Document 5baKeqdDwowG5VnJpX2Z5mp2V

SnTrhnniTinr PiD 1flfft"" a I. ABSTRACT Introduction Asbestos Siu.iers.i3 5. B^neri-mcntal Methods IT. 6 ItA SICPERB3EJPEAL AS5IST0SIS I 6. Species Susceptibility 7- Peculiar Characteristics of Asbestos 8. Rate of Tissue Reaction to Asbestos Fibers 9- Asoestosis Bodies 12. *- . X. rio^vs King*3 Floats Aefceott-o--iusx----. --, BJgHEOMSKr^ AsWsis 5>us4 _ _ -_ Dusting Materiel 13- .111 Dust Conmositiori Dust Concentration 1?. 16. Reaction in Animals Guinea Pigs 17- Rate and Type of Reaction .18 Progression 15. .20 21. 22. Infection. Coincident isith Dust Inhalation Infection after Dust Inhalation Asbestosis Bodies Rabbits 23- 2k. Rcits Sursnary and Interpretation f2zT S^tei L-fibai--Ao'pc?otr>ir Piv^t ` 26. Busting llatarlsl 21- Bust Coopersit.ion `~ 23. Bust Concentration 29. Size--frequency of Bust 30. Reaction in Animals 3132. Guinea Pigs Rate and Type of Reaction 33. Progression 3h. Asbestosls Bodies 35. Rats 36. Rate and Type of Reaction 37. Cats 38. Rate and Type of Reaction 39. X-ray Changes ho. Asbestosls Bodies hi. Rabbits 1x2. Rate and Type of Reaction 1x3. Asbestosls Bodies uu. -e:5- Summary and Interpretation -scuv 100 Per Cent Bal 1-cc' Tea lJ543U5'fe ifliR! U6. Busting Material j oAH L7. Bust Composition ii.8* Bust Concentration li9. Size--frequency of Dust '' l!i lh '"-"15 15 15 15 15 15 17 17 18 16 19 19 19 20 20 2C 20 21 21 21 22 22 23 50. Reaction in Animals 51- 52- Gui nea Figs Rata and Type of Reaction 535U. 5556. Progression lymph Node Involvement Asbestosis Bodies Bats and ILce 57- -5&x- Stannary and Interpretation ___ lv iom. UQMC-nw. w Bong fiVior1 Aabcatoj Pact - -- * ~Tm-- 5960. Busting ISaterial Dust Composition 61. Dust Concentration 62. Sime--frequency of Dust 63. 6U- Reaction in. Animals Guinea Figs 65. .66 67- 68. 69. Bate and Type of Reaction Progression Lymph Node Involvement Asbestosis Bodies Cats 70. Rate and Type of Reaction 71 - X-ray Changes 72. 73- Rats Rate and Type of Reaction -p 23 21 2i 21 25 26 26 26 26 27 27 27 27 25 29 29 30 30 31 7y. 75. ^?6- ili.ce ____.. -f-c-i--x^d '*2".J--^--.-.. cl h;i:ioo-.or. * 3J1C i. rtrr.r'r . .ou A/" i * t ->--1---i t~ 75 r Intratracheal Ehcporfronts 79- f-ocrerrisor: of Plbrous or: 80. C-cirrcarlson of ^-cions 1..:: 31. Corparison. of Lcnr,--filer .no 'l-v-'-t-rio-r,- ]>-ats 32. Intravenous Erperirierits Intraperitoneal 'I-rDcriner-ts 35- lijociv. ariiz- zT'O^iiehts ' T~ t t--c-r-^-i Protective Action ci Alnir.Iniiis Cc&r-ou.'r- 36. Fonoation of Asbestoses Pcdias 39. Infection 90. Susceptibility to Tuberculous Iniaciion 91. Susceptibility tc Hon--tuberculous Infection >3 xcnfl. ^te=fce>..^:k- XClf/. TA13L5S 3j *>T 32 32 32 33 31a 3li 35 35 35 35 36 3? 37 35 33 3? iiO io - 71 ABSTRACT ir-H; Asbestosis is a pulmonary disease caused iy the inhalation of asbestos dust. In animpla it' is "Characterize 1 by ^peribronchiolar fibrosis vhich s. V~ ~/ i ( seems to b^the _re. sult AC mechanical, arather than^chemical, irritation of V"* ^ u*>\ > 7^ the tissue by asbestos fibers. the long fibers produce a typical reaction; short fibers (are relative; y inert^n The filamented structure of the fibers is an essential factorAn th l. mechanism of irritation*. dt-character- /^ rJ \ istic tissue response can he produci d^by non-eiliceous (as--well as siliceous^ fibrous minerals. Inhalation of aal estos dust ^pparentlj does not alter significantly the ----------L ----------*..1---------------------------i--j_ --iine^ pigs. The^ H- {% A asbestosis body, tdiian is a specific concomitant of asbestosis and forms soon after the entrance of the asbes bos fiber into the lnng, is bUlldVM^GT prevent further -S' re to the tissue by the fiber and thus tar limit pro gression of the/reaction vhen exposure ceases. Aluminum does not exert a protective actcLon against the tissue irritation of asbestos fibers as it does age 'that of quartz particle 2. --xntroclu cfci .sbestosis is a form of pneumoconiosis resulting from prolonged i.-Jiala tion of asbestos dust. The name asbestos, literally "unburnable, :1 is not that of a particular mineral but is a term applied to a number of different ninsrals Taiose characteristic feature is a structure composed of long, par allel, flexible fibers- This structure is unique bacause the fibers are capable of repeated longitudinal subdivision to units of molecular proportions In length the .fibers vary .from a few microns to six or mors inches. Some varieties are staffer than others but many are sufficiently flexible to be spun into yam and -woven on modified textile machinery. 3- Asbestos Minerals The asbestos minerals are silicates of variable composition and belong to the serpentine and the amphibole groups. Listed belonr are the mare common varieties. Amphibole Group Anthophyllite (Kg, Fe) silicate AEC3ite (Iig3 Fe, Al) silicate Amphibole (Ca, Vg, Fe, Al, Ha, X) silicate Treanolibe (Ca, Kg) silicate Actinolite (Ca, tig, Fe) silicate Crocidolite (Na, Fe) silicate Fe silicate Chrysotile Kg silicate (hydrous) -2- The bulk of tha as.*&2wC& of corscnrca is chrycofile, 3'SgC-2S102-2IioG, vhich is mined in the Thetford region cf trie Province of Quebec. Crocido- lite and amosite are also nsec coEoiarciaHy but in Enoch smllor amounts. Chrysotile occurs as veins iu serpentine, s. mineral similar in chemical com position to chrysotile -rhich exists in massive form an\is made-uo of . microscopic fibers vritiout the parallel orientation characteristic of chrysc- tile. The massive blue bl&ck serpentire, which is smooth and soapy to the touch, is traversed by veins of fibrous cfcrysotila varying in width from a barely perceptible line to six or more inches. The fibers run across the vein and not lengthwise -with the formation. Attention is directed to the mineral brucite, jUgO-HgO, "which. is often found in the same formations -with serpentine and chrysotiln and may be fibrous in structure. It has no commercial value at present because its fibers ere not sufficiently flexible to be used in textiles but they are capable of repeated longitudinal subdivision. Unlike other asbestiform minerals, brucite is not a silicate and for this reason it has been a valuable tool in an. experimental evaluation of the action of fibrous cinerals upon lung tissue. IU_ EjCFEHlisESTAL ASBESTOSIS . ----------+ -t- T I-- For many years studies have been carried on-by the Saranac laboratory in an investigation of the cause, nature and development of asbestosis. The present re port is devoted to Experimental Asbestosis. In it are described the an-imad ex periments with various kinds of asbestos dust- Another report, to be prepared and issued later, vd.ll bo conceruse with dune;: acossuosia ane will cov the uatitl aspects of -norkers uho have been exposed to asbestos dust in an industrial environment. -3- Although csbastosie in' man is 2 chrome disease vv.ct reci.uxros years to develop, it is possible to reproduce in one or marc- species cf aninai character istic tissue etiangeo vhich are similar to the lesiens 01 human sobestasis. Since the life-span of the experimental animal is relatively alert; it is not possible to'develop the characteristic lesions in animals vrder the usual -nfcrtss"i.rial--gssditions. Consequently, to obtain a complete avaluc.ti.on ci` the tissue response to inhaled particulate and fibrous material, it is necessary to accelerate the reaction by employing higher concentrations of dust, than vrouid ordinarily be en countered in industry. V.hile conditions of exposure are thus different, the information yielded by experiments -pith animals is invaluable in furnishing a better understanding of the reaction of the human ergavnism to inhaled asbestos duat. 5. Experimental Methods For investigating the biological reaction of the experimental animal to the various asbestos dinarala, two types of technique have bson employed, namely, the inhalation method and the injection, method. In inhalation experiments, groups of animals -- up to ICO or more guinea pigs and sometimes smaller numbers of rabbits, cats, dogs, rats or mice -- ore kept for eight hours a day in a cubical dust room, eight feet in dimension, in which a cloud of asbestos dust is maintained. At intervals during the experiment, a few animals tire sacrificed and the tissue examined to determine the nature and extent of the dust reaction. Sons animal3 are exposed for periods up to three years. The infection experiments, in which the dust, either dry or suspended in fluid, is introduced into the nimal by the intravenous, intraperitoneal or intratracheal procedure, are used to determine whether or not a particular dust has a potential capacity to produce tissue reaction. -h- Long--tern inhalation experiments furnish information upon -rrtiich great reli ance is placed 'when e3tinia+<ing the degree to which a dust, might be hazardous to industrial workers- blether cr not atmospheric dust, even though potentially dangerous, can be inhaled, pass the natural defense barriers and reach tne pul monary tissue in quantities sufficient to cause damage can be determined only by inhalation procedures- Injection experiments are useful because in them contact between the dust particles and tissues is assured and the potential capacity of the dust to produce reaction can be estimated accurately. TShen dealing with fi brous minerals like asbestos, the intratracheal method is valuable since it per mits observing the effect of the fibers on pulmonary tissue. 