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4/ r if ^ ^ p ^ ^ -^ ifiT ri i"d f iff it iff iiM y -tm T h ^ i ir'^ ^ i Ann. N.Y. Acad. S c i . , 1 3 2 :4 5 6 -4 8 8 , 1965. TESTS FOR CARCIXOGEXICITY OF ASBESTOS* \Ym. E. Smith. Llonas Miller. R. E. Elsasser. D.D. H ubert Health Research Institute, Fairleigh Dickinson University Madison, X . J. Reports in this Annal and elsewhere associate exposure to asbestos with numerous cases of pulmonary carcinomas and mesotheliomas in man. E arly observations on this association were reviewed by one of us.! Re | cently. Selikotf. C h u rg and H am m ond- reported studies on a group of .632 insulaton workers exposed to asbestos for 20 or more years. In these men, pulmonary carcinomas were found at six times the expected incidence and carcinomas of the gastrointestinal tract at three times the expected inci dence. Pleural mesothelioma, a rare tumor, was found in three men and P peritoneal mesothelioma in another. - In contrast to statistical association between exposure to asbestos and development of cancer in man. a large literature on experimental studies has failed to furnish any definite evidence for induction of m alignant tu mors in animals exposed to various varieties and preparations of asbestos by inhalation or intratracheal injection. Recently, W agner1 reported meso theliomas in rats after intrapleural injection of chrysotile and crocidolite varieties of asbestos, and our group reported fibrosis and development of mesothelioma in ham sters after intrapleural injection of the amosite variety of asbestos.4 Detailed accounts of mesotheliomas in animals and associated tissue reactions have not as yet been presented. These are offered in the present paper together with reports of intratracheal and feeding tests with asbestos. In extensive experimentation at the Saranac Laboratory, reviewed by Yorwald et a!..' and by others reviewed by Behrens.'' no cancers were found in guinea pigs, rats, rabbits or cats examined after long-continued inhalation exposures or after long maintenance following one or two in tratracheal injections. Xordman and Sorge; renorted lung tumors in two mice after inhalation of chrysotile asbestos over a period of nine months. Their claim for induction of cancer with this material must be evaluated, however, with the consideration that one of the tumors reported by them w as a n a d e n o m a to u s type not uncom m on as a sp o n ta n e o u s t u m o r iri mouse lungs, and their photograph of the other lesion leaves a question as to w hether it may have been an area of squamous metaplasia rath er than neoplasia. Lynch et at/ exposed mice to a commercial preparation of chrys- otiie asbestos by in h a latio n f o r 8 to 12 h ours daiiy f o r five d ay s p e r week This work was supported by L'. S. Public Health Service Research G ra nt CA 07081 from the National Cancer Institute and by a g r a n t from, the Deerir.g Miiliken Foundation. 456 i Smit beginning at two m 24 months of age. found that the pern mas was the same individuals bearing mice (18 per c e n t' any of the tumors i W ith known care, between first expos able proportion of t creases with decrea genicity of materia over periods approx the test. The experil to meet this requir his associates, corn served over period studies of this nau experimental expos riods for study of required for adeqi monkeys. In exper days were required carcinogenic petrol r a b b it s a n d TO day tests with rats or for carcinogenicity In designing tes' form minerals, we been previously err life span. The gold tracheal injection Characteristic^ consideration. In crease in particle of chrysotile with sis in the lungs of terial was lost whe therefore of inter* exposures in the . cent of fibers long* a- "grains." The " that had surround ^ ' * k. Smith r-t o !.: Tests for Carcinogenicity 457 ASBESTOS* | b e g in n in g a t tw o m o n th s of a g e an d sacrificing f o r ex a m in a tio n a t U.t or 24 m onths o f age. In 127 exposed mice compared to 222 controls, they , D.D. H ubert ton University % found t h a t th e p e r c e n ta g e of in d iv id u als b e a r in g single p u lm o n a ry ad e n o i m a s w a s t h e sa m e ( 2'6 p e r cent < in both groups, b u t th e p e r c e n ta g e of >osure to asbestos with lesotheliomas in man. red by one of us.1 Re dies on a group of 632 e y e a rs. In th e s e m e n ixpec-ted incidence and mes th e expected inc-ijnd in three men and osure to asbestos and experimental studies :tion of malignant tujparations of asbestos agner3 reported mesorsotile and crocidolite dsis and development jetion of the amosite omas in animals and ted. These are offered tracheal and feeding ! i ! 5 ; : ; i , i ; individuals bearing multiple pulmonary adenomas was greater in exposed mice t l S p e r c e n t , th a n in th e controls eg per cent n T hey did n ot re g a rd any of the tum ors in th eir animals as carcinomas. W ith known carcinogens, it has often been found that the latent period between first exposure and development of tumor can occupy a consider able proportion of the life span of a test species, and that this period in creases with decrea-e in potency or dose of care nogen. T est' for carcino genicity of materials of weak or unknown potency m ust therefore extend over periods approxim ating the natural life span of the species selected for the test. The experiments by Lynch and his associates on mice were designed to meet this requirement. In the inhalation experiments by Yorwald and his associates, considerable numbers of guinea pigs and rats were ob served over periods approximating their natural life spans. Long-term studies of this nature have not yet been reported for other species given experimental exposures to asbestos and sacrificed at relatively early pe riods for study of asbestosis.'* A considerable period of years m ight be required for adequate tests in long-lived species such as cats, dogs or monkeys. In e x p e r im e n ts r e p o r te d by one of us a n d a s so c ia te s.1" 1.373 days were required to induce cancer in the rhesus monkey with a highly carcinogenic petroleum derivative th a t elicited cancer w ithin 411 days in rabbits and 70-days in mice. This same material did not elicit cancer in tests with rats or guinea pigs, an illustration of another aspect of tests fo r carcinogenicity i.e., v ariatio n in susceptibility of different species. oratory, reviewed by is.'' no cancers were after long-continued >wing one or two in lung tumors in two riod of nine months, il must be evaluated. >rs reported by them eous tumor in mouse .'es a Question as to taplasia rather than reparation of chrvsr five days per week rvice Research Grant ant from the Leering In designing tests to explore the question of carcinogenicity of asbestiform minerals, we therefore determined to utilize a species that had not been previously employed for the purpose, and that had a relatively short life span. The golden Syrian ham ster met these requirements. Also, intra tracheal injection proved to be conveniently accomplished in hamsters. Characteristics of samples selected for the tests presented a fu rth er consideration. In contrast to increasing fibrogenicity of silica with de crease in particle size, it had been reported that intratracheal injections of chrysotile w ith average fiber length of 20 to 50 microns induced fibro sis in the lungs of guinea pigs but that the fibrogenic action of this m a te ria l w a s lost w hen milled to a v e r a g e le n g th s of 3 m ic ro n s o r less.-' It w as therefore of interest to note that a principal sample used for inhalation exposures in th e S a ra n a c ex perim ents w as said to contain only 6.7 p er cent of fibers longer th a n 10 m ic rons a n d 20 p e r cent o f particle s described a s `grains." T h e " g r a i n s " m a y not have been asbestos but d u s t fro m rock that had surrounded veins of asbestos in the deposits from which it had I mm I nilii<*ilfltW mrii<M`l t riftitii-v ^ ',rriiatMMigi>iftlariirnffiiiitrtini inin^.fTHtfriBiiiMTi 45S Annals New York Academy of Sciences been mined. Other samples used in inhalation exposures by the Saranac group were said to have an even smaller content of fibrous asbestos. The sample used for the inhalation experiments by Lynch's group was said to be composed to the extent of 95.5 per cent of material that "appears to be mostly irregular pieces of granular serpentine rock." Intratracheal injections of long-fiber preparations of a variety of asbestiform minerals were done in the Saranac experiments with guinea pigs and rats, but these experiments were not designed to test for carcinogenicity and ani mals were sacrificed at relatively early times to study development of asbestosis. . A further requirement for tests for carcinogenicity of asbestos was therefore to prepare samples consisting principally of asbestos fibers freed insofar as possible of rock dust and other impurities. A method for this p u r p o se w a s developed in th is la b o r a to r y by Badoliet and G a n t t . 