecutively counted particles to JO ranges of dimension was a compromise which gave us a fair sampling of size distribution In the optical range. This distribution Is tabulated In table 1, Part A. The preparations that were counted are Illustrated! at low magnification In figures 1-18. Extending these ranges to ^particles of submlcroscopIc size proved futile because of the film!ted sample size that could be evaluated by electron microscopy^ f- ...-------------T^rr>'----------- However, our Impressions^can be sunraarlzed as follows: In all 7 asbestos and glass samples, fibers (I.e,, particles with ratios of diameter to length of 1:3 or greater} extended nearly to the limits of electron microscopic resolution, fibers less than 1,0^ In diameter were far more numerous In asbestos samples than In glass samples. Both^the large and small cl imps of asbestos that appeared non-fIbrous[gtas0in the optical range were composed of clunps of mlcroflbrlis ?n the range of 0.05-0.2 u X 0.6-2.0 y (fig. 8). In contrast, non-fibrous glass particles were Irregular sol id masses. ^ ^^i'^^iHelther the silica nor metal samples contained fibrous particles. 1- , h . *- 7 Nickel particles were irregular, sharply angulated splney crystal; both types of silica were smooth surfaced spheres. <> 25 an estimate of the number of particles In each dimensional range per unit weight by taking?density Into consideration. Part 8 of table I represents this estimate, assuming that the fibrous particles were cylinders and that the particles In each dimensional range were distributed uniformly about the mean volume for each range. In the ranges of large sized particles, particularly those with few particles, these assumptions were liable to error. Furthermore, the whole fibrous glass used as a vehicle could not be treated this way since the length of most particles exceeded limits that could be measured accurately. Put despite this defect In methodology, we felt that such estimates might be revealing If only broad differences in carcinogenic response and particle distribution were compared. 02 215 0759 2$ Considered In this way, 3 of the k conventionally allied crocldollte preparations with equally high tueor Incidences had nearly comparable distributions of particles. The one deviation was UICC-SRAS crocldollte which had fewer large particles and a consequent excess of particles In the finer ranges because of more complete milling. Similarly, the UICC-SRAS amoslte and chrysotMe preparations with comparably high tunor Incidences, contained few particles of large size, but here the lumber of small particles per unit weight was far greater because of the lesser density of these asbestoses when compared with crocidollte. From this data we could conclude either that fibrous particles In excess of 5 p in diameter were not essential to the induction of tumors or that these large fibers were reduced to finer fibers In vivo so that the ultimate distribution of fibers would be the same. 02 215 076<? J27 Th*"Critical comparison scaled*to be between the excessively stifled, partially pulverized crocldotltd with Its reduced Incidence of tutors and the conventionally pH led crocldolltes with their high tiraor Incidences. Table I, fart B Indicates that prolonged milling ted pulverized the crocIdoMte until no particles were 4 greater than 2.5 p In diameter or 20 4 In length. Slightly more than half of the sample by weight (84% of the ntfnber of particles) consisted of minute clumps not exceeding 2.5 ^ Jn either dimension that were non-fjbrous by optical standards. However, these clumps, like those occurring less frequently In other asbestos samples, were composed largely of submicroscopIe fibrils (figs. 7 and 8). Since the number of these submfcroscoplc fibrils far exceeded those In the conventionally milled croc I dollte samples It was reasonably to assume that they were less carcinogenic than larger fibers and served to reduce tumor Incidence by simply diluting the dose. Presumably, the 20-32% tumor Incidences resulting from this sample were related to those particles which retained their fibrous structure In the optical range. Interestingly, the three dimensional ranges of fibers represented In this low tumor yield sample, each contained more fibers than their counterparts In the samples that