6. Species Susceptibility Unlike free silica, asbestos does not exert its specific effect in an organa of all species of animal (Tells 1)* Injection of fine quartz into various organs of the guinea pig, rabbit, rat, cat, dog, chicken and even tadpole will produce silicotic nodules. However, similar injections of long or short fiber asbestos have resulted in a fibrous reaction in the lung and, to a lesser extent, in the peritoneum bub cot in other organs ^gS . JU^ is*-ft uAuit wismm Vt~ * - r To 7. Peculiar Characteristics of Asbestos h^t/ ****** ++-* u&I* t o#\i*>VW &**** u *** SrrotinT-/* po Experience has demonstrated that most of the particulate matter inhaled into the lungs of man and animal is 10 microns or less In maximum diameter- Larger particles apparently are excluded by the protective mechanism of the upper respir atory tract- In the case of fibrous materials, however, this restriction does not apply and fibers 1O0 and even 200 microns in length have been found in the termin al air spaces of human lungs- In small laboratory animals exposed to asbestos dust the maximum length of fiber found in the lung rarely exceeds 6o microns Not every kind of fibrous material is inhaled with equal readiness; for example. the synthetic Titers of `reel u-'pvjrently arr. roc inflsrcLble to -jess easily through the nose, pharynze, fracrea azul bronchi and seldom reach the terminal bronchioles and alveoli. Inhaled particulate matter cornea to rest thro-zgheut the terminal sir spaces (alveolar duct, atr-ia,-alveoli) in all parts of the innyze*.- inhaleS^ttabeetea--fibers are first retained in the respiratory bronchioles. These very email tubes are immediately distal to bronchioles lined by ciliated epithelium. Their otun essen tial lining is a lav cuboids! type of apitholiun but, ay their name implies, they actually function in respiration through lateral alveoli given off as pouches along their walls. Either these pouches, or v.hs abrupt change in the character of the lining epithelium, or the decrease in diameter of the tubo, or perhaps tha combination of all three factors is responsible for local retention of the inhaled fiber. Only after asbestosis is well established are appreciable numbers of fi bers carried into the more peripheral sir spaces. 8. Rate of Tissue Reaction to lsbe3tos Fibers The rate of tissue reaction to asbestos is much more rapid than to an active dust IdUce quarts. Evidences of tissue response appear as soon as fibers have localized in sufficient concentration in specific areas. In rats receiving asbes tos fibers by intratracheal injection this evidence is visible as early as two weeks after injection; for quartz dust the latent period might be two mori'ae or more. The behavior of the tissue reaction to inhaled cust after the termination cf exposure is not the same in 3ilicosi3 as in asbestosis. 7n silicosis, the young nodule8 become larger; in asbestosis, young scar tissuejthat may have formed,con tracts and becomes more dense but the area of involvement decreases in size. Xf exposure to asbestos dust is terminated after a brief period, the recently-inhaled I fibers in the lung may cause the fibrous tipuua response to continue for & short time, until the fibers have been coated. This progression is of only a slight degree and of little significance. 9. Asbestosis Bpdiea . ^ .-- _________ The peculiar structure known as the asbestosis body or curious body is a specific concomitant of asbestosis. The typical body is a golden-yellow, beaded or hanstrated rod "which nay be either straight or curved. Often one or both ends are bulbous like a dumb-bell. The bodies vary considerably in length, and di mensions up to 250 microns have been recorded. It is believed that asbestosis bodies are due to a deposit of protein and iron pigment upon the surface of inhaled fibers. In guinea pigs they form after about 60 days of contact "with the tissue. They are abundant in man and the guinea pig (see Table l) but are much larger in the farmer, probably because the larger- sized sir tubes admit fibers of greater dimension. In cats, rabbits and mice there is an atypical coating of a few of the fibers after much longer residence in the lungs. In ruts and dogs no bodies could be discovered. Although the evidence Is incomplete, it appears that the formation of the asbestosis body prevents dsnege X, INHALATION EXPERIMENTS i I- 1 s - v -. -w * -\ W' Four comprehensive inhalation experiments have been conducted at the Saranac Laboratory with various forms of asbestos duet. In each of these investigations more than 160 animals -were used and the experiments were carried on far periods ranging from 2 to mare than 5 years. The four kinds of asbestos dust employed are identified as King's floats, short fiber, 100 per cent ball-milled, and long-fiber asbestos dust. rJtf'iLji'tr'-i-t Etrpqrirq^.t Td-th^ "King ^ Flpat^s" ^A3ber-to3 ^Diia^t The .first inhalation experiment conducted at the Saranac laboratory with asbestos dust -was begun in 1928. Animals inhaled, the dust for periods up to nearly three years and some guinea pigs lived for about four years after their first exposure to dust. A praliniDary report giving observations after 29 months of exposure appeared in the February, 1931 issue of THE JOtRNAL OF INDU STRIAL HYGIENE.-* At that time observations covered a period of only 2~l/h years and the conclusions as to the ultimate effects of inhaled asbestos duet were provisional. Results of the completed study shot- that most of the conclusions drawn in the preliminary report -wore substantiated. A complete review of this experiment follows. 12. The dusting material -was a commercial variety of asbestos dust known as King's floats and was composed of short fibers and particles of variable size. It was obtained from the Thetford, Quebec plant of the Asbestos Corporation of America. 13. The dust composition (Table 2) reveals that the amount of fibrous chryso-- tile was only lit per cent, a rather low value- However, there was suf ficient fibrous material to produce a characteristic fihrosis. ill. The dust concentration at first was quite low and for impinger samples taken soon after the experiment was started, the average light field count-- by the standard technique was only 6.0 million particles per cubic foot of air. An appreciable number of large particles (or fibers) also were present, as -^STUDIES ON EXPERIMENTAL PNEUMOCONIOSIS. VI. Inhalation of Asbestos Dust., Gardner, L.U., and Cunmrings, D.E. J. Ind. Hyg., 13: 6$-81, 97--Uii, 1931. -8- ahcrnn by an average corat of c-.d raiiiioc for psrticj.es greater than 10 morons. After the inhalation experiment had been under wsy for about. two years, the spaed of tha rotating paddle in the dusting machine r-as increased and for the remain ing 9 or lO months of tha experiment considerably rioro dust -sras dispersed into the-atmosphere. Average-dust counts for impinger samples 'collect'd?! 'fter''tYrIs change were 53-7 million for the usual light--field method, and 1-6 million for particles larger than 10 microns. 15. Reaction in Animals to Inhaled "King's Floats * Asbestos Dust. Results of the investigations briefly summarized in Table 3, ohow that inhala tion of King's float3 asbestos dust produced a typical peribronchiolar fibrosis in guinea pigs but not in rabbits or rats. 16. Guinea Pigs. Seven groups of guinea pigs -were used. In three groups the effect of a continuous and of an interrupted dust exposure -was studied* in two other groups the relationship between infection and dust exposure whs investigated. The remaining two groups were infection controls. 17. Rate and Type of Reaction. Guinea pigs inhaling this dust for periods up to 33 months developed a characteristic fibrosis occurring in conical patches about the respiratory bronchioles- During this exposure the peripheral alveoli were not involved. The particulate elementshin the dust were transported to the lymphatic system where they caused no signi ficant reaction; the fibrous elements remained fixed at the site of original localisation and -were seldom detected in the lymphoid tissue. Pleurisy and fi brosis in the septa were observed only when Infection complicated the process. After exposure of approximately a year, a small asount of cellular reaction had been produced about many respiratory bronchioles. As more dust was inhaled, it continued to accumulate in the Same location and later stages of the disease . s^T* consisted of extensions cf ths original lesions- Ra-v craas were not involved. Apparently, the inhaled fibers '-'ere caught in ths poeket-liica alveoli that are given off from the Inters! nails of the respiratory bronchioles. There they wore phagocytized and many of them were carried into the nail by migratory ceils, itonorraoiear leucocytes attracted to the area caused an appraci^Xe^EHicEbffing- of the bronchial call. After 16 months a delicate fibrosis made its appearance. The process evolved 60 gradually that mitotic division of fibroblasts could rarely be discovered; nevertheless, the number of fine intercellular collagenous fibers (fibrosis) steadily increased. As this fibrosis contracted, it partially cloaed the alveoli, and with this atelectasis the lining epithelium assumed its embryonic cuboicel form. The result was the adenoma--like appearance that hillis described in guinea pigs inhaling silicon carbide. The longer exposures resulted only in more thickening of the walls of the air spaces, largely due to an in crease in the amount of fibrosis- The fibrous tissue always remained cellular and never shewed the hyalinization characteristic of silicosis. 