11 Since any effects of fiber lengths upon possible carcinogenicity may differ from effects upon fibrogenicitv, preparations with a broad range of fiber lengths were employed for our animal tests. A review o f e x p e rim e n ta l s tu d i e s o f lu n g c a n ce r h as been pub lish ed .11 In the present work, pleural mesotheliomas were found in ham sters after intrapleural injection of amosite and of harsh chrysotile but not after intrapleural injection of soft chrysotile. Diagnosis of these tumors and comparison of them with mesotheliomas in man has been discussed else where." Tumors were also found in the gastrointestinal tract and liver of hamsters fed asbestos but these may have been spontaneous growths unrelated to asbestos. Xo tumors have as yet resulted from intratracheal injections of asbestos, but many animals injected by this route are still living. From necropsy findings in these experiments, some theoretical con siderations arose in regard to genesis of tum ors a fte r exposure to asbes tos, These will be discussed. Methods F o r in tr a p le u r a l -ar.-i in t r a t r a c h e a l inje ctio n s, s o f t ch ry so tile S am ple 2, harsh chrysotile Sample 1 and arr.osite Sample 7 prepared by Badoliet and G an tt'1 were used. Distribution of fibers in various size ranges in these samples are detailed in their paper. The per cent of fibers between 5 and 100 m icrons in these sam ples was 90, S2.5 and S7 p e r cent, respec tively. A v erage fiber lengths w ere 67, 36 an d 17.6 microns, respectively. For feeding, new batches of soft chrysotile and of arnosite were from tim e to time prepared with characteristics roughly approxim ating those of Sam ple 2 and Sample 7, respectively. For intrapleural injection, the hair over the rig h t chest was clipped away and the skin was swabbed w ith tincture of iodine and then with 70 -, Ht'mm?*3 Smi per cent alcohol. T pointed 25 gauge ne later, injections we needle was ground the pleural space r? inoculum occasional found instead in th, there evidence that For intratracheal fo r h am sters was fi The ham sters we Diet for H am sters." apples, carrots or s libitum. When asbe purchased as an unp was mixed into it. a moistened with w at in a n oven a t 64 C. the daily regimen of Weight records of 1 carrots or spinach a comparable to that i For all experimen (Lakeview Hamster of age at the start o cages. T hey w ere we 20 per cent or more rificed for examinat natural life spans, formed that were u not exami ned. The larynx-, trachea and with the lungs under vations were record* right and left lungs cut vertically throug ings required additb indic ated by g ro ss fi.' diets, and in untreat ach distended with 2 As this needle was t pulled tig h t. T h e di quently bisected for Sciences Smith ft al.: Tests for Carcinogenicity 459 rposures by the Saranac - per cent alcohol. The right chest was stabbed six times with a .-harp- of fibrous asbestos. The , pointed 25 g au g e r.eedle wit.-, object of in d u c in g p n e u m o th o r a x ." Tw o days L y n c h 's g ro u p w as said ' la ter, in je ctions w e re m ade v.-itr. an 18 ga..ge r.eedle. T he bevel of this ' materia) th a t "appears 1 needle was gro .nd short ar.d rounded to favor depo-ition of inoculum in line rock." Intratracheal the pleural space rath e r than into the lung. As described in the text, the of asbestiform m inerals inoculum occasionally failed to he deposited in the pleural space and was inea pigs and rats, but ; found instead in the ti.-sue of the chest wall, hut in only one instance was carcinogenicity and ani- there evidence that the inoculum had been injected into the lung. o study development of j For intratracheal injection, the technique used by Herrold and D u rh am " j for ham sters was followed, using Nembutal anesthesia. enicity of asbestos was T h e ham.-ter.s w e re m a in ta in e d on a com mercial pelleted feed 1" G L F v of asbestos fibers freed D iet fo r Ham. t e r n " \V. F. F l r h e r . Bound B rock N, .T. supplem ented w ith ities. A m ethod f o r t h is j apples, carrots or spinach three times a week. W ater was avauable ad lollet and G antt." Since i libitum. When asbestos was added to the diet, the commercial feed was renicity may differ from 1 purchased as an unpelieted powder and one per cent of the asbestos sample ad range of fiber lengths j 1 was mixed into it. adding three per cent dextrin as a binder. The mix was moistened w ith w ater and molded into blocks which were dried cvem ight ;er h a s been p ublished.1- j in a n oven a t 64 C. T h ese blocks th e n replaced th e com mercial pellets in found in ham sters after | the daily regimen of ham sters designated to receive asbestos in their feed. chrysotile but not after ! W eight records cf ham sters fed these blocks, supplemented with apples, sis of these tum ors and { carrots or spinach as above, showed gain and maintenance of body weight has been discussed else- i comparable to that of ham sters on the diet without asbestos. ntestinal tract and liver j F o r all experiments, male ham sters obtained from a commercial breeder en s p o n ta n eo u s g r o w th s i (Lakeview H am ster Colony. Newneld. X. J. i who were about two months lilted from intratracheal i of age at the sta rt of the tests were used. They were housed in open mesh d by this route are still cages. T hey Were weighed biweekly. Animais exhibiting persisting loss of its, some theoretical con- j 20 per cent or more of body weight or who appeared moribund were sac after exposure to ashes- ; rificed for examination. Otherwise, they were allowed to live out their natural life spans. At sacrifice or natural death, necropsies were per formed that were complete except that the brain and nasal sinuses were not examined. The oropharynx and cheek pouches were inspected. The larynx, trachea and bronchi were opened by dorsal incision, ana examined oft c hrysotile S am ple 2, with the lungs under a dissecting microscope at 7 x magnification. Obser 7 prepared by Badollet vations were recorded and tissues taken for histological examination. The : various size ranges in right and left lungs were taken separately and routinely saved. They were r cent of fibers between cut vertically through the hilum except where tumors or other gross find and 87 per cent, respec- ings required additional blocks. Blocks of other tissues were taken where .6 microns, respectively, indicated by gross findings. In animals that had received asbestos in their d of amosite were from diets. and in u n trea ted c uitrols. the esophagus was tied off and the stom tly approxim ating those ach distended w ith 2 ml. formalin by a needle run in from the duodenum. As this needle was withdrawn, a ligature laid around the-duodenum was right chest was clipped pulled tight. The distended stomach was placed in formalin and subse iodine and th en w ith 70 quently bisected for examination by a cut along the lesser and greater s' i \ :i i *w Annals N ew York Academy of Sciences Sn curvatures. The small and large intestines, the cecum and rectum were opened, examined, coiled on cardboard for th e ir full lengths and fixed. AH tis s u e s w e r e fixed in 10 p e r cent f o rm a lin c o n t a in i n g 2 p e r cent so dium acetate. Hematoxylin and eosin were used for routine staining. Mallory s connective tissue stain was used for special studies. Amosite and harsh chrysotiie fibers could often be seen in tissue sections by reduced illumination through closing the diaphragm of the microscope condenser. This procedure seldom revealed fibers in tissues treated with soft chryso tiie. In the course of this work, a method for visualizing asbestos fibers in tis s u e sections w ith o u t d e s tro y in g tis su e r e la tio n sh ip s was developed.15 F indings Erpcrimrnt A : one per cent amos treated with asbes G roup 1. T h r e e injection. In two. in the chest wall around it. In the t to the parietal plet firm g r a y zone or. of this lobe was a formed a densely 1 w ith p ro m in e n t pi seen in the adhesi This experiment comprised 60 ham sters divided into four treatm ent groups of 15 anim als each. E ach anim al in th re e of these groups w as given a single intrapleural injection of 25 mg. of a sample of asbestos suspended in 0.5 ml. of a 0.9 p er cent sodium chloride solution. Those in Group 1 received h arsh chrysotiie Sample 1; those in G--"up 2 received soft chrysotiie S am ple 2 : those in Group 2 received a m o site Sam ple 7. Groups 1 and 2 received one per cent soft chrysotiie in the diet. Group 2 received 1 ; i i ' ' k JP-' F igv?.:-: 2. H cr.