yielded high Incidences of mesotheliomas. Furthermore, If one totalled all of the fibers In the low-tunor-yleld partially pulverized crocldollte, they numbered more than the totalled fibers In the high-tumor-yield crocIdolJtcs. Thus, It was evident that the Incidence of mesotheliomas did not relate directly to either the number .of fibers In a particular dlnvenslonal range or the total number of 02 215 0761  28 fibers implanted. However, this reasoning <fld not consider the fate of the particles after Implantation. Asbestos fibers are composed of bundles of microfibers which readily separate through fragmentation of the fiber* Touch preparations from the lesions suggested that fragmentation frequently occurred In vivo* It, therefore, seemed reasonable to consider the nunber of fibers present 2n the rat If all fibers had been reduced to a lesser dimension. * 02 215 0762 29 9 Reduction-of all particles to their ultimate submicroscople fibrillar dimension would, of course, result In equal lumbers of the low-tumor-yield submlcroscoplc fibrils In all groups. But eliminating these submlcroscoplc fibrils from the calculations on the assumption that their minute size rendered them Inert a yielded different results. The smallest fibers that could be recognized by light microscopy and counted were those with ; ?\ diameters of approximately 1*2$ u and consequent lengths of 3.75 -- the approximate mean dimension of-our smallest category of optically visible fibers. Converting all of the particles In excess of 1.25 X 3.75 V to this mlcroflber ^ ^v^3"i*cnslon and excluding alVyibrlls and particles of lesser dimension resulted In the calculation at the bottom of table I. The potential number of microfibers per unit weight was roughly equal for not only the four high-tunor-Incidence conventionally milled croc I doll te samples, but also the high-tumor'Incidence amosite and chrysotMe samples as well. Conversely, the 1 owtunor-incldense partially pulverized crocidolite sample, diluted by the submlcroscoplc fibril clumps of lesser dimension, could yield only half as many mltroflbers as the high-tirtor-Incidence groups. The same reasoning w *. then used to consider the glass samples. We assigned that glass fibers could fragment transversely as easl.ly as asbestos, but since glass was not composed of bundles of ralcrofibers, longitudinal fragmentation would be a rare event. Consequently, only transverse fragmentation of all fibers to lengths of 3*75 p was calculated for the glass samples.. _---t--------r~ . 02 215 076; for the two fine glass samples that Induced 12-18% mesotheliomas the total .number of calculated mleroflbers was approximately half that of the partially pulverized croclOolite that yielded 20-52% mesotheliomas, and approximately one fourth to one fifth that of the conventionally milled asbestos that yield 58 to 75% mespthellomas. The three coarse fibrous glass samples that yielded negligible numbers of tisnors had comparably 4ow ` |.'*/K $*?*? numbers of calculated mlcrof ibers that totalled less than a fifth the number of microfibers In the fine glasses and less than 5% the number of microfibers In the_hih-tumor-Incidence asbestos samples. The mlcrofIbers In the whole glass vehicle could be assumed to be less than those In the milled coarse glass unless the process of fragmentation was perfectly efficient. 02 215 0764 31 Thus, mesothelioma Incidences for both the asbestoses and glasses correlated reasonably weU with the number of mlcroflbers In the range of 1.25 X 3.75 * that theoretically could result from the fragmentation of larger fibers j_n vivo. Whether the Induction of mesotheliomas Is Indeed closely related to this relatively narrow median range of fiber sizes would seem amenable to further Investigations. 