18. Progression. The reaction produced in exposed guinea pigs did not pro gress significantly during a subsequent period of 3? months when the animals lived in a normal atmosphere. Between 8 and 11 months after exposure ceased, the cellular reaction had been completely replaced by thin strands of fibrous tissue. Observed still longer, the scar tissue decreased in amount but in the last animal sacrificed, 37 months after discontinuing dust exposure, some fibrosis was 3till visible. 19. Infection coincident with Oust- Inhalation. Of the group of 1*0 guinea pigs infected with atten uated tubercle bacilli (R^ strain) 31 died or were sacrificed before two years of dust exposure and were reported in the paper by Gardner and Cummings mentioned above- Seventeen of these died from intarcurrent pneumonia- Briefly, the re sults were as follows: 10 revealed some evidence of spread of the tuberculous urocess; in 6 of these it raa confined to tha lungs and in the other k the abdominal viscera also were involved. Usually a slight local extension of the tuberculous infection--had occurred but subsequent healing hac-'feseitaed""in"`fi> brosis of both ths pulmonary lesions and the secondary lesions in other organsThe healed pulmonary lesions shoved more fibrosis than is characteristic of either tuberculosis or osbestosis alone - The 9 mimala -sciich -were still alive after two years of dust exposure were sacrificed at intervals during the following year* In U of them the primary foci of infection had healed with fibrosis and even calcification and there was no evidence of progression- In the other 9 the tuberculous foci showed evidence of having previously spread locally: in it of them it had healed3 by tha time of autopsy, with excessive fibrosis; in the other animal there was a generalized chronic tuberculous pneumonia in one lobe and isolated primary tubercles, which were still active but had not spread, in the other lobes* Evidence of extension of the infection was first seen after 7 months of dust inhalation; during the next 20 months more than half of the animals showed an actively spreading tuberculosis and in 3 of them small cavities had developed. During the last 9 months no animals exhibited ary evidence of active infection although in half of them the healed fibrous scars of previous extensions were ob vious. Sixty per cent of the guinea pigs with spreading pulmonary tuberculosis showed tuberculosis of the spleen and liver. 20. Infection S uperimposed Qpon an Established inbeotosis. Twelve guinea pigs, after in baling asbestos dust for nearly 26 months, ware infected with tubercle bacilli and then removed to normal air- The subpleural tubercles in tha dusted animals were -11- no aore.nuwrous than in ncn-dnsted controls; b.vt a considerablp. number T?ere found in the depths of the lung about foci of asbestosis. The reaction to infec tion showed only slight local extension, about the original aiton in the lungs and tracheobronchial lymph nodes. The abdominal viscera none involved in only one animal. Caseation was founa in tubercles 1-1/2 months old' but by 5-1/2 months it had conplstel" rlf.rrr.rj'i-o'U'1.. s.~".r tis.-- - '" t; ' r-.ttor still persisted In the last animal, -which -ras killed 1L iaonths al'ter infection. 21. Asbestosis Bodies. Moderate numbers cf asbestosis bodies occurred in. the lungs of the guins-a pigs., becoming more numerous and more distinctly- segmented in later months - 22. Rabbits. Rabbits exposed to the asbestos dust for periods up to 19 month* developed a low-grade foreign-body type cf reaction but no fibrosis. Although their lungs contained particulate elements of the dust, fibers were not present, indicating that the upper respiratory mechanism of the rabbit is adequate to exclude fibrous foreign bodies. Tco rabbits, after inhaling dust for 6 and 18 months, lived in nonoal air for more than too years. At autopsy neither animni showed any evidence of cellular reaction or fibrosis in the term inal bronchioles nor were there ary asbestosis bodies. 23- Rata. All the white rats had acquired an infection; resulting in. the formation of pulmonary abscesses, before they came to autopsy. Apparently, so much heavy mucus obstructed their bronchi that very few fibers could have entered their lungs. In a few of the rats, an occasional asbestosis body was discovered but there was no fibrosis. -12- 21. Smeary and Interpretation of Inlialailon Experiment with King *s I^loata Dust. The findinga in "the exnerincnt with Sing's floats dust fan be summar ized under three headings. A. Effect of the inhaled dust on normal animals. Ths King's floats dust caused a charac- teristic peribronchiolar fibrosis in guinea pigs but not in rabbits or rats. The fibrosis did rot progress after the dust exposure T?as discontinued and the guinea pigs transferred to normal air. B. Effect of the inhaled dust on tuberculosis in guinea pigs. In guinea pigs in fected with attenuated tubercle bacilli and then placed in the dust roam, the results were more variable than is usual in an experiment of this type. A few aniron showed no sign of ^progression; in most of them there sas evidence of tem porary progression with subsequent healing; in one animal there was continuous progression to death. In contrast, when guinea pigs, after being infected, are exposed to quarts instead of asbestos dust, the infectious process continues to progress and eventually causes the death of the animals. On the other hand, ex posure of Infected animals to a harmless dust like calcite or gypsum does not lead to ary progression of the infection. Guinea pigs infected with attenuated tubercle bacilli following the termination of about two years ' exposure to asbestos dust did not develop progressive disease. The only modification of the infection was in its localization, a few bacilli being retained in the fibrous terminal bronchioles and forming tubercles there in addition to the usual foci beneath the pleura. ' In view of this variability, the unusual nature of the response and the high proportion of deaths from intercurrent pneumonia it is felt that definite concluoions rs to the influence of this--dust on the course of tuberculous infection are not justified. / when tha particles are 3 microns and less in size, an inhalation experiment "was carried on to determine whether this condition in true also Tor asbestos dust. It was thought that toy using a short--fiber asbestos dust consisting almost entirely of fibers and particles smaller than 3 microns an accelerated tissue response might be initiated and an advanced reaction obtained in a short time- The previous inhalation experiment with King's floats asbestos, which contained fibers from 1 Tnm- to 1 micron or less in length as well as a great deal of particulate matter aiid which produced a typical peribronchiolar fibrosis in exposed guinea pigs, served as a basis of comparison. 26. The dusting material for this experiment was forwarded from the KaxxvfHe plant of the Jbfans-lfanvUla Corporation. It was the remains of fibers collected in dust bins after a carding operation and screened to pass 200 mesh. Since the material as received contained many long fibers, it was ground in a steel ball Tiril i to reduce practically all the particles to 3 microns, or less in size. ^Khen used alone in a standard dusting machine, this finely-ground asbestos tended, to pack in the hopper and it became necessary to mix one volume of the unground material with three volumes of the ground to generate a satisfactory du3t cloud. The addition of the smell quantity of ungronnd asbestos was unfortunate because it confused the interpretation of results. Probably the minor amount of reaction that developed was due to the long fibers in the mixture although the data or this experiment do not prove tho point. 27. The composition of the short-fiber asbestos as received is disclosed by the chemical and petrographic analyses given in Table ii. Samples taken before and after grinding yielded about the same values ..^n. analyses.^, indi anting that there was no contamination from the mill or los3 ox vrater content. 28. The dust concentration varied somewhat during the experiment and light field counts for atmospheric samples collected inside the animal cages with the impinger apparatus ranged from 83 million to 182 million. The average of counts TO3 130 million for the first year of the experiment, 13U million for the second year and lliO million for the third year- 29. Siae--frequency measurements of air-floated dust from Inside the cages at a magnification of 130QX revealed a great preponderance of fine par ticles (Table 5). Nearly 90 per cent of the particles seen were smaller than 3 microns. 30. Reaction An Animals to Inhaled Short-Fiber Asbestos Dust. Four species of ftn-tmalw -- guinea pigs, shite rats, cate and rabbits -- sere used In this experiment. The results of the dust exposure, which are summarized in Table 6, will be considered more in detail below. 