-.ei Most o: me c d l; ar ^VclefctV - ` C;-!7 On the l*7th d ?m . in v/c; gr.t a r/ cen.-'fcyV c.``>herer.t t cavity v.a s 0olite lu n g a r.n 1 : r a c * 7 - 2 rr_. (\ \ -C' io T h e i:r, *-r y<r - j r f a- 7 .arhi p e r k a r d i mr. f-.r.ri t Sciences cecum and rectum were full lengths and fixed, containing 2 per cent -o ed for routine staining. :dal -tudie-. Amosite and -sue sections by reduced he microscope condenser, tre a te d with .soft chrysoisualizing asbestos fibers ionships was developed. ded into four treatm ent :>f th e se g ro u p s w as given sample of asbestos sus ?solution. Those in Group n Group 2 received soft imosite Sam ple 7. G roups he diet. Group 2 received 1 of harsh chrysotile. Alvemuscle bundles of the chest er right corner. The space thick adhesion. The large are blood vessels. 100 x . Smith ft ai.: Tests for Carcinogenicity m one pel' cent a r r o - i te in th e diet, fjr o u p .7 was m a in ta in e d as a control u n treated with a-ee-tos. Re-.Ji- v ere as follow-. ft roup 7. Tr ree hart-ter.- were examined between 07 and 102 davs a fte r injection. In two. tn e injected a-ve-to.s wa.-. found to have been deposited in the che-t wab hn-tead of the pleura! cat ity. There was little reaction aro u n d it. In t i e th ird , all lone- of th e rig h t lung '.cere bound oy adhesions to the parietal pleura. The thiche-t area of adr.e.-ior.s was a 7 / 3 c 2 mm. firm g r a y zone on th e rr.edi&j .-urface of the r ig h t lower lobe. T he base of th is lobe wa.- a d h e re n t to th e d ia p h ra g m . Ili-toiogicaily. th e adhesions formed a den.-eiy cedular and highly vascular granulom atous iml a tarnation with prominent pleural thickening ' figures 1 ar.d 2 . Asbestos fibers were in trie a c e - .o n -, but a-oe-to-- oodles were r. : found. r* " e O V.v " ^ (t ' , ^ v# <m3 * * V" * ,,S - v .*, : *v -- * * ' ^ : r*'' < . ,v 1 * Figure: 2. Higher power view of an area of the adhesion shown in figure 1. Most of the cells are macrophages, some of wh.ch are coalescing to form multinucleated giant ceils. 400 \ . On the lS7th day. a ham ster was sacrificed because of the loss of 40 gra. in weight, and deform ity of the chest. All lobes of the rig h t lung were densely adherent to each other and to the parietal pleura. The right pleural cavity was obliterated by these adhesions, which compressed the right lu n g an d con tracted the r ig h t chest wall inw ard. A firm g r a y -w h ite 10 x 7 \ 2 mm. discoid lesion bound the base of the right lung to the diaphragm. The inferior surface of the heart was bound by dense adhesions to the parietal pericardium and diaphragm. There were adhesions between the pericardium and the left lung. The adhesions in this animal differed from w pw w appjp 4t52 Annals New York Academy of Sciences those in the preceding animal in that they contained many well-developed r.vultinucleated giant cells often w ith a dozen small nuclei a rran g e d in a circle around the periphery. The discoid lesion was a thick adhesion with a necrotic center that contained granules of calcium. Five ham sters died or were sacrificed between 231 and 398 days. In two of these, nothing remarkable was found aside from a nodule around a subcutaneous deposit of asbestos. The other three had dense pleural ad hesions involving the right lung, delicate adhesions involving some areas of the left lung. and. in two. obliteration of the pericardial cavity by adhesions. As in the preceding animals, the lobes of the lungs stood out as pink and air-containing though compressed to varying degrees by the adhesions on their surfaces. In the 393-day animal, the adhesions were sparsely cellular fibrillar connective tissue with only occasional giant cells. T h e r e w ere calcium dep o sits in n ecro tic foci <fig u r e 3 >. A m a s s m e a s u r i n g 15 - 9 x 5 mm. lay in th e m esentery of the small intestine. Microscopically, it proved to be a malignant lymphoma. On th e 419th day. a h a m s te r >H 12 > w as sacrificed because o f p a ra ly sis of both hind legs. The lobes of the right lung were pir.k and*ai-r-corttaining b u t com pressed by a solid tu m o r t h a t m e a su re d 30 ' 20 x 15 mm. an d lay between the chest wall and right lung from which it could not be sepa r a te d ( FIGURE 4 ' . T h e c u t s u r f a c e of th e tu m o r w as g ray -w h ite, firm, and F 2. Pleural aor.esion 352 cays af te r injection of harsh ehry.-or;V. F i bre.-: t.- s - e s c r r o c r c ; an area of r.ocro.-is in v.i,;ch there- i a larger oepo.ut of >rr. I black;. 100 x . Fror-RF. 4. Coronr a pleural mesothell is the dark mass oi The right lower lobXote that the tumc the lung. The base diaphragm is the i hesions show as lij between the lung are W -F > .% U T i Y; ~t-, -*7, - -. '.-fYVYY - ^ : r'< S&7.y :' - ;.;;*: C F igfke 5. H ig h e r tYt*? r ; ^ h t . Thf; corr.p is fibrous trijck:nir.v' Sciences ned many v.eH-developed .a}] nuclei a r r a n g e d in a a* a thi'-'k a d h e -io n w ith ium. i'il and 398 days. Iri two mm a nodule around a e had dense pleural ad os in v o k in g sorr.e areas e pericardial cavity by of the lungs stood out varying degrees by the tal, the adhesion= were y occasional giant cells. i- 3 i. A m a ss m e a s u r in g '.es.tine. Micro.-copically, ed because of paralysis pink and air-contain ing x 20 x 15 mm. and lay h it could not be sepa s gray-w hite, firm, and J F igure 4. Coronal section of thoracic organs from a ham ster th at developed a pleural mesothelioma 419 days after injection of harsh chrysotile. The tumor is the dark mass on the right. The ventricles of the heart are seen on the left. The right lower lobe of the lung is compressed between the tumor ar.d the heart. Note that the tumor has extended along the p eurai surface without invading the lung. The base of the lung is bound by an adhesion to the diaphragm. The diaphragm is the inverted U-shaped structure beneath the lung. Pleural ad hesions show as light gray zones between the lung and the diaphragm, and between the lung and the tumor. 3 x . f harsh chrysolite. Fi'c is a large deposit of F igure 5. H igher magnification of an are a from figure 4. The tum or is on the right. The compressed lung is on the left. The light gray zone between them is fibrous thickening of the pleura. 100 x . ' IIWMJHWB'. wni i. i-.iw u f w w m >ji,* , t- ~ lifi-ifitr i I 1 464 Annals New York Academy of Sciences r __Jr-> * ^ * * * -,? v ,^ k c i^ J * " ' * 0 * ' , - . . ., . (Sk. ^ V 1 - .e "/- -js*4 r ' "V c - r u zt r p t t j & , " Jit*' f f ' " >7* V ' V k 'W# **' X'** 1" jfcV * -- rJ ' s i $ M . ^ aw ` r>T V ^ a 5 w s^i - F igure 6. Anaplastic, elongated cells of the tumor shown in figures 4 a n d ' 5. There are several mitoses. 4wo x . * appeared living granular materia seen to contain a the areas of neei fiammation and i arisen in the \va basophilic, elong: filled the cells i f pressed but did i thoracic vertebra tween the lung ai irskSfC^uV^,; ^ w: - V i :. F igure 7. Section o f the thorax o f a ham ster th at developed a pleural meso thelioma o'2. cays afte r inject,on of harsh chrysotile. The tumor is the dark mass. Intercostal muscles ar.o r.bs are seer. :n cross section on the right. The tumor has ir.vaued ar.d d es tro y three intercostal muscles and two ribs. Alveolar lung tissue is .r. tne center of the photograp.o. The heart is on the left. The gray zone between the lung and tne .oeart is a thick pleural adhesion. A / . Figure 8. Juncth does not invade the in which asbesto were rare. Pieces of this the right poster! adult female ham curred in th e mic plant sites in eac' large tum ors 50 t Among the sui ficed because of 1 textured, hard, wl imr#***'* .Sciences >. 2 *?' ` *> < - * re * -I. ? , v f **-/ {_ . v/ .-,./ ;> ;:. - -S G-^'V ' hr .* I-o'.