02 215 0765 32 Korehologic Observations: The primary response of the pleura to all, Of the asbestos Implants vaa a vigorous granulomateua reaction ending in the formation of dense fibrous coats firmly adherent to the visceral pleura and pericardium. This fibrous tissue closely invested residual particulate matter and was composed of avascular. Interlacing, lumatuta spindle cells and abundant collagen interspersed with foci of hyalin and liquifactive necrosis (figs* 19.20). Pleural fibrosis in each rat vaa assessed, grossly and histologically, as extensive, moderate, or slight. Using this simple evaluation it was evident that for each group the extend of fibrosis roughly coreelated vith the incidence of pleural neoplasms not only in the high and low tumor incidence groups, but also in the medium tumor incidence groups treated vith Xov doses of asbestos or fine pulverised glass. Ope of the purposes of the glass as a control vaa to determine whether pleural cell proliferation in the milieu of efi inflammatory effusl6ni might act as an in vivo cell culture vlth~fi-liigh potential for non-specific neoplastic transformation. Ideally. 6n would expect such a control to yield a reactive fibrosis as extensive as that of the asbestos, however this was not the case. In all of the experiments that yielded fev or no mesotheliomas, irrespective of the type of material used, fibrosis was negligible. Reactive sites is these groups had an abundance of mononuclear.leukocytes, foreign body giant cells, end capillary vessels, but a papcity of collagen and connective tissue (fig. 21). The dense fibrous plaques which characterized the primary reaction to asbestos and pulverized fine glass had many of 'the qualities of neoplasia (fig. 20)., but true neoplastic 02 215 07R 33 development by our criteria vaa distinguished by abrupt conversion of tbe proliferating collagenous connective tissue to masses of closely packed, atypical disoriented cells that extended beyond tbe residual foreign particulates and the reactive fibrosis to them. In general, abundant mitoses suggested a rapidly lethal course for these neoplasms. However, this may mot have been tbe case since tbe data indicate* that a large proportion of the neoplasms became evident only when the rats were killed at the end of the experiment (text-figs. 1-7). The latter neoplasms were not structurally different from those that killed rata < earlier in the experiment, and sioce only 13Z of the neoplasms metastasized or invaded structures adjacent to the thorax (table 3), the most reasonable explanation is that most neoplasms were relatively slow-growing and remained confined to local sites for long periods of time. It is perhaps significant that a similar slow course often characterizes the mesotheliomas of man (22). 02 215 076? The histologic characteristics of the 170 neoplasms were not unlike those previously described (2,7)* Hone could he conceived as arising from tissues other than the reactive pleural mesenchyme, hut interesting variations in structure were evident (table 3). The dominant cells had. either a spindle or pleomorphic structure sometimes intermixed - a phenomenon common to most ooo-epicbeliai neoplasms in the rat. Additionally, some contained giant aulticucleate cells, ocher osteogenic foci, and a few had tubulopaplliary patterns suggesting surface covering features of mesotheliua (figs. 22-26). Efforts to identify hyaluronic acid were equivocal in selected neoplasms, although some contained neutral and add mucopolysaccharides. The 14 neoplasms with foci of osteogenesis appear to be unique to our strain of rats. There was no apparent difference in structural types between tumors induced by asbestos end.tumor induced by glass. The single tumor induced by silica was a simple fibrogenic spindle cell sarcoma. 02 215 0768 35 Table 3.--Structural types of the 170 saotheliomas induced by asbestos, glass and silica. Spindle cell types: Ttbrogfeoic Osteogenic Giant cell Pleomorphic cell types: Medullary Tubu1op api1lary Total Asbestos 105 (13)* 12 9 (2) 23 (3) 8 (2); 157 (20) Class & Silica 10 (1) * O) 1 13 (2) figures in parentheses are those neoplasms that metastasized or invaded structures outside the thorax. 02 215 076f The present experiments were not designed to exanlne all parameters of the potential carcinogenic Hazard of asbestos or glass to f&an. In fact, the direct application of our results to the problems In man would be unwise since the method of application and the high doses employed arc remote from the usual exposure of man to fibers. However, the means by which these materials exert their carcinogenic effect Is a critical problem and the present data along with that of other reports seem adequate to develop preliminary conclusions. The present studies confirm that convent 1 na 11 y-mII1 ed asbestoses have a high Intrinsic capacity for Inducing neoplasms and that this capacity Is virtually equal regardless of the type^. * of asbestos or Its source. Furthermore, fibrous glass when >. reduced It. approaching ttyat of "" conventionally milted asbestos Is also.in-vt*r-i*n. sically--carcinogenic. U* ^ atXfttrvr.