31. Gtrippa Pigs. Eighty guinea pigs were originally placed in the dust room but 21 of them were Inter eliminated from the experiment a-pd killed because of enlarged lymph nodes. Of the other 59 animals, li6 remained in the dust room until they were sacrificed or died from natural cesses and 13, after being exposed to dust for 20 months, were transferred to a normal atmosphere. 32. Rate and Type of Reaction. The type of tissue reaction to the inhaled shcri--fiber asbestos '.ais ess-s-ivtix.ilr the saca s-3 that already observed In the experiment with King's -floats asbestos. The rate 01 reaction also was approxi mately the aasas but the extent of involvement- trith the short--fiber dust was very much less and after 2.6 to 2L months of e:cpo3ure only & very few small foci of 'reaction, which generally required microscopic examination for^detoctibnj~wer3 produced in the guinea pigs. Until exposures had continued for approximately on year, there was little tendency for dust--containing phagocytes to collect into clumps. By 16 months phagocytes had begun to collect about the walls of a few of the respiratory bron chioles with a little proliferation cr infiltration of mononuclear cells in these walls. There were also some uniltinucleated cells but they were always of the inert foreign-body type. At 2C to 2k months the cellular clumps were sometimes quite marked and sometimes changes in the epithelium resulted in the adenoma--like or "adenomatoid" appearance previously described in Section 17. In most of the subsequent members of the series, the reaction remained cellular in type. In a few, however, fibrous elements dominated the picture. In the latter case, the collagen was pale in color and tenuous with no heavy swollen hyali ni aatdon. As in the rats described below, the alveolar walls night be made up of a band of collagen supporting a layer of epithelium, but with no contained capillaries. In the tracheobronchial lymph nodes the reaction was more pronounced in this experi ment than in the previous one with King's floats asbestos, probably because of the transportation of an excess of fine particles to the nodes in animals inhaling short--fiber asbestos. The reaction wa3 essentially an increase in reticulum, rather than a fibrosis, with preservation cf the original cells between the thickened reticular fibers. Diffuse chronic pleurisy -without evidence of pulmonary infection was present in a few animals. -16- 33- Progression. In the 13-3/U months folios!ng the cessation of 20 months1 exyosurff to dust, progression of disease ras not definitely demonstrated but neither could it be absolutely disproved, owing to the varia bility of the response in different animals. At the end of the dust exposure of 20 months two pigs were read as * and one as 2-*-. Among the" 13 removed 'from duct the findings were variable: in 2 the reaction was x: in h it war- *-, in 3 it was 3-'-; In 3 it was k+; and In one animal it was 5*. It 3 quite possible that those with the most marked changes had already developed more reaction than the remain der by the time exposure ceased. Since the more severe reactions occurred spora dically and bore no relationship to the length of time after cessation of expo sure the differences were attributed to variation in individual susceptibility. This view received support from the chemical analyses (Table 7), which often re vealed comparable amounts of ash and silica in lungs with widely different amounts of tissue change. For example, the ash and silica values were quite similar for three animals in dust 20 months and then in normal air 13-3/lt months, yet the tis sue reaction for one animal was U-*-; for another, <; and for the third, only . 3It. Asbestosia Bodies. The formation of asbestosia bodies was at first ex tremely limited. After 5> months5 exposure only a very rare short body could be found, usually inside of cells. Around the finest intracellular particles there were yellow deposits having the same color as the asbestosio body. Exposure of one year had permitted an accumulation of many longer fibers about which the asbestosis--body coating developed. Ifost of these were still short enough to be partially or entirely within phagocytic cells. By the 20th month and thereafter, they were comparatively numerous although still rare in comparison with the findings in the King*3 floats experiment. *7_ 3?- Tvhite Rats. Seventy--three rrhita rcio ware exposed to atmospheric short- fiboi* asbestos dust for periods up to 32 months. Saeri-- ficings during the first 10 straths -sere r&de bimonthly s/ad for the remainder of the.eocperl sent at less frequent ir.tprvsls. . .. . --, .....-- 36. Rate and Type of Reaction. The dust cells until S months were widely scattered and existed in foci only sporadi cally. Reaction was limited to occasional slight thickenings of the septa about. small accifluulations of dust cells. In a few rats at IO months, there -was a sug gestion of early fibrosis but ilia change was so slight that it would probably be overlooked -without the clump of dust cells to attract attention to the area. Only 10 animals were exposed from 12 to 32 months. In each of them the lungs showed minute patches of -Bell-defined fibrosis distributed like that of asbestosis but -without asbestosis bodies. The lesions, -risible only at a magnification of ISO diameters or more, consisted of patches along alveolar ducts in which the walls of the air spaces were very thick:, due to swollen collagen framework. Connective tissue rri Foot--Bielschowski silver preparations revealed complete loss of capil lary bed locally. Outside the collagen was a thin layer of epithelial oalls. This d5d not resentile the "adenomatoid" change characteristic of guinea pig as-- bestosia. So pleurisy was present. Hear the lesions the air spaces were filled with, phagocytes containing grey to yellow particulate dust and a rare long naked asbestos fiber- Careful search failed to reveal even a suggestion of an asbesto-- sis body. The tracheobronchial nodes showed compact focal collections of mono cytic cells at 12 months and, at 20 months, some diffuse thickening of the reticulum. In a few rats there was definite fibrosis along the margins of the node extending into the mediastinal areolar tissue. Cospared with the response to active dusts like quartz and chert the reaction to short--fiber asbestos was negligible- Results of chentLcal analyser r.yle on. h>e T2u.be rate- 572 given in Tables 3 and the average vaiue3 have been tabulated ir. Table f' for comparison -with similar values fbr rats inhaling other dusts. The concentration of atrs)spheric particles to which the animals 'srore exposed -was approximately tha same for asbestos and quarts; for the gypsuni-qoartz rirfcxrre; it was about tvics an higft' ajicPfor'chert'' five tines as high- It roll be noted that the percentages for asbestos are lower than those for quanta or chert but -are similar be those for the gypaim-quarta mixture, in which atmospheric agglutination bonded to reduce the astonne of dust inhaled. It night be inferred that the total quantity- of asbestos dust inhale^ was low or that it had been eliminated from or diseolvsd -within the lungs. In the present state of our knowledge evaluation of thssa hypotheses is not possible. 37. Cats. Twenty cats -wars used in this inhalation experiment -with the short-fiber asbestos. Eighteen -sere kept in the dust room until death, the exposure period ranging from cue month to nearly ii-1/2 years, and two, after a dust exposure of 31-1/2 months, were removed to normal air. One of these -was sacrificed 5 months, and the other 2ii months, later. 38. Rate and Type of Reaction, The reaction -was essentially that to an inert dust, even after more than li years of expo sure. The tissue response in this species -was confined to microscopic foci of fibrosis in the walls of groups of subpleural alveoli, rather than in the peri bronchiolar areas. In one animal the change -was extensive enough to be visualized on gross inspection of the section. 39. X-Ray Changes. Only in the animal -with the longest exposure did the X-rgr Tvrvaal definitely abnormal shadows. After 29--3fh month6 the picture ws negative; after It5 months a faint mottling could be detected throughout both lungs. At autopsy,. 3 soi'-t.hs later, there r,as only microscopic fibrosis in ibc rri.bcia'irsi ZZOT*? brcnchioles . iyvrohocry-tic inflltrati-r.n ebcut. rr.c-ii tiO. Acbestocia Rodigs. On prolonged search a fee? velicv atypical asfeestoois - .