-I* '2 > g n % i 5 f ^ i 5"1*5 - , T E * :*, r V % 1 -'l- - i iA t ^ , - j n in figures 4 a n d 5. Smith t t nL: Tests for Carcinogenicity 4o5 appeared ..v.r. g t r .r v jg r .c w. e:-:r.e p t f o r a -rr.nl) centra! a r e a of fria ble , g ra n u la r m a te r,a ; -orne or rvr.;ch wa= c r a ro ir e d in a c ro p of w a te r ar.d seen to cor.Oa.r,`a.-oe-to? fiver-.. T h is grar. d a r c e n te r was r em in isce n t of the area- of r.ecro-is with calcification surrounded by granulomatous in flammation a r c ::oro-i- seer, :r preceding" .mimals. The tum or may have arisen in the wa.l o: such a .e-;on. It wa = composed of densely packed. basophiiic, e > r.g a te d cells tvr ' se nuclei v a r ie d g r e a tly in size b u t n e a rly filled th e cei!^ nc-u?.f;s 5 ar.<i t ' . T h e r e w ere occasional m itoses. It cor.upressed but did not invade th e lung. It had invaded and replaced several thoracic v ertebrae. Adhesicr. s .oetween th e tu m o r and the hung, and between t h e lur.g ar.d h e a rt, we; sparse.;.- ce.. b ar. fibrillar connective ti.-s..e I a pleural r tumor is the |n on the ri^ht. f (i two ribs. AIvi Pn the left. The fusion. 3 x . F igit.e S. Junction of the tum or of figure T and the lung. Note th a t the tumor does not invade tr.e lung but .5 seperated from it by thickened pleura, loo \ . in which asbestos fibers, but no asbestos bodies, were seen. Giant cells were rare. Pieces of this tum or were transplanted aseptieally with a trocar into the right posterior thigh muscles of three adult male hamsters, three a d u lt fem a le h a m s te r s an d t h r e e new -b o rn B A L E C mice. X'o g r o w th oc curred in the mice. Progressively growing tum ors developed at the tran s plant sites in each ot the six ham sters, and all of these anim als died w ith la rg e tu m o r s 50 to 70 mm. in d ia m e te r w ith in 10 to 15 weeks. A m o n g th e s u r v iv in g a n im a ls of th e o r ig in a l G roup 1. one w as s a c r i ficed because of labored b rea th in g on the 527th day. It had a solid, finete x tu r e d . h ard , w h ite tu m o r m e a s u r in g 20 x 15 \ 10 mm. w hich lay behind i ; 466 Annals New York Academy of Sciences Smi i F igure 9. Spindle cells of the tumor of figures 7 and 8. Two mitoses are near , the center of the photograph. 400 x . I the heart. The lungs were bound to the chest wall, mediastinum and dia ! phragm by adhesions or by the tum or (figures 7 and 8). The pericardial sac was obliterated by adhesions. The liver contained a 6 x 4 x 3 mm. grape-like cluster of cysts w ith paper-thin walls, filled w ith clear fluid. The t u m o r w as composed of spindle cells ('figure 9 . Another animal of the original group was sacrificed on the 539th day. In it, there were adhesions in the right pleura! cavity and pericardial sac. Three hamsters of Group 1 survive at the present writing. Group 2. Two h a m s te rs w ere exam ined 200 days a f t e r in'V-*:on. In one. theie v.as no evidence oi asbestos or reaction to it in the pleural cavities or lungs. In the other, all lobes of the rig h t lung w ere broadly bound to each other and to the chest wall by thick adhesions. The base of the right lower lobe was bound to the diaphragm by a firm, gray 6 ^ 6 / 4 mm. mass w ith an ill-defined edge. This proved to 1e an area of necrosis surrounded by fibrous tissue containing macrophages and rnultir. id e a te d giant cells. The adnesions elsewhere also showed foci of necrosis, but were for the most part composed of sparsely cellular fibrillar connective tissue. On the 255th day, a ham ster dying of "wet tail" v.is found to have the rigr.t p.eura! cavity entirely obliterated by firm adhesions which sur rounded and encased the lobes of the right lung. The thickest adhesions were between the base of the right lower lobe ar.d the diaphragm, where MWl'WPS*! F igure 10. Pleural lung is on the left. Tr contains many discrete tissue. Some of these : 13. 100 x . l 5.;.*- * , F igi:rf. l l . J u r . c t i o n in fiocrf. *. The 1+''> o f the a t h * re ; pie^ra wr..ch are .-een i fe n c e s Sm ith ft. oi.z Tu-ts fo r Carcinogenicity 467 f e .v :':!A s-. / i> * * ; /.1 X i ' ; . - - * v . y 1y . v - v - . >' - . v y . , ; . * ;. y ' i ' / i Tr -. ' } ,,,-, -i. : vv-'i-- V - . v . t -* ^ -* y -<r *; ',,r^S H o y / . W y V ; . y ! ' ' * ' ''^Ai 'x jj .'*/ >'&'** %'r*'*&'~* Ai V <- C' "># V"/ s\ ^ : ^ 3 Two mitoses are near I'd iastin u m and dia8 ). The pericardial a 6 x 4 x 3 mm. *-ith clear fluid. The 1 ; i F igure 10. Pleural adhesion 2oo days a f te r injection of soft chrysotile. The lung is on the left. The rest of the field is filled with the thick adhesion, which contains many discrete, dark-gray isiands of celis lying amid fibrillar connective tissue. Some ot these islands are shown at higher magnification in figures 11-- 13. 100 x . 3 on the 539th day. and pericardial sac. w ritin g. . I *r injection. In one, the pleural cavities re broadly bound to he base of the right > x 6 x 4 mm. mass necrosis surrounded ideated giant cells. 5. but w ere fo r th e tive tissue, was found to have Ihesions which surthickest adhesions diaphragm, where ; i F igure 11. Junction of the lung and pleural adhesion of the specimen shown in FIGURE 10. Tr.e lung is on the left. In the area between the iur.g and the center o f the adhesion, there is hypertrophy and hyperplasia of mesotheiial cells of the pleura which are seen as plump, rounded or cuKndal cells. 400 \ . WHIP* W W W ^ 1iW U M W M W Jw J M K g W W I T , I "M "WIHMI.I. *-'.! JP jj^WH W i k ....... ...... - - --Titj-Kftrr 46S Annals Xew York Academy of Sciences two n odular lesions. 7 x 7 x 3 mm. a nd 6 x 6 x 5 mm., w ere located. On the right side of the pericardial sac. there was an adhesion 4 mm. wide and mm. thick between parietal and visceral pericardium. Sections of the lesions at the base of the right lower lobe and of the adhesions else where resembled those described in the preceding animal, but the nodular masses contained much finely granular material that stained blue with hematoxylin and was considered to be calcium. Also, the adhesions con tained many islands of euboidal cells which lay in clusters or lined cleft like spaces. They resembled cells of the thickened pleura (figures 10-13). In a h am ster sacrificed on the 352nd day because of weight loss, the lateral surfaces of the right upper and middle lobes were bound by ad hesions to the chest wall and their medial surfaces to the mediastinum. F igure 12. Mesothohai cells lining a cleft deep within the adhesion o'" figure 10. 41 > x . The dorsal surface of the heart was adherent to the parietal pericardium in an area continuous with the adhesion of the right lung to the medias tin u m . A t th e base o f the r ig h t lower !che, th e re w as a 13 * 4 4 mm. yellow mass containing necrotic tissue and grar. b ar material surrounded by a capsule 1 rr.m. thick. Microscopically, th e gran,,I.r m a te ria ! w a - found to be calcium, and tr.e capsule was fibrillar connective tissue lightly infil tra te d by round cells. In it. a few asbestos finer.- were -eem Tr.e re w ere occasional mult nucleated giant ce..s. but the-e were degenerating. Occa sional islands of euboidal cells lay in the adhesion.- n ea r the p.eural surface. Sciences 5 mm., were located. On an adhesion 4 mm. wide il p eric ard iu m . Sections nd of th e adh esio n s eleanimal, but the nodular that stained blue with \Iso, the adhesions cona clusters or lined cleftpleura 0'IGt/KE.S 1 0 - 1 3 i. mse of weight loss, the ibes were bound by ad es to the mediastinum. r' V 1 I Smith <t a l.: Tests for Carcinogenicity 469 A h a m s te r t h a t died on th e 3%1st day had thick pleural adhe sio n s s u r rounding- all lopes of th e r ig h t long except the in fra c a rd ia c lobe. In a ham.-ter killed on the 427th day. a boggy, yellow mass m easuring 10 / 12 / 12 rr.m. replaced th e r ig h t u p p er and middle lobe-. Tr.e c u t s u r face showed it to be .-oft, ca-eoo.s m aterial surrounded by a th in capsule. A.-bestos fiber.-, w e re seen in some of t h i s m a te ria l .suspended in a d rop of water. It was concluded th at the injected asbestos in this animal had been deposited into the lung instead of into the pleura! space. The tissue re sponse to this injection contrasted markedly with the previously described granulomatous and fibrotie responses to intrapleural injection-. On th e 434*.h (idy, a nam .-ter .-acrif.ced because of v.eignt .uss had a 10 s 8 y g mm. yellow mass in the rig h t costophrenic angle. It lay in the w - / * ? *' 'V/*- - - !