-------- ------This capacity to induce neoplasms ^can be related to 1) contamination by carcinogenic hydrocarbons and metals, (2) the carcinogenic constituents of the firbrous mineral Itself, or (3) the structural character of the fiber. 02 215 0770 57 Both natural contaminating oils and minerals and those Introduced In the milling and packaging of asbestos have been Investigated as causes of asbestos carcinogenicity (2, 6, Mj 23. 2b, 25), These studies indicate chat asbestos from which ells have been rigorously extracted Is as carcinogenic as the non-extracted asbestos, and that these contaminating hydrocarbons are not.present In amounts sufficient to account for asbestos carcinogenic!ty* Experiments reported here concern the role that contaminating metals might play, an hypothesis suggested by the findings of other investigators (13* jj** 16, 21)* Such metats retain a carcinogenic potential of their own, but they seem unlikely causes of asbestos carcinogenicity because of the minute amounts Involved* The present experiments demonstrate that at levels far exceeding those that might contaminate asbestos, neither finely particulate nickel metal nor nickel-chrome Steel are sufficiently carcinogenic to account for the mesotheliomas Induced in the rat with asbestos. This observation would not exclude a synergistic or catalytic action of contaminating metals. But such an hypothesis is not supported by the experiments which show that handcobbed crocldolite ore, milled without exposure to metal induces numbers of mesotheliomas at graded dose levels equal to the cumbers of fftesotheUomas Induced either by the same ore milled in a steel bail-mill or by mine-milled UICC-SRAS crocldolite. These experiments would not exclude the potential carcinogenicity of the variety of heavy metals or other complex-------------------------- - constituents of tf^e asbestos or glass fiber Itself. Yet, with the exception of silicon there are remarkable differences In the composition of asbestoses of different types and these differences do not seem to alter the Intrinsic carcinogenicity of the .asbestos fiber as Indicated In our experiments or those reported previously (, j 11.* 22) Silica, the major constituent of both asbestos and glass proved to be relatively Inert In our experiments^ However, this result must be reconciled with the high Incidence of Intrathoraclc reticulum cell sarcomas^ -c absence of such neoplasms In our experiments Is related to differences in the physical structure of the silicas employed by us and by Vagner. Because we were Interested In testing the smallest particle of silica, we used sMicas prepared by flame hydrolysis. These silicas were composed of agglutinated clumps of particles 5-150 nm In diameter that were spherical In shape. Neither type caused the extensive reactive fibrosis characteristic of freshly fractured quartz silica - a quality that may be relevant to f1brogenes1s and In turn to carcinogenesis Further evidence against the Idea that Intrinsic constituents 02 215 0772 of the fiber ere responsible for the neoplasms ere the ________ experiments with partially pulverized crocldoilte. Prolonged mining not only reduced the fibers to clumps of submlcroscoplc fibrils, but also exposed this residue to additional metal contamination from the mil 1. The resultant loss In carcinogenicity Implies that the structural Integrity of the asbestos fiber above that of the finest submlcroscoplc fibril Is essential to carcinogenicity, and that neither contaminants nor elemental components are 11 feeIy factors In this process. 