-- bedies, a mootii and without bn ttro animals exposed for more than a year. 1x1. Rabbits - Sight rabbits -were exposed to dust for periods extending from one to cora than fire years. The last eni&al rras rswred from the dust room and loft in normal air 6 months before being sacrificed. U2*. Rate and Type of Reaction. There was never enough fibrosis to be de tected grossly and there was no chronic adhesive pleurisy. Microscopic evidence of alveolar vail thickening vra3 first detected after about 3 years of exposure and was seen in all five animals ereaniied thereafter- In one animal that died of paralysis after nearly four years of ex posure the reaction was extensive enough to be visible on gross inspection of tissue nections. The possibility of pulmonary infection in this animal could not be excluded. However, in another animal dying two years later the focal fibrosis was not nearly as obvious or as advanced- Areas of invdvement, which were largely visualised because of phagocytic reaction within the sir spaces, tended microscopically to become more fibrous with the passage of time but there was never much encroachment upon the lumen of air spaces and the architecture of the lung was prese'ved- 1x3. Asbestosi3 Bodies. Asbestos bodies were not detected in rabbits that died early in the experiment but were seen in all animals that had been exposed to the dust for more than three years. iiii - ?n~nrjry and Irtierp'reta.th.on. Tba Oi'icp.'ns.l. purpose or t-hs e.'cparimeni S33 to evaluate the chemical theory or the pathogenesis of esbeetosis. it vas felt that if the tissue ras.oiion to asbestos -sere chemical in origin an accelerated or accentuated response would resiilt Traza exposure to finely-divided asbestos, es is the case with quarts. This experiment, in vSiich the reaction vns elccsr and less extensive than with King's floats, indicates that the reaction QjrotsSbl^ is not primarily cheaical in 1/ nature. Of the Tour speciea exposed, in this experiment only the guinea pig and rat reacted with chsractsristic peribronchiolar fibrosis. The cat reacted with atypical sub-plsural fibrosis and in the rabbit the fibrosis which occurred could not be positively attributed to the dust because of a strong possibility of pulmonary Infection. XUV^l^t*UU4T'GsM- 1" ' TnhaTp.tion l^perdm^it^-wl'^h IgO P^r Cen^ BaX^-lil^ed Aa^estoa^Dust In the inhalation exparinent with short-fiber asbestos dust a small quantity of unground asbestos was nixed with ground material in order to produce a suitable dust cloud, then evidence of a dust reaction appeared in the guinea pigs during the experiment, it was not dear whether this was a tissue response to tits smaH number of 1f>ng fibers in the onground asbestos or was a delayed effect of the more abundant fine dust. Consequently, another inhalation experiment was started, in which no unground material was used. 1*6. The dusting material was the ground short-fiber asbestos used in the previous inhalation experiment but no unground material was mixed with it. To obtain a sufficient amount of atmospheric dust, the design of the dusting apparatus was changed to sn opsn type of hopper and fresh dust was added daily. Owing to the cendsncy of the iratet-i* i ;.~ i'-oi-r.i spherules 'rV.ici rrcvented much of the fibrous portion fron freer.ir..~ out of the hopper, the -dispersal of the dust tvas not entirely oatisfactory and efter ? months of operation. the dust ing machine TTo.s reconverted to its criminal design. To prevent "pilling" or the formation of spherules of asbestos, steel nj.re brushas rare attached to the in side surface of the hopper and to the rotating paddle - '"'bis e~'STfanient gave satisfactory results and was usee, for the remaining J. months of the experiment. hi. The composicier of the ~'3& matarial (short--fiber asbestos) and cf atmos pheric dust liberated from the ball-uLiled product in the dusting ma chine is given in Table 10. These values are based upon petrographic study and X-ray diffraction analysis. The acmesphoric sample vac collected with an elec trostatic precipitator after vine brushes had been installed in the dusting machine. Previous to this- the chrysotile content of the air-suspended material was undoubtedly less than the 15 per cent value given in Table 10. In an inter im report, it was stated that the air-borne dust contained about 5 per cent of chrysotile before the wire brushes isere used and up to 6 per cent afterwards, but these values were probably low. Quantitative estimates on ball--milled asbestos dust may be somewhat inaccurate because it is difficult to determine heir much of a dust sample is fibrous chrysotile and hertr much is non--fibrous serpentine. IiB. The dust concentration for the first 7 months of the experiment was about 100 mjLllion particles per cubic foot of air. After the wire brushes had been installed, the dust counts pore a little higher and the overall average for the first year was 106 million. The average of counts for the second year vas 163 million and for the third year 1L5 million. h.9. The size--froq?iegey of the components c.X .atx-esphsric bust collected hasIdo the animal ca^es Tilth the electrostatic apparatus is reported in Table 11. TVo samples x/ere takenr one before the vrire brashes trere installed and one after. It will be noted that after the "Hire brushes were in use a greater proportion of very fine particles and also of loiigar fibers was released into the air. SJO- Reaction in Animals to Inhaled ICO Per Cent Ball -Billed Asbestos gust. Guinea pigs, rats and nice were used in the inhalation experiment with the 100 per cent bail-milled asbestos dust. The results are summarized in Table 12. 51. Guinna Plga. The experiment teas started with 100 guinea pigs. As the dust exposure proceeded, there were 39 accidental deaths, 32 of pneumonia in an epidemic. After 28 months of dusting the 16 surviving guinea pigs were transferred to normal air. 52. Rate and Type of Reaction. For the first year of exposure practically the only reaction to the dust was the pre sence of scattered phagocytes and an occasional minute asbestosis body. At 16 and 20 months no gross response was visible on the tissue section but microscopically peribronchiolar foci of inflammatory cells could be seen. At 2k months there was still no change large enough to be seen with a hand lens although microscopic examination revealed cellular accumulations about terminal bronchioles and many more asbestosis bodies, chiefly within cells. Chemical analyses of the lungs (Table 13} reveal that in spite of the limited tissue reaction considerable dust had been retained in the lung. ''v -* 53- Progression. The lungs of aninalc exposed for the Tull dusting period (26 months) and than living in normal air for 2 months revealed the changes described above and also very slight peribronchiolar fibro sis- After 8 raontlis in normal air the findings mere similar but at 12 months 3 of ii animals showed gros3ly-visible characteristic peribronchiolar fibrosis with adenomatoid change. 5U- Lymph Bode Involvement - The tracheobronchial nodes were essentially negative until exposure had been continued for more than a year and a half. Animals sacrificed at 12 months and 16 months revealed a few minute collections of phagocytes containing particles hut prac tically no fibers large enough to be recognized as such. After 20 months of exposure many monocytes filled with yellow granules were present. At JO months there had been a slight increase in reticulum hut no fibrosis. No further changes occurred in the nodes. Asbestosis bodies were not seen in the nodes of any of the guinea pigs. 55. Asbestosis Bodies. Minute asbestosis bodies were observed as early as 3 months after exposure began, but they did not be come numerous until 16 months had elapsed. The bodies were short and practically all wore intracellular, although at 20 months some were long enough to project beyond the cell borders. It is important to note that in the later months of exposure there was a dis tinct increase in the number of long fibers (ftp to 70 microns in length) in the lungs and that after exposure ceased characteristic long asbestosis bodies were seen. 56. White Rats and Mice. In this experiment h0 rats were exposed for periods up to 20 months and 2h mice for periods up to 12 months. Neither species developed even a suggestion of asbestosis and reaction, was limited to phagocytosis of iXihaled particles fay -widely-scattered dust cells which remained free in air spaces or -were transported to the tracheobronchial lymph nodes. Mo asbestosi-s bodies were found in the rats but in the mice there were a very few small non-haustrated forms 'aithin phagocytes. - In 21 mouse lungs sectioned there were 3 instances of pulmonary adenoma Qli). 57. Summary ana Interpretation. The tissue reactions observed in this experiment -ware much less extensive and slower in development than in. the previous investigation with, short-fiber asbestos. Since presumably there were fewer fibers longer than 3 microns in the material used in this experiment, the results tend to confirm the interpretation made in Section iUi of the short-fiber experiment that the reaction fieMNy is not primarily chemical in nature. The finding of long asbestosis bodies in animals inhaling the ball--milled material is an example of the difficulty of completely eliminating long fibers from an asbestos preparation. In regard to progression of reaction after removal from dust, liilch was ob served in this experiment but not in the others, the following interpretation is offered: T?hen the reaction is well-developed at the termination of exposure,the contraction of the fibrous tissue would obscure any possible progression. In this experiment, however, since only the earliest stage of reaction was present at the time of removal from dust, its subsequent progress was apparent. It should be noted that the degree of progression was so slight that it can have little, if any, practical significance. K yitt sfa u m O' Xn^lat^or^gcporlmant ld.th bong^f l^ t Di'pt. After animals inhaling shcr^>--fiber asbesios dust for more than a year had failed to develop significant reaction, the hypothesis that asbestosis is pro duced by the mechanical irritation of long fibers was given added support. Since the King's floats asbestos used in the first inhalation experiment had a rather low content of fibrous clirysotHa and contained considerable serpentine and other impurities, it was decided to conduct a new inhalation experiment with a purer form of chrysotile which would be richer in long fibers. 