*. *^i +Sr i '8 37** - n * , . v- . '+S* .r * 'ce- ' & ' I - - > . / VT ' r - V - ' 11 ' ^ *< b i the adhesion of figure e parietal pericardium ht lung to the medias.as a 13 x 4 x 4 mm. r material surrounded ar material was found ive tissu e lightly infilrere seen. There were e degenerating. Oeeaar the pleural surface. F igure 13. Msothlial cells arranged in discrete islands deep within the ad hesion of figure 1". This adhesion was attached to the chest wall and the block w as decalcified. The appearance of the nuclei m FIGURES 12 and 13 is ar. artifact of dcalcification. 400 x . pleural space, compressing the right lower and middle lobes and binding them to the chest wall and diaphragm. The cut surface revealed granular yellowish-white material lying in a fibrous mesh-work surrounded by a tough gray wall 1 to 3 mm. thick. An ulcer was present in the forestomach. It was a thickening 5 mm. in diameter with a central crater. A large, weii-developed ham ster that had seemed in good condition was fo u n d dead on th e 440th day. In th e r ig h t axiila. th e re w as a 20 x 10 x 6 mm. encapsulated mass containing thin, greenish-yellow pus. The pleural I I mm -i <"ifn.tM.Hri.iY.-, - .ff, 4 4 ! 1^ Annals New York Academy of Sciences cavities each contained about 2 nil. o f clear, th in tluid. T h e re were no pleural adhesions. The lung's were small, dull and boggy. The fluid in the pleural cavities was attributed to congestive failure. The liver contained numer ous tnin-walled cysts tilled w ith clear tluid. The median lobe was largely replaced by a solid tum or which proved histologically to be a cholangiocareinoma. On the 455th day. a ham ster was sacrificed because of a 10 mm. mass in the right axilla. It communicated by a wide extension beneath the clavicle w ith a 20 mm. mass in the subcutaneous tissue on the rig h t side of the neck. The mass in the axilla was composed of tough fibrous tissue witn white, granular deposits of calcium. The mass in the neck was a thinwalled sac filled w ith moist, white caseous m aterial. The pleural cavities, lungs and other organs were not remarkable. A ham ster sacrificed because of labored breathing on the 476th day had ex ten siv e pleural ad h e sio n s <figu re 14 i on th e r i g h t w ith a 10 mm. mass extending from the right pleural cavity through an intercostal space into the subcutaneous tissue of the right chest where it communicated with a thin-walled cyst filled w ith colorless fluid. F our blocks from differ ent areas of this specimen revealed no tumor. The parietal and visceral layers of pericardium were bound together by adhesions continuous with adhesions binding the right lung to the mediastinum. The left lung was F i ' 14. Pleura! adhesion 476 days af te r injection of soft chrysotile. The lur.sr i- n a t tne to,o. The center of the adr.esion is r f-fro!.c and contains def*o~ of caicium w.o.ch show as h.ack areas. The denosit on the right s surroum ed by fibrosis. Ido x . Sn not remarkable, b the peritoneum, si diameter, were fou An animal sacri x 4 mm. yellow m surface of the latf granular, white m nation of th is m; showed occasional neoplastic change ; In an anim al foi were bound to eacl areas where there 5 mm. in diameter, diaphragm . The lit 11 mm. in diameti scending colon, the scopically to be an studied by electron ciated with it. One animal of Gi Group 3. In th is i 181 and 248 days a to an endemic disc these animals were or hem orrhage in g no tum ors. The plei in the livers. Xor u Group 3 that died t unable to determim at the present writii Eight untreated i ik e d a t ages of 48i not remarkable. Tin at any sites. In hamsters obta not involving asbesi tumor of the adreu. tourinary tract of ; Group U. A large labored respiration 3 i f Sciences fluid. Th ere were no pieural ry. The fluid in the pieural he liver contained num er e median lobe was largely ugicallv to be a cholangio- because of a 10 mm. mass de extension beneath, the ;s tissue on the right side ed of tough fibrous tissue a-cs in t h e neck w as a thin;rial. The pleural cavities, ;athing on the 476th day th e r i g h t w ith a 10 mm. 'ough an intercostal space t where it communicated . F our blocks from differThe parietal and visceral dhesions continuous with tinum. The left lung was , 1 ' ; ; f [ m of soft chrysotile. The necrotic and contains de posit on the right is sur- Smith f t a l.: Tests for Carcinogenicity 471 not remarkable, but the left pleura! cavity contained w atery fluid as did the perito n eu m , s u g g e - ti n g congestive failu re . Seven cysts, 2 to 12 mm. in diameter, were found in the liver. An anim al .-acrifieed on th e 057th d ay had, in th e r ig h t axilla, a 10 / 7 y 4 mm. yellow nodule w ith a co n tig u o u s 11 mm. - th e r ic a i mass. T he cut surface of the latter -bowed a 2 mm. thick, tough rind surrounding dry, gran,.!ar, white material that grated under the knife. Microscopic exami nation of this material in a drop of w ater with reduced illumination showed occasional asbestos fibers. The rind of this mass had undergone neoplastic change and proved to be a sarcoma. In an anim al found dying on th e 563rd day, all lobes of the rig h t lung were bound to each other by adhesions that were delicate except for four areas where there were spherical or ovoid yellow, opaque masses about 5 mm. in diameter. Two of these masses bound the right lower lobe to the d ia p h ra g m . The liver contained th r e e fluid-filled cysts t h a t w ere 2, 9 an d 11 mm. in d ia m eter, respectively. In th e m e se n te ry a d ja c e n t to th e de scending colon, there was an 8 x 3 mm. discoid lesion which proved m icro scopically to be an anaplastic malignant neoplasm. This lesion is being stu d ie d by electronic a s h i n g '"' to le a r n w h e th e r as b e sto s fibers w ere asso ciated w ith it. One animal of Group 2 escaped on the 157th day. Two survive at present. Group 3. In th is u n trea ted control group th e re w ere nine d eaths between 181 and 248 days a fte r th e s ta rt of th e test. These deaths were attrib u ted to an endemic disease of ham sters known as "wet tail." The tissues of these animals were not remarkable aside from injection of blood vessels or hemorrhage in gut. liver and lungs attributed to infection. There were no tumors. The pleura! cavities were not remarkable. Xo cysts were found in the livers. Xor were any anatomical lesions found in three ham sters of Group 3 th a t died on the 375th, 4S0th and 555th days of causes we were unable to determine. The three remaining animals of this group survive at the present writing. Eight untreated control hamsters from another experiment were sacri ficed at ages of 4S0 days. -- 30 days. The tissues of these anim als were not remarkable. There were no cysts in their livers. Xo tumors were found at any sites. In ham sters obtained front the same colony but used for experiments not involving asbestos, we found cysts in the livers of four individuals, a tum or of the adrenal cortex in one. a m alignant tum or in the lower geni tourinary tract of another, and gastric ulcers in six. Group 4. A la rg e tu m o r w as found in a h a m s te r sacrificed because o f labored respiration 244 days after injection. On opening the abdomen, a is mm 472 Annals New York Academy of Sciences F igure 15. Mesothelioma in a pleural adhesion 244 days afte r injection of amosite. The fibrillar connective tissue (light gray) of the adhesion contrasts with the darker islands and sheets of tumor cells. 100 x . Figure 16. Another area of the turner of figure 15. Here, it is invading the diaphragm. The pleomorr.h-c polygonal ee..s of the tumor are growing between muscle bundles of the d.aphragm. There are several mitoses. 400 x . tencas '::' -' A` A h A /V"r i 'V. V" ' i''Y-V-t?'-;. ../. /> -J-. = ;, r* t . -V- S 'h * '* C 7 ' / - 7 * * V' V :- < :, ' w ' , >-*r Kf y ^- ' , ,\ >i : - ."V f ki - V V-V -*i - '' yr,^ -h ' . i ? M k J ays after injection of he adhesion contrasts Smith 't al.: Tests for Carcinogenicity 473 F igure IT. Pleural adhesion between lung and chest wall in another ham ster 244 days after injection of amosite. Air spaces of the lung are seen on the right. This adhesion was very vascular. The dark round or elongated structures are blood vessels. This adhesion contained great numbers of muitmucleated giant cells packed with amosite fibers. 