02 215 0773 If the physical structure of asbestos rather than Its chemical constituents ere critical to carcinogenicity, then similar particles, W sufficiently durable, should Induce similar tunors. On this reasoning rested Mir attempts to reduce both coarse and fine flbered glass to a range of sizes comparable to those of milled asbestos. Our attempts to reduce 3 types .if" of coarse flbered glass resulted In a range of particle sizes that spanned the range of asbestos, but quantitatively had $ far t&ore large fibers and, consequently, few of the minute fibers so abundant In asbestos, in contrast to the Intact fibrous glass all four experiments with partially fragmented coarse glass yielded single mesothellomas, Implicating this fragmented A fibrous glass as a carcinogen, but these experiments were Jj\'- jJ. I sufficient to show that reduction In size was the causative factor. However, the Idea that fiber sl2e is critical was supported by the experiments with the partially fragmented AAA-glasses of especially fine diameter, for here both the Incidence of neoplasms and the range in fibrous particle sizes A F\> more closely matched that of the asbestoses, furthermore, by assuulng^that larger fibers tended to be reduced in vWo~^) 7 to a narrow range of sizes just above the Halt of optical r resolution. It was possible to correlate the number of microfibers potentially present In vivo with the Incidence of msesothel Icmas, Irrespective of the type of asbestos or glass employed. 02 215 0774 k\ The stalest explanation that can be derived thus far Is that the Intrinsic carcinogenicity of asbestos and glass Is not related to any (contaminating or Inherent physicochemical quality, but rather to Its fibrous structure perhaps In a narrow range of sites. This conclusion Is not new for Bryon and Blscboff argue that asbestos carcinogenicity Is simply another'form of `solid state carcinogenesis11, the key factor y being the formation of a unique avascular fibrous tissue In response to chemically Inert materials (28). These experiments offer no challenge to this argument for it was our Impression that the Incidence of mesotheliomas correlated well with the extent of fibrous tissue In the lesions just as they did with numbers of mfcroflbers potentially present In the lesions. Ml of the experiments reported here require further confirmation and controls. Of particular significance In examining our hypothesis will be the testing of both glass and asbestos In narrow, selective ranges of fiber siie and * totally reduced to non-flbrous fora. Additionally, the experlments indicate that similar microfIbers of other types of durable material, particularly those unrelated chemically to asbestos or glass, should be tested. **' W 02 215 0775 VMM Li *m f`m-- fW Iwiium mm MtutMia b*. aM im< W *<-*ail n--rat i .x*UaiMUa at UB (a*a*Ma gi n*iB Mmila luab M rlMU MM......... m Nana *m*m MM Mt luwt a XN * **.' A * U u m M MR u N4 *4. MMB BB, MM a MU RUM L I Ut Ml CMC. m m Ml Rat ana mm nac am. Mas. Nil. rue aac. Mar. sw. aM Ml M au M Mas. law. MB. m*. mm MU an IM ftm Bb * at ii* > w an m tra IM MS IM M Ut MS m M m M t* II u M > K a t* *** a it a t* -B*ua t a .ismib 1 1 al t > tl II K rt A U u .!. ** M aa f MS MB. pm. law. m 4B 4S a MUU M BOB mar auaB NM BBT. U Mart mm Ml Iff in a) 1* U a* SM 14 Ma* ir iaa |y .sa-aa MB i 1 >a>Mi *g ti * ) t > *!* >* * - .t>M ,B>M ** IM* MrU* - -m >M ** >*** M'W i j > l > 1 1 t( *H- I*M* At ** * mA I.l ut 1.1 'MM* ............................ W >1 * * .fc * * M > i.l .* *.\ .MM .m *IMB # * **M IB kiadi .M *< l. hM - ia<a6 * >giaa * *a-wa |h * ,)W LXH **** .t . U* B A a* i.l tfc a.a i i.i a .a IB M n - a t it #.* ra ft i** ni n M It I.l )t . I. v. l.t 1,2 i.a M tn t.a 1.* M i.a . i-> .* 1 * j t* B u 1 i an t 1 IS M at U * Ml i IM m m tl Ml jt i vv t s IM* M M.t i i> it IM i i * aa* a. i i sa i)m ( ua ii ! / a. 11|** tm mi Ml *W M ii MMa iaa ua M ll *< 12 tat I.l IM IM ii > .1 I.l a. i 11 t ta* -- aaaaa i 11 > t m *. r V i. t. i e >. > t a %> ta m t.t 14 .l r * .. t* a i f.i a.i > ! t i.t . 4. t l.t 1.* a.> l.t >a i> . a.t i.t i: a. i >. t ai , i.t a.t in tM If lit I a> -i ... ai a.. m rti n stat > in n t*Ut ---Ht-- MMlitaiSaifl m IB m Mi *U m IIIH m IM ii 4 IS It M til *t t it i IM r"*M*** i.n *nU* tfk IMHU MHMHI MM !** mi n MM 1 nM >WI nst w M MH H* II .a t a a # a it a IIM| *BM*Ur*a iMtan. -ana MIHUl M (M Han-- MX^X MUHkUn B--aia H* twna < M-M.ll 'HrIUI Trfl W air M Hiitita amt m a arm * taa Mill aHB ' u* 9flB rwM, --U lU.rw. M| <* an .U--n * I. -- <ln mm Mil >otm - uiir MmXm IUI ! ItM.I lna>a JJI Qg M MM b^UI ( t.f^ aaUMMI fM*. MnuiH UB^kT A IB IMIlWMill > MM *1 `t xc^:vrzsT TEST MATERIAL 7JHPABATI0S csociPOLm Asagros SOUSCI Of BOS* TEST MATERIAL SUSGERT i BATS ! sacs:?: DA?I * Uftepened fibers, Cap ScooSlue cabbed ore Tally pulverised in CD CreeldelIte Fulverited 10 minute* in Spex sill 2ad Serlee UICC Hone nJroSrSoTile - 40 *g 2nd SeHct tJlCC Bone "CrocidoTita .. 