59- The dusting material employed in this investigation was obtained from the Manville plant of the lohns-Jfenville Corporation. Samples of se veral varieties of asbestos dust were first submitted to the Saranac laboratory for examination and one kind, identified as Z<ot D, which was low in magnetite and chromite and bad a fibrous content estimated to be about 75 per cent, was selected as most suitable. Steel wire brushes were fastened to the inside sur face of the hopper and to the rotating paddle in order to open up the bundles of asbestos and liberate more fibers into the atmosphere. 60. The composition of the long--fiber asbestos used in this experiment is indicated by the chemical and petrographic analyses given in Table lU. It appears that this material was a much purer form of asbestos than the shortfiber dust used in other experiments. This is borne out by comparing the approx imate analyses of the long--fiber and short--fiber idust in Table 15- i 61. The dust concentration as revealed by impinger samples taken inside the animal cages was much lower than the concentration for the experiments with short--fiber or ball-milled dust. For the first year of the experiment with i long-fiber asbestos the average of the light field counts was 32 million; for r the second year, ii8 million; i'cr the third year, 31' million? v-nc for the foi:rch year, Li3 million. Examination of the impinger samplea 7>itn ciark field illumin-- ation disclosed that many fine particlea less than one micron in size accompanied the larger particles and dark field counts -were, on the average, about 5 or 6 times larger than the Eight field counts. ' c `"" ' ' --------- 62. The sine--frequency of aticospheric samples of the long-fiber asbestos dust and of the ball-nil? od dust is 3horui in Table 16. Both samples were collected with the electrostatic precipitator. It vd.Il be noted that there was far more fibrous material in the long-fiber dust. 63. Reaction in Animals to Inhaled Long--Fiber Asbestos Dust. Guinea pigs, cats, rats end mice were employed in the inhalation experiment vd.th long-fiber asbestos. Results of the experiment, summarized in Table 17, are described in greater detail below. 6i. Guinea figs. The experiment was started with 10O guinea pigs. After exposure had been carried on for a year, a severe epidemic of pneumonia arose in the dust room and about one--third of the animal a died or Here killed. To replace them, 38 more guinea pigs were added to the surviving group in the. dust room. 6^. Hate and Type of Reaction. Histological examination revealed grossly visible lesions in the lungs after 6 months of exposure to dust, consisting of cellular infiltration about the terminal bronchioles. At 12 months, there were adenomatoid changes in the air spaces and by the 16th month a definite fibrosis was present in these areas in half the anisals. The fibrous lesion could be seen macro3copcaULy at 20 months. Prom this time on the reaction increased in- extent and in the amount of collagen and j;r BOtith, it h&i out into the carencfctT&. The lesions ^re rather eharply localised and the extensions from diffarc-nt- bronchioles showed no tendency to lass, even ir. animals exposed Tor the nairinmn period (3 years). Although the intra--ptfLmonary reaction sonatiraes reached the pleura^. there was no involvement of" tliat membrane - No emphysema was visible at any point. Some thickening oi' the larger bronchi 'frith a chronic inflaunatory infiltration etas re vealed, but it probably was no mors than would be produced by a similar exposure to any dust. For the first $ months the phagocytes consisted of monocytes or very snail giant cells: later, giant cell formation was more prominent. After 16 months the giant cells were large, filled with ye7\bowish-hraan pigment and sometimes vacuolated. An occasional animal showed an admixture of polymorpho nuclear leukocytes ana, in guinea pigs exposed for a considerable period, eosinophiles. The reaction was at first entirely cellular but by- 16 months fi brous tissue fomation was definite. However, it never attained a stage of byalinisation suggestive of silicosis. A moderate individual variation occurred among the exposed ai Inals, both in the rate of developing lesions and in the stage of development attained at the end of exposure. Analyses of the lungs (Table 18) disclosed that although the tissue response was much greater in these guinea pigs than in those exposed to either short--fiber or ball-milled asbestos, the amount of mineral matter in the lung ash was l^ee. 66, Progression. In guinea pigs exposed to the dust for 20 months and than removed to normal air, there was a marked tendency for cellular inflammatory reaction to clear. This effect, accompanied by contraction of the fibrous tissue, resulted in a diminishing size of the focal lesions. None of these animals, killed at- various periods -rp i-c Hi Tuon tls after exposure, revealed lesions as large as those in she group sacrificed at the _enc of the 20-month exposure period or those in animals which regained in the dust room for more than 20 months- Fourteen norths after dust exposure cessed- the foci in four of the six remaining guinea pigs were so small that they were visible only Tri-th a hand lens. Reaction in the group exposed for 27 months and then transferred to a normal atmosphere was quite similar to the response in the 20-ccr.th exposure animals mentioned above. However., small foci were always visible on gross inspection of sections of all guinea pig3 of ths 27-month series but in no instance was there evidence of extension of the reaction. 67. lymph Rode Involvement. Reaction in the tracheobronchial lymph nodes was first visible at the third month of ex posure. At the 6th month patches of cellular connective tissue began to appear in the medulla and by the liith month most of the node had been replaced by cellular connective tissue. This picture, which resembled that in early silicosis, persisted to the end of the experiment. Some animals, as a variant, showed heavy sheets of diffuse monocyt3 and large active giant cells but there was never any necrosis or hyaline formation. The spindle-shaped new cells were yellowish in color from fine pigment granules that stained for iron. Ho fibers or asbestosis bodies were 6sen. 68. A3be3toais 3odie3. Although asbestosis bodies were seen as early as one month after exposure began, they were rare and hard to find. At 5 months more were visible, chiefly coiled inside giant cells, and at 8 months many bodies wore free In connective tissue. They became fairly abundant as exposure progreoeed although in some later animals the asbestosis bodies were only moderately numerous- S?. CCaattss. lour cats totaled the long--fibsr asbestos dust i"or periods ox III. 25, 33 and U2 months, respectively, and were immediately sacrificed- Tto ether cate, after being exposed to dust for 16 months, lived in a normal atmosphere for a:a additional 21* months. 70. Rate and Tyne of Reaction. Exposure for li* months was sufficient to produce cellular accumulations of phagocytes around terminal bronchioles and peripheral arterioles together with compact col lections of similar cells in the tracheobronchial lymph nodes. At that time there were no typical asbestosis bodies, but smooth pointed yellow fibers were seen very rarely. With continued exposure, up to k2 months, reaction in the locations noted progressed to the formation of cellular connective tissue which made well-defined sheaths about the respiratory bronchioles and arterioles, marked lymphoid hyper plasia and lymphoid infiltration of bronchiolar walls. The bronchiolar epith elium was low and flattened, giving the tubes a smooth contour. Typical asbesto sis bodies were not formed although there was an occasional yellow, smooth, pointed fiber. No pleurisy was present. The reaction was similar in location to that in the guinea pigs, but fibrosis was much slower in development and had not reached the same degree of maturity. (j_. 1--ttay Changes. Roentgenograms of three cats were made after exposure periods of 2S', 33 and li2 months, but tissue changes were not dense enough to be seen on an X--rsy film. 72. Rats. Although 20 rats wore placed in the dust room, many died from pneu monia and were not suitable for study. Five animals, of which one was exposed for 19 months and four for 2 months, ware free from pulmonary infec tion and offer a basis for conclusions. 73- Rats end Tyos or Reaction. All four animals sacrificed at 85 months shewed a well--narked peribronchiolar fibrosis. In. the 19-month animal, reaction was iust beginning. Asbestosis bodies were practically absent at both 19 and 25 maths althcragh two small smooth, bodies were fdtad in the 19-month animal ax ter a lorirr; search. Thus,-these ..animals exhibited fibrosis without asbestosis bodies. 7h- Hice. Oat of 20 white mice U33d in this experiment, 11 lived a year or acre in dust and died or were killed without showing an appre ciable degree of pulmonary infection. 