100 x . re* it is invading: the 3re growing between ses. 400 x . w F igure IS. Higher magnification of multinucleated giant cells from the lesion shown in figure 17. Innumerable amosite fibers lie within these cells and in the surrounding tissue. The fibers are best seen by examining the photograph with a hand lens. The large, dark structures are blood vessels. 400 x . j I *_"-*?**>-* '* I-mfl I.........n---- '1---------- --------- ------ --- ' " ..u<IT-*llt>r * Annals New York Academy of Sciences pinkish-white mass was seen extending from the chest through the dia phragm. It was surrounded by a delicate capsule which had perforated at the lower pole, revealing the contents of the mass as soft, lumpy tissue. On opening the chest, this mass was seen to be part of a tumor that meas u r e d 40 x 20 x 15 mm. a n d w as composed of soft, so m e w h a t tr a n s l u c e n t, light-gray tissue th a t largely filled both the rig h t and left pleural cavities, displacing the lungs posteriorly, and covering the diaphragm with a sheet of gray tissue 3 mm. thick which had the soft texture of the tum or in contrast to the tough, yellowish pleural adhesions described in preceding animals. The tum or was composed of plump pleomorphic polygonal cells a n d some a r e a s o f spindle cells (FIGURES 15 a n d 1 6 '. T h ey had a b u n d a n t, moderately acidophilic cytoplasm and large, oval, vesicular nuclei. In a Smith ' jt fw ,tr V i * .V : .* * ft v ^ . . \ '* i ' . * ^ F igure 19. Asbestos granuloma in an interlobular fissure of the right lung 2 .2 days a f te r :r.;ec:ior. of amoslte. Alveolar Itr.g t.s-'ue s seer at corners of tr.e pr.otograph. 1 m s granuloma is densely speckled with giant ceds. 100 y . ~ fibrous adhesion between the right lung and the tumor, many multinuc.eated g.ant cells were packed w ith asbestos fibers. L arge num bers of a s bestos fibers lay in the tum or in the aret adjacent to this adhesion. M eta static ceposits of tum or celis were found in the liver and lymph nodes, -"xhb.c.or.al pr.otograpr.s of th is tu m o r and a m e ta s ta sis of it a r e contained In a prelim inary publication.'' i r . r e e otr.er h a m s te r s o f th is g ro u p died, p re su m a b ly o f ``w et ta il," b^t^erin tn e 2-4th and 252nd day. In one of these, th e lungs w ere not remar.-taole, and a yellowish nodule on the outer side of the thorax showed F igure 20. Higher m FIGURE 19. Innumerable with a hand lens. Mam t h a t th e injected asb* pleural space. In the S i and lower lobes to eac third, thick adhesions i inferior surface of th few delicate adhesioi i between the apex of t i around the right lung l i n u m b e rs of m ultinucl 18-. In an adhesion 1 I islands of cuboidal ce of the pleura. A ham ster sacrifici gm. in w eig h t over t, hesions up to 1 mm. t) in the left. Both righ The pericardial cavit There was a 3 mm. ) lu n g and a n o th e r si scopically, these nodu multinucieated giant **w.uv m **n WJ> ' Sciences je chest through the diaiie which had perforated s soft, lumpy tissue. & tum or that meas- ft, somewhat tran-Iucent, and left pleural cavities, : diaphragm with a sheet texture of the tum or in s described in preceding omorphic polygonal cells 16,. They had abundant, 1, vesicular nuclei. In a ' ''T.>-, C*. *Ar-, i 3 r>* fissure of the right lung issue is seen at opposite speckled with giant cells. tumor, many multinu. Large numbers of as*o this adhesion. Metaiver and lymph nodes, isis of it are contained timablv of "wet tail," e, the lungs were not ?of the thorax showed f 1i i Smith ft a i : Tests for Carcinogenicity 475 &V -t.4t *Sj .' (S- ? ' a V- * ` *^ * tr s t? # . *>*. ~ " * #*' 'f % , ' '> ' *. f - C. - *. , L -,C, ' .. ' v . t, - - - y * j , ,- f vs <3* tt. % " ,, t o " . '* ! ;* . > i^ 5 - *- . * v- ,;T ; -* "* . #?. ' * . . i. v *> * * , > >5 * f. _ ST* ` .1 > ..<*" _ a "ss*. . -r- ... ' v .,,5 , ` :f fct ' T *-i * W MK F igure 20. Higher magnification of multinucleated giant ceils in the lesion of FIGURE 19. Innumerable amosite fibers can be seen by examining the photograph with a hand lens. Many are packed into the giant cells. 400 x . that the injected asbestos had been deposited there rath er than into the pleural space. In the second, pleural adhesions bound the right middle and lower lobes to each other, and to the chest wall and diaphragm. In the third, thick adhesions encased the lobes of the right lung and bound the in f e r io r s u r f a c e of t h e h e a r t to th e d ia p h r a g m I FIGURE 17 j . T h e r e w e r e a few delicate adhesions between the left lung and the diaphragm, and between the apex of the left lung and parietal pleura. The thick adhesions around the right lung were highly vascular and densely cellular with great n u m b e r s of m u ltin u clea ted g i a n t cells loaded w ith a s b esto s fibers (FIGURE 18). In an adhesion between the right lung and heart, there were many islands of cuboidal cells th a t appeared to be sequestrated mesothelial cells of the pleura. A h a m s te r sacrificed on th e 272nd day because of gradual loss of 60 gm. in weight over the preceding three months had extensive yellow ad hesions up to 1 mm. thick in the right pleural cavity and delicate adhesions in the left. Both right and left lungs were adherent to the mediastinum. The pericardial cavity was obliterated by adhesions up to 3 mm. thick. T h e r e w a s a 3 m m . round. .1 firm yellow nodule a t t h e apex o f th e r i g h t lung and another similar nodule adjoining the right ventricle. Micro scopically. these nodules were fibrous tissue studded with large numbers'of m u ltin u c le a te d g ia n t cells (FIGURE 1 9 '. M any a s b esto s fibers lay both , .I.o ^ n in iu 1I.J.,,'Ju^a.; f'JWj iJWl^ . i i WMji,iii.n-?.uAi 476 Annals Xew York Academy of Sciences w i t h i n and o u ts id e th e g i a n t cells (.figure 2 0 ' . In th e th ic k en e d p le u ra , there were num erous clefts lined by cuboidal or rounded cells w ith small vesicular nuclei. Firm and extensive yellowish adhesions up to 2 mm. thick encased both the right and left lungs and the heart of a ham ster dying on the 275th day. To search for possible early stages of neoplastic change, serial sec tions were cut on a block from an area where the right lower lobe was densely adherent to the diaphragm. These showed granulomatous inflam mation. The area between lung and diaphragm, and between lung and chest wall was filled w ith dense fibrous tissue that stained a brilliant blue w ith M allory's connective t is s u e s t a i n (.figu re 21 >. T h is t i s s u e c o n tain e d m any multinueleated giant cells, macrophages, lymphocytes, fibroblasts, and some plasma cells. Innum erable asbestos fibers were present, many w ithin the giant cells. A lymph node contained many macrophages and many asbestos fibers. The lung itself was partly collapsed and markedly congested, but otherwise not remarkable. Many places in the scar near the lung showed islands of cuboidal cells in acinar arrangem ents. F rom the scar tongues of fibrous tissue extended between muscle bundles of the dia phragm and chest wall. These tongues of tissue stained blue with Mal lo r y 's connective tis s u e stain. X o cells w ere fo u n d t h a t could be recognized as neoplastic. Sll On the 358th d which felt boggy shown a sudden p domen showed la necropsy, dense ye of the left lung an face of the heart, adhesions. On the lobe there was a i inal cavity. This n white tumor tissie nodules of such ti: were greatly distc the level of the I nodules. There alt F igure 21. F ibrot.e pleura 1 adhesion between toe rig h t lower ioh-e and the diaphragm 270 days a fte r inject.or. of aroo =.te. A ir spaces of tr.e ;ur.g are in the upper left corner. T.hs sect.or. was sta.r.ed w.-.h M a.lory's connect.ve tissue stair.. The thick p.eural a/ir.eslon stam en a br.ihar.t hire, loo / . Figurk 22. Meto*l and a thick .vibe lor photograph) at. 2 >3 over the pleural sur f Jacob ( linin'. vA nnal,n f n n i i `.hed s t e r fFIClJKMU 22 27 RiffIII la II'I H " 1 rounded by 11 b* ciences the thickened pleura, >unded cells with small r.m. thick encased both 1er d y in g on th e 275th stic change, serial sece right lower lobe was granulomatous inflam and between lung and stained a brilliant blue . This tissue contained mphocytes. fibroblasts, rs were present, many nany macrophages and collapsed and markedly ees in th e scar n e a r the (rangements. From the jscle bundles of the diastained blue with Malthat could be recognized Sm ith t f oK'. T ests tor C arcinogenicity 4t( On the 353th day, a ham -ter was noted to have a distended abdomen which felt boggy and gave a dull sour, a on percussion. Thus aroma! had shown a sudden gain of 30 gm. in v,eight. Sm ear of fluid from the ab domen .snowed la rg e round tu m o r ce. . w ith la rg e v esicn .ar nuclei. .