20 mg Cape Aibeetd 40 ag South Africa (Fty.) LimitAd U3CC 40 mg UICC 60 me UICC so &g 5/27/706/1/70 6/1/72 3/17/71 ; 3/17/7 6/a/n 6/8/73 6/9/71 ' 4/9/?3 2nd Series UICC . PulVirlted is Spa* grocTdoiite partialis* sill 3 minute* pulverised 'UICC 40 Bg 6/16/71 6/16/7' Hicro fibrillar fraction Sedimentation and UICC UICC Crecidollie centrifugation in both water and alcohol Finely ground Crocide ite Fulverited in Sptx `Johne-Manvlli 4106-22-1B sill with plattie hilt 10 houre Crocidollte, long, fine >10*.* .5, # 4106-22-4 * Cape Blue crude fiber sheared in Variag blender, screened in special bottle Johna-Kurlll Cr&cldolite, hfcort, 5llfc" -5- Cape Blue crude fiber beared in Varisg blender, *creened in epecial bottle `Johne-Mwelll . Hacrofiber fraction UICC Crociddllt# Sedimentation and Greening in water and detergent UICC 40 eg 5/6/71 40 mg 55/71 : 40 mg ; S/7/71 40 eg i 11 i ' t 5/6/71 40 ng 5/6/73 5/5/73 S/J/TZ 5/6/73 Received from Gem ral ludkin Friday* Cm* 11* 971 who in turn received it res Ed Dotgen. <rv-.3J4 ,js^` 02 215 0777 * ^riPnil.AS2ST0a Alr-de*ned Jeffrey Hose ChtysotUe - 6320 Johoi-Kanville 40 mg Air-cleaned Jeffrey Hose Oijyeetile - 6320 'Johse-KanviHe 20 ft* Mr-denned Jeffrey Hose CHryeotiie - 6320 Mr-desced Jeffrey Hose ChryeotiXe - 6B20 Johne-Manvi11* 10 eg ljohn-44ftnTine 5 Bg Air-cleaat4 Jeffrey ChryaoUle - 6320 fully pulverised S'ul^eTlaed in $pex Bllljohse-Kanville for 10 nicutet j Cefctad crude Chryeotile Hone oro fro* Jeffrey i ainee f Jeffrey Biaee.j Attar.toe, Quo.| kaanda i 40 ag 40 ag Air-cleaned Jeffrey Pulverited ia $ptx BlllJohoe-Manville' 40 mg Chryeotile - 6320 for 3 minutei perUally pulverise 7/21-22/70 7/22/72 7/31/70 7/31/72 8/2/70 8/2/72 8/3-4/70 8/4/72 U/23/70 11/23/72 4/13/71 4/13/73 02 215 077!  * mtRlKBlt mm test material FKPASAT10S souses or xosz TEST KATSSAL SURGES? BITS sacritic ' EATS KSMCS CLASS Whole fiber uncoated fine fiberglas Fane v> Owens-Coralng 40 Eg 4/14/70 &/l&/72 Claes fibers Claes fibers 45^x3^ Sheared 1& Waring * blender, sedimented, and pulverised la Spex sill Sheared la Waring blender, sedimented, and pulverised In Spex eill Johns-Hanvllle 40 ag Johne-Kanvi5le 40 mg 8/31/70 r 9/6/70 6/31/72 S/672 Claee fibers y 10*, x Clans fibers ?i/uxy. Sheared in Waring Johns-Manrillt blender and sedimented twice, filtered. 40 Eg Sheared in Waring Johns-MeaTille 40 Eg blender a&d sedimented twice, filtered 9/13/70 9/19/70 9/13/72 9/19/72 Code 106 Micro-fiber Hone glass Johnt-^anvi lie ho Be 12/1/70 12/1/72 Sheared uncoated fine Sheared in Waring fiberglss - 40 mg blender 10 alautes Owens-Corning 40 mg 12/29-30/70 22/30/7* Sheared uncoated fine Sheared in Waring fiberglas - 00 ng blender 10 minutes Owene-Corning 80 mg 12/22-23/70 12/23/71 Sheared uncoated fine Sheared In Waring fiberglas - 160 mg blendor 10 minutes Owens-Corning 160 eg 1/15/71 1/15/73 Glass fibers, long, fine *4106-27-5 Code 100 sicro-fiber gists (4?5 glass) Sheared In Warlag blendor. cont-rlfused in Sharpies euper- cen tri fuge, screened Johne--Kanvi 11 .. Tul iy\uiv er t* ed fine uncoated fiberglas Pulverised 10 -minutes Owena-Coraing in Spex mlU 40 mg 5/13/71 fa ag j Partially pulverised fine uncoated fiberglas Pulverised 2 minute* in Spex alii Oweni-Corning 40 mg | i 1 r j 5/13/73 ) i I j i 02 215 0779 \ '* WftSAlfcb PREPARATION related riasopg amp vxtmik'Lk nguecs ur TEST MATERIAL iMS surds** BATE J SACSXUi PAT2 Aatigorite Pulverised Is Spex sill Snithsonina l| slnut#* Institution Alaau.ua oxide vbisker : Hone Artech Corp. Alusjmas oxide vbitkert Nose Alusioua oxide whiskerl Hone Attach Ccrp. Artech Corp. Alucisua oxide vfaiskeru Hose Attech torp. Linda Corp. Alualna 0.3'S' Bone - Attepulglt* *4418-100-1 Paste fora, 18,yf> ^UAa.i ojo^Ht* oiide, originally obtained froa Engel* hard Research Labs. Artech Corp. Johns-Xnnvii 1 t 40 g 40 ag 20 oe 10 ag 5 ag 40 &g 40 ng 7/6-13/70 9/29-30-70 10/12/70 10/19/70 10/20/70 12/18/70 5/12/71 7/13/72 9/J0-7? 10/12/' 10/19/' 10/30/: 12/18/7 5/12/7: Tuncftton carbide whisker* Bone Originally fros Suva Seikoeha Co., Ltd,, Japan .Metala A Method*, Lt< 40 ag 5/17-24/71 5/29/73 02 215 0780