75 Rate and Type of Reaction. Reaction was limited to phagocytosis by mononuclear cells. Usually these were wide ly scattered through the air spaces; a limited number were grouped about the ter minal bronchioles producing some thickening of their walls. There was no sug- 1 gestion of fibre ais. fThU^kr-iking "fosjxtJr^ of the experimerft was that 9 c/it of the fee (8^per cent)j exposed fojf a year or mare flowed pulaonaj^y- tumors, (Gaily a/enomatous in /ype*. These lesions did not contain dust or asbestosis Numerous asbestosis bodies were observed in animals killed late in the experi ment. Thus, these animals exhibited asbestosis bodies without fibrosis. 76. Suanaxy and Interpretation. The purpose of this experiment was to evaluate the importance of long fibers in the tissue response to inhaled asbestos. The results indicate strongly that long fibers are chiefly responsible for the reaction. Thus, in guinea pigs reaction developed earlier and became more extensive than in previous experiments in spite of a smaller concentration of atmospheric dust and a lower mineral con tent in the lungs. Furthermore, a typical peribronchiolar fibrosis was produced in cats although in a previous experiment/with abort--fiber dust it did not develop in this species- The cause of the cellular fibrosis,in the lymph node3 of the guinea pigs is not clear. It did not occur in other inhalation experiments with asbestos. In order to determine to what extent the various fibrous minerals possess the' capacity to produce tissue damage, numerous injection experiments were per formed. In these experiments guinea pigs and rabbits were used and the mineral dust was injected by the intratracheal, intraperitoneal and intravenous tech niques. For the purpose of simplification the findings in each series of testa have been condensed end reported in tables, to which reference will be made later. 78. Experiments Using Intratracheal Technique. Since the asbestos minerals do not cause a typical advanced fibrosis in extra-pulmonary tissue, the intratracheal technique is the perferred way of in troducing fibrous dust into the experimental animal. In this method thd dust suspension is injected by means of a special needle or catheter deep into the trachea, from which it flows into the lungs. 79. Comparison of Fibrous and yon--Fibrous Dusts. To demonstrate that the ability of asbestos to produce fibrosis resides in its fibrous character, the series of injection experi ments reported in Table 19 were performed. The tests vrere made with unheated long--fiber chrysotile and with chrysotlle that had been ignited to destroy its -32- xlegible structure or bell ~ billed bo reduce the lor.-th ox liber i-c 3 :or.c.'cr.r. xud less. At t-he can's tine oor.ti'cl tests were swde with snrpisntiue- which has -.ii.-? sans chemical cornposition. ss chrysotile but is r.on--fibrous. A review oi the findings reveals that, only the unheated lon^--fiber- chrysotile produced flbrcsis. f*ibsr3 subjected to ignition or shortened by ball-willing imd 'last thsir capacity to causa serious tissue dauage. Ignition produced important changes in the ebrysotils fibers, among them being io3s of water, an alteration fror. fiyxifcie t-o a brittle structure and rocsibly other changes. 80. Comparison of lar-ions Long-Fiber Dusts. Some very interesting findings are disclosed by t-he results ex the experiments included in Table 20. First, all the long--fiber asbestos minerals tested, with the exception of anthophyllite, produced a typical fibrosis. It is not entirely clear xrhy anthophyllite behaved differently from the other asbestos minerals, ifafortunately, ? of 8 animals died of pneumonia within the first tiro weeks of the experiment anti the remaining animals ware sacrificed at 1, 8 and 12 months} thus observations irsre not made at the optimum periods of 2 and It months. Second, -with the mineral, brucite, which is not a silicate but is a fibrous form of magnesium hydroxide, a characteristic fibrosis Ilka that of the asbestos minerals was obtained. Sines the brucite used contained only 0.90 per cent silica (as an inrourity), it is obvious that a siliceous component is not an essential factor in the development of asbestosis. Third, no fibrosis resulted from the injection of glens wool fibers, even though glass wool resembles asbestos in souse ways. There are fundamental, differ ences, however. A glass woo', fiber 3 microns in diameter is a solid rod and, in short lengths, is fairly rigid. Voile an asbestos fiber of the sane diameter is a bundle of extreiEsly fine xllauents which impart to the fiber a high degree of flex ibility. It would 3i=25! that this structure and the associated flexibility are important factors governing the capacity of a mineral to produce peribronchiolar fibrosis. 81. Comparison of Long-Fiber and Short-Fiber Dusts. With quartz dust it has - --- bean1 dSSnstrated^that the smaller the particles, the more intense is the tissue reaction, and that there is little reaction to particles larger than 3 microns in diameter. In the case of asbestos, however, the reverse is true and apparently only long fibers have ary specific effect. This is confirmed by the data of Table 21, in which a series of tests with fibrous minerals 13 reported, Rhen the injected dust consisted of fibers 20 to 50 microns long, all the minerals tested (except anthopbyllite, as noted in Section 60) produced a fibrosis; when the material was prepared by first grinding the fibrous dust until the length of fibers was reduced to 20 microns and less (or, in some cases, 3 microns and less), none of the injected minsral dusts caused fibrosis. 82. Experiments Using Intravenous Technique. The experinxmts, described in Table 22, in which the intravenous method of injection was employed, show that the asbestos minerals are far different from quartz in their action on tissue. It has been repeatedly demonstrated that intra venous injection of quart2 particles 3 microns and less in diameter will, cause a typical tissue reaction with the development of, fibrefn extrspulaonary sites, <\C such as the liver and spleen. Asbestos minerals, however, on intravenous injection generally produce only an inert type of reaction, as is revealed by the results given in the table. The reason for the early deaths in the experiment with chrysotile particles is not clear; it may have been caused by silicic acid liberated by the finely-ground mineral. i 83. Experiments Using Intraperitooeal Technique. The results of injection experiments with. the intraperitoneel technique are given in Table 23. It trill be noted that the long-fiber dusts produced a fibrous reaction 'while dusts composed of particles 3 microns and las3 in size caused only an inert type of response- These experiments Indicate also* that the fibrosis initiated by the irritation of asbestos fibers is not restricted to the lungs, as was formerly assumed, but can be produced in the peritoneum as well. I A number of additional experiments were conducted to throw more light on specific phases of the asbestosia problem. 85. Protective Action of Aluminum Compounds. Intratracheal injection of a suspension of long-fiber chrysotila to which colloidal aluminum hydroxide bad been added revealed that the addition of the aluminum compound did not prevent the tissue irritation produced by chryaotile. If anything, the acute inflaamatory response to the injected fibrous mineral was accelerated. One month after the last injection of the dust armprmart on the bronchiolitis was becoming fibrous- 86. Formation of Asbestosia Bodies. The iron in the coating of the asbestosia body appears to b e derived from blood or tissue elements and not, as has been suggested, from the mineral fiber. Following subcutaneous injection of two kinds of chrysotdle into the groin of guinea pigs -- one kind containing 2 per cent and the other 0.2 per cent the asbestosia bodies were equally numerous at both sites of injection. An attempt to produce asbestosis bodies in guinea pigs by Implantation of three silk bags containing fibrous chrysotile ms unsuccessful- One bag planted subcutaneously in the abdominal rail disappeared; the other two bags, placed in the peritoneal cavity, produced a little foreign body reaction but no asbestosis bodies in a year. Intratracheal injection into guinea pigs of asbestosis bodies recovered from human lung tissue failed to produce the typical tissue reaction to asbestos fibers. The injected material was obtained by digesting with sodium hypochlorite solution lung tissue removed at autopsy from, an asbestos worker. The asbestosis bodies could be seen in the guinea, pigs far at least a year after injection. This experiment shows that the asbestosis body has a rather resistant coating which Is not destroyed by moderate hypochlorite treatment and may be maintained in vivo fen* a year or longer. LU1VH. THEORY -OF IRRITANT -ACTION OF -ASBESTOS .MINERALS^ n-v-n i iv--r--r----C- ^ -v >< Two hypotheses have been proposed to explain the tissue irritation and reac tion ceased by asbestos fibers: tee chemical and the mechanical. In the chemical theory, which is based upon experience with quarts, it is assumed that tee as bestos minerals dissolve In the body fluids and that in this process their bases are leached aw^y to leave silica in a form capable of ir-fcltatlng tissues. Accord ing to this hypothesis, asbestosis. would bo merely an indirect silicosis. Several facts make the chemical theory untenable i (l) intratracheal injections of bruclte fibers, which had a silica content of only 0.90 per cent, caused a typical fibrosis like that produced by the-asbestos minerals; (2) freo-elllca particles increase in potency as the particle site becomes less, but asbestos fibers shorter than about 10 to 20 microns are relatively innocuous; (3) aluminum 'r hydroxide neutralizes the irritating effect o quarts but not of asbestos 5 (li) serpentine has the sane chemical composition as long-fiber chrysotile but it does not produce the same kind of tisane reaction; (?) there is a ride range in the chemical composition of the minerals which do cause asbestosis (see Table 2ii) In view of this evidence it seems more likely that asbestosis is caused by an unusual mechanical irritation from long asbestos fibers 1 Probably this irritation related to the peculiar fi lamented structure of the fiber and the associated flexibility, which are possessed by no other foreign body. For example, ignition of chrysotile fibers changed their structure and made them inert whila the sane fibers, before being heated, would produce fibrosis (see Table 19). Further support for the theory of mechanical irritation i3 that asbestosis occurs in an organ of nigh nobility - the lung - and that a fibrous reaction can be produced by injection of asbestos fibers into the peritoneum, where there is also a degree of mobility, but not in other extrapulaonary organs. unmn. cpE^cayaiis The experimental investigation with asbestos minerals was concerned primarily with the effect of the dust on normal tissue but some attention was given to other phases, such as susceptibility to infection and occurrence of malignancy. 89. Infection. The only experiment in which the effect of inhaled asbestos dust on a pul monary infection was studied was the first inhalation experiment, carried on with "King's floats" dtist. It is, perhaps, unfortunate that infection atidies were not made in the other inhalation experiments also. 5*0. Susceptibility to Tuberculous Infection. The development of a tuber culous process'initiated at the beginning of exposure to asbestos dust, and also of an infection superimposed, upon an established asbestcsis, vas described in Sections 19 and 20 _of this re port. It -will be noted that asbestos, -when classified according to the effect of a dust on tuberculous infection, would be placed below an active dust like quartz but above inert dusts, such as calcite and gypsum. In animals infected with attenuated tubercle bacilli, quartz will cause the infectious process to progress until the animal dies of tuberculosis. Inert dusts will have no effect on the infection and the lesions will usually heal and the disease disappear. Asbestos dust is in a different category. T>hen the fibrous dust was being in haled during the evolution of the infection, there was a spreading of the tuber culous process for a time but usually the stimulus for continued proliferation of the tubercle bacilli was not sustained, the progression was arrested and healing followed. In guinea pigs infected with attenuated tubercle bacilli following the completion of nearly .three years of exposure to asbestos dust, progressive disease did not develop. The only modification of the infection was one of localisation, a few bacilli being retained In the fibrous terminal bronchioles and forming tubercles there in addition to the usual foci beneath the pleura. Such tubercles healed in a few months and there was nothing to suggest any influence on the course of the disease. 91_ Susceptible ty .to Non--Tuberculous Infection. There was no pointed ex periment concerning the effect of inhaled asbestos dust on non-tuberculous infection. Intercurrent pneu monia among animals exposed to asbestos dust was rather common, the frequency in guinea-pigs exposed in the four inhalation experiments ranging from 16 to 39 per cent. This incidental evidence ru'^errts the possibility of an effect of asbestos dust on non--tuberculous infection. Nevertheless, since such epidenies are not uncommon in inhalation experiments vrith other dusts and even in the colony of normal animals, it is felt- that the inhalation of asbestos dust docs not asert a significant effect on the susceptibility to non-tuberculous pulmonary infection. 92. Ifeop c. asbestos f- r~~l c t~* * *<. -- r lalatdon of observations on this 'graph on asbestosis submitted by In it he called atten- tion to the In his experimeu long-fiber asbestos, ese lesions es adenomas, There is an and cancer vhich should be made clear. A cand f local invasion and de- struction of tissue, lymphatics or blood stream to produce is lop. This phenomenon of dissemination is .bits it is a malignant growth, o the other hand, is a so-called benign, mf not be capable of local In order to clarify the logical mater- ial is being carefully examined benefit of Doctor Vor-wald's judgment, a review of the data on this subject is bWng post poned until after hia return from Europe. Rather than delay the entire report further discussion Trill be reserved for\a supplement to be issued later. lunge of guinea pigs, rats, eats and rabbits but not of mice and dogs. Both inhalation and injection experiments provide ample support for this conclusion/ figures $ and 6 short the reaction to two different ktnidd?i of asbestos mineral. B. The mode of actior^Cap ^ea^j^nube-^rS'aeri.ly aechnnical rather than |ioX{t chemical in nature. ; I ^ The evidence is given in section LXJXVTT, Figures I, 2, 3, U and 7 illustrate the important points. The fibrous fidanentsi structure of asbeatd3(wpeTac^j pla/' an essential part in the irritating action, since the solid fibers of glass wool do not produce fihrosis {bee Figure 8). C. Short asbestos fibers}do not produce fibrosis, (h t. --* > h The tumi-laniun is |jnpHed in the evidence mentioned in para graph B above. Er>eriments Tihich further support this finding are rcperiea ir. T; bles 21 and 23. D. Topical fibrosis can be produced by an atmospheric suspension of asbestos du3t contaiiing only an extremely small proportion of long fibers. In the inhalaticT experiment with 100 per cent ball-milled O' 'tw' asbestos dust a typical, though delayed, fibrosis was V* obtained no;; Tibis Id;, although less than i per cent of tie atmospheric wist consisted of fibers longer than 10 microns. ~s is storm In Tabic 11- In contrast, the Ictrs.- trachcal infaction experiment -cibh fine =sites dust con-- iaining ::r long fibers Toiled tc produce fibrosis.. (see Taol; cl). Inhalation ol t-sbestos dust -eppareiit-lj dees not alter significantly the ^ <4-^ .. courae ax experiEentai .tuberculosis in guinea This goacDttsiar* 3 tentative since ttje evidence on which it i3 based does not conform mth cur upual experience- Refer" ence to Table 3 will ahorr that Then infection was coincident with onsa '- of dust exposure there tits temporary progressior of the disease Kith subsequent healingj when infection was initiated after 2> 3/k months of dust exposure the course of the tuberculous disease was not appreciably altered. In contrast,it has been observed in experiments with mixed du3ts containing quartz that if there is a slight >rogression of the tubercu losis when infection and dust exp >strre are coincident, this effect is more narked (instead oi less, as with asbestos} Then infection is initiated after a p; riod of dust exposure. This conclusion concerning the effect of inhaled asbestos dust- on tuberculosis seeas jusui.Iad because in the more sensi tive test {infection initiated after a period of dust exposure) there was no appreciable increase in susceptibility to the tuberculov.-s Infection. 'loTfever since the findings in ths JF 7* since the du3ii'h:-' r^ateri^l ussdyiu iiw experisaaiii PLAINTIFF'S EXHIBIT 4170.1 AFFIDAVIT WASHINGTON ) ) ss. DISTRICT OF COLUMBIA ) BEFORE ME, the undersigned Notary Public, personally came and appeared MICHAEL RHODE, a person of the full age of majority, being of sound mind and body, who, after being first duly sworn by me, did depose and say: I, MICHAEL RHODE, am an employee of the Armed Forces Institute of Pathology, Washington, D.c. I am custodian of the original archives of Dr. Arthur J. Vorwald. Pages 1 through V attached hereto are true, correct and authentic copies of documents from Dr. Arthur vorwald's archives. The pages attached are _______Revised version of ASBESTOSIS - Experimental Studies bv The Saranac Laboratory. Report to the Johns-Manville Corporation, dated January 31. 1949 Dr. Vorwald was the Director of the Saranac Laboratory of the Trudeau Institute. Upon his death. Dr. Vorwald's archives were donated to the Armed Forces Institute of pathology by his widow. Dr. Vorwald's archives are in such condition as to raise no suspicion concerning their authenticity; they were received by the Armed Forces Institute of Pathology from Dr. Vorwald's widow after RHODE AFFIDAVIT - 12 Dr. Vorwald's death; they are presently on file as authorized by law in the National Museum of Health & Medicine, Armed Forces Institute of Pathology, Building 54, Walter Reed Army Medical Center, Washington, ,.D-C. 20306^6000; and the archives have^-been-in existence for 20 (twenty) or more years. SWORN TO AND SUBSCRIBED before me, the undersigned Notary Public, on this the c 'lday of April, 1992. My Commission^Expires: NOTARY PUBLIC RHODE AFFIDAVIT - Pace 2 of 1