-,.t necropsy, derme yellow acr.t-;o n s were found on the d ia p h ra g m atic surface of the left lung and delicate colorless adhesions on the diaphragm atic su r face of the heart. The right lung was completely encased by dense yellow adhesion-. On the posterior and diaphragm atic aspect of the right lower lobe th ere was a mass extending through the diaphragm into the abdom inal cavity. This mass, 25 mrn. in diameter, was composed of soft, grayish white, tu m o r ti =<ue. In th e m e se n te ry of the bowel, th e re w ere a t least 50 nodules of such tis su e 1 tu i l mm. in c..a.r.etci.'L acteals r. tn e m e sen tery were greatly distended. There was a 6 mm. mass adjacent to the aorta at th e level of the kidneys. The liver had several small, superficial, g ray nodules. There also was a gastric ulcer. right lower lobe and the spaces of the lung are in Mallory's connective tissue e. 100 x . U-.;> t~ Sl'-.L*: F igure 22. Mesothelioma cells (dark gray) lying between the right lung (top) and a thick adhesion (light gray) that bounc this lung to the heart (bottom of photograph) at 35S days after injection of amosite. This tumor spread widely over the pleural surface of the lung. 100 x . Jacob Chm-g, who describes cases of human mesotheliomas in this A n n a l.1' f u r n is h e d th e enclosed note on his stu d y of blocks f r o m th is h a m s t e r (FIGURES 22-27 >. Right lung and tumor. The lung is partly collapsed and is partly su r rounded by a layer of granulation tissue containing asbestos fibers, '1PliiJSH HWfftSMPBp! rnmm !IHiT'`tiJiliitftl'lt'''ift stufladii 47S Annals New York Academv o f Sciences .J i ; ^ V 1 x , .^- v -A 4 F igure 2-3. H igher magnification of an area in figure 22. Polyhedral or irregular*Y-shaped tumor cells occupy the upper half of the p r.x o g ra th . The lower half shows the fibrous oleural adhesion with two niuitinucleateci giant ceil*1. 400 x . F igure 24. Lung with two metastases of the tumor ~rr>~ .r. FIGURES 22 23. 100 x . *5"W Sciences '? V' x i > - i 46J . ^rCrr* * , 7 / A ... * . J&< %** 0 \ . - *V"< * ^ K V ''1 Vb. -/ < *1 - >Kff .*O'"A t 1V ^ %%4 ^> : . *** i ^ 1 ., jr. i> i 22. Polyhedral or irreg e photograph. The lower rltinucleated giant cells. )wn in figures 22 and Smith t-f l.: Te.st.s for Carcinogenicity 479 ^ '*** \ ) r > ) i ./ j '# ib- i ..v * ' /r e ; f> (D v - . ; . * -.V ' ' _*iv - * ;> * '- J. A;* * f* > 3 *' . . < * * ' < .^ 4tv -. * *';* - v * . . - - . h^ o. , i ; ' \ <# * - S ** ^ ^ . / ^ f r w r. ' ? ' ^ * r r ': V -s* e * i Ui .v- , rr rr..' . >f4-o, . ; ?' -. V...'- >V< ' - - : r t V ,' y ` y <' ' 1_* * s ', . o ,. r v v -- -. ... .'-.. y> ^ - * 4 fe, J ^ V iL'r' ' -i*. . ',*> F igure 25. Hieher magnification of the edge of one of the metastases shown in figure 2-4. The compact mass of large tumor cells (right) abuts open alveolar lung tissue (l ef t ). 400 x . macrophages, lymphocytes, plasma cells, numerous fibroblasts and dense collagen. The tum or is located mainly on the diaphragmatic and poste rior surfaces of the lung. It consists of polyhedral or irregularly shaped. .sometimes elongated cellswith variable, generally light nuclei and often prom inent nucleoli. The cytoplasm is distinctly eosinophilic. Man}' mi toses are seen. Asbestos fibers and even occasional asbestos bodies are found between the tum or cells. The tum or infiltrates the base of the lung. A small independent plaque of tum or is found on the lung surface be tween lung tissue and asbestos granulation tissue. An independent nod ule of tum or is found near the trachea. This apparently represents a lymph node metastasis. In addition to the tum or and the rem nants of lymphoid tissue, this nodule contains a considerable amount of iron pigment and a smaller amount of asbestos fibers. Occasional foreign body g ia n t cell is seen. Lung and heart. Asbestos granulation tissue with numerous asbestos fibers is found adherent to the lung and also extending into the inter lobular fissure. Granulation tissue is also adherent to the heart, appar ently having invaded the pericardium. It also extends into the dia phragm. Small nodules of metastatic tumors are found in the lung and in the diaphragm. Metastasis. Metastases are found both in the chest and abdomen. They g^ P P W P 8 P < P i j p i||pWlli.iJii . - u n inm.1iMfwnmujp WWWiPfWWlW W W W 1 imMinrtiftliiiinaiiltii ^rttftfciiiiiiiiiM8aiaM8iMMI^>i<M^il im tum ttHla 4S0 Annals N ew York Academy of Sciences replace the thymus, are seen in the retroperitoneal space, in the liver, kidney, mesentery and omentum, around the pancreas and adrenal, at tached to the serosa of the large and small intestine and the stomach. No tum or metastasis was found in the vertebral column. yjt Stom ach. An ulcer of considerable size is found in th e stomach. It is ih;? q u ite a c u te w ith n ec rosis o f th e m uc osa a n d d e n s e i n n it r a tio n o f t h e '-1 subm ucosa by poly m o rp h o n u c lea r leucocytes. A sb es to s bodies a r e fo u n d in one area at the base of the mucosa distant from the ulcerated area. A ham ster sacrificed on the 423rd day a fte r gradual loss of 55 gm. in weight over a period of six months had ail lobes of the rig h t lung bound to each other and to the parietal pleura by gray adhesions 1 to 2 mm. thick. The right iower lobe and the heart were bound to the diaphragm by a broad, continuous, yellow adhesion 2 mm. thick. These were fibrous ad hesions w ith g reat num bers of giant cells containing asbestos fibers. There were many asbestos bodies. Search was made for areas of beginning neo plasia but none were identified. Some small spaces in the adhesions were lined by cuboidal cells. In a ham ster dying on the 453rd day of an unknown cause, the in jected asbestos evidently had failed to reach the pleura! space but had been deposited partly in the subcutaneous tissue of the chest wall and p a r tl y in in te rco sta l muscle. In th e su b c u tan e o u s a re a , t h e r e was a n 11 mm. spherical nodule in the muscle. On the 4S2nd gain of 52 gm. : erated by adhes dicated congest! the mediastinun each other and t the rig h t lower 8 x 8 mm., lesio which proved to of calcium. Adjt F iqcbe 26. I:-' ` ' ` v.hth metastasis of the t .roor of FIGr/KE 22. Intestinal glar.C-S are or. tre right. Muscle coats of the ir.test.r.e are seer, as a narro-*' band separaneig the giar.-is from the mass of the metastasis. lOo o . F igure 27. Higi are seen intestinal neoplastic spindl graphs, see Refer On the 514th c loss showed equa u re had n o t set ii almost identical prominent lesion angle. Another h hesions in the ri Sciences oneal space, in the liver, ancreas and adrenal, at testine and the stomach, a] column. md in the stomach. It is lense infiltration of the sbestos bodies are found rom the ulcerated area. adual loss of 55 gm. in j f the right lung bound lesions 1 to 2 mm. thick, to the diaphragm by a These were fibrous ad Z asbestos fibers. There reas of beginning neo in the adhesions were known cause, the in pleural space but had o f the chest wall and a, there was an 11 mm. Hr wm m I ; i . r v/z- >; ' - ir - I n c r s E 22. Intestinal "e'en as a n a rro w band 1 1 ! .Smith et nL: T ests for C arcinogenicity 481 spherical nodule of fibrous tissue which was attached to a 5 mm. nodule in the r.uuscie. On the 432r,d day, a ham ster was sacrificed because of edema w ith a gain o f 52 grr.. in w e ig h t in tw o weeks. T h e p e ric ard ia l cavity w as obJit erated by adhesions. Anasarca, ascites and enlargement of the liver in dicated congestive heart failure. The left lung was delicately adherent to the m ediastinum. All lobes of the rig h t lung were delicately adherent to each other and to the parietal pleura with a prominent thickening between the r i g h t lower lobe and the d iaphragm . T his was an ovoid-shaped, 10 x g x 8 mm., lesion with a tough, thick rind surrounding granular material which proved to be necrotic tis su e c o n ta in in g asbestos ftoc-rs and gran u le s of calcium. A djoining the wall of this lesion, a small tum or composed of F igure 27. Higher magnification of an area in figure 26. From right to left are seen intestinal glands, intestinal wail, then the cells of the metastasis. 400 x . neoplastic spindle cells was found in the microscopic sections (for photo graphs, see R eferen ce 13, FIGURES 7 and 8 >. On the 514th day, a hamster sacrificed because of weakness and weight loss showed equally extensive obliterative pericarditis but congestive fa il ure had not set in. Gross findings in the right and left pleural cavities were almost identical w ith those described for the preceding animal, the most prominent lesion being a 6 x 4 x 4 mm. nodule in the right costophrenic angle. A nother hamster, that died on the same day. had very sim ilar ad hesions in the right and left pleural cavities w ith a 4 mm. firm granular m ' l""1111' U I K W H W J mm i 4 4S2 Annals New York Academy of Sciences nodule in the right costophrenic angle. There was a 6 mm. grape-like clus te r of thin-walled, fluid-filled cysts in the median lobe of the liver. An S mm. thin-walled cyst was in the median lobe of the liver of a ham ster sacrificed on the 552nd day because of congestive failure associated with obliterative pericarditis. Both lungs were densely adherent to the mediastinum. The base of the left lung was bound to the diaphragm by a wide m at of yellow adhesions 2 mm. thick. All lobes of the rig h t lung w ere bound to each other and to the parietal pleura by dense adhesions. There was a 7 x 5 x 5 mm. mass of yellow adhesions in the right costophrenic angle. One animal o f Group 4 w as lost. T he last of the 15 ham sters in th is group was sacrificed 559 days afte r injection. He was a big animal in excellent condition. There was delicate adhesive pericarditis. Both lungs were adherent to the mediastinum. Delicate adhesions bound the left lung in some places to the chest wall and the diaphragm . All the lobes of the right lung were adherent to each other and to the parietal pleura, the thickest adhesion being a 1 mm. yellow mat, between the right middle and rig h t lower lobes, and a 7 x 3 x 3 mm. yellow m at between the rig h t lower lobe and the diaphragm. The liver contained ten thin-walled cysts 1 to 11 mm. in d ia m e t e r filled w i t h clea r iluid. It will be apparent that in the last four ham sters described, the asbes tos had evidently been injected into the most suitable position for induc tion of pleural mesothelioma, and these four animals had lived 270 to 315 days longer than the time of detection of the first mesothelioma in Group 4, yet no tu m o r resulted in them. Intratracheal Injections To gain experience with the technique for intratracheal injections of hamsters-' trials were made with suspensions of India ink in saline. The animals used in these trials were sacrficed immediately after injection, and examined for site of deposition of the ink. In the first groups, the ink was sometimes found in the lungs and sometimes in the stomach, the latter event showing that, the pipette had been introduced into the esophagus rather than the trachea. As experience a as gained, it was found that ink could be reliably deposited into the lungs by delivering the injection only when pressure on the chest wall caused a rise in fluid level in th e pipette, showing that the pipette was positioned in the trachea rath e r than in the esophagus. T hese tests also showed th a t a volume of 0.1 mi. of saline could be safely injected, but th at a volume of 0.2 ml. caused deaths immediately after injection, evidently from asphyxiation. In undertaking tests for carcinogenicity of samples of asbestos by the intratracheal route, a first requirement v.as to learn the amounts of these materials that could be injected into ham sters without unacceptable losses of animals. Fc had been used and harsh chr the soft chrys led to deaths ; three samples ; were then util \ administered 1 , have been mail ; tumors. J j j F o r ty - tw o h. [ on weekly intr; \ sotile Sample \ 0.1 ml. T h e ami ! the description j in an effort to < in groups treai \ hamsters each G roup 5. T w mg. harsh chrj mediately after animals died. F 1.25 mg. T hree doses which we four survived a 12.5 mg. of har respectively, af' A second coi tions of 1.25 m pending rr.ediur; in this cohort, series of 24 injt tile. Of these, I a f te r th e first ir the animals that o f t h e iiver. Pe tum or in any of G roup fj. Of 1 soft chry^otile F jection and aii o A second coho Ai'TViwrti I "Sciences s a 6 mm. grape-iike du?m lobe of the liver. obe of the liver of a hamfestive failure associated densely adherent to the id to the d ia p h ra g m bv a >es of the r i g h t lu n g w ere v dense adhesions. There in the rig h t costophrenic th e 15 h a m s te r s in th is w as a big anim al ir. ex pericarditis. Both lungs dons bound the left lung gm. All the lobes of the the parietal pleura, the itween the right middle >v m a t between th e r i g h t ed ten thin-walled cysts ;rs described, the asbesable position for inducals had lived 270 to 315 mesothelioma in Group ; , . : . > 1 i | ; 1 \ { j *s j * atracheal injections of India ink in saline. The tely after injection, and rst groups, the ink was the stom ach, the latter ;ed into the esophagus , it was found that ink ring the injection only tid level in the pipette, diea rather than in the f 0.1 ml. o f saline could ied deaths im m ediately lies of asbestos by the 5 the amounts of these nit unacceptable losses j ! | | j j | Sm ith f-t of. : T ests for C arcinogenicity 483 of anim als. F o r th is work, we u.-ed th e same th r e e samples of asbestos th a t had been used in the preceding tests by the intrapleural route. The arr.osite and harsh chrysotile fibers settled and resuspended readily in saline, but the soft chrysotile fibers tended to form a gel which choked anim als and led to deaths immediately a fte r intratracheal injections. Doses of these three samples which caused only occasional losses after single injection were then utilized for E xperim ent B to explore amounts th at could be administered by repeated weekly injections. Survivors of E xperim ent B have been maintained throughout their lives to observe for development of tumors. Experiment B Forty-two hamsters divided into three treatm ent groups were started on w eekly in t r a t r a c h e a l in je c tio n s of h a r s h chry so tile S am ple 1, s o f t c h r y sotile S am p le 2, o r am o site S am ple 7. T he volume of each injection was 0.1 ml. T h e am o u n t of asbestos and th e su sp e n d in g m e dium is sta te d u n d er the descriptions of these groups. Several suspending media were utilized in an effort to reduce losses from injections. Because of high initial losses in g ro u p s tr e a te d w ith both ch ry so tile samples, ad d itio n a l cohorts of 15 ham sters each w ere started on these samples. Results were as follows. Group 5. Twelve h am ste rs w ere given an in tra tra ch e al injection of 2.5 mg. h a rsh chrysotile Sample 1 suspended in 0.1 ml. saline. One died im mediately after injection. This dose was repeated one week later, and two animals died. For subsequent weekly injections, the dose was reduced to 1.25 mg. Three animals died immediately after the first of these reduced doses which were adm inistered in casein suspension instead of saline. Only four survived a total of eight injections, each thereby receiving a total of 12.5 mg. of h a rsh chrysotile. These lived until 181, 216, 356 and 357 days, respectively, after the first injection. Xo tumors were found in them. A second cohort of 15 h a m ste rs w as given weekly in tra tra c h e a l injec tions of 1.25 mg. of this sample, but hum an plasma was used as the sus p e n d in g m edium . A f t e r 12 weekly injections, th e re had been only one dea th in this cohort. The injections were continued. Xine animals survived a series of 24 injections, each thus receiving a total of 30 mg. harsh chryso tile. O f these, five died a t 173. 175, 314. 321 and 404 days, respectively, afte r the first injection, and four are still living. Xo tumors were found in the animals that have thus far died in this cohort aside from a hemangioma o f th e liver. Roentgenologi al exam ination of the chest does not show tum or in any of the four survivors. G roup i>. O f 15 h a m s te r s g iven a n in t r a t r a c h e a l in je ction of 2.5 mg. of soft chrysotile Sample 2 in 0.1 ml. saline, four died immediately a fte r in jection and all of the others died within the following week. A second cohort of 15 ham sters w as then given an in tratrach eal injec- TT**" .ivjiijmimi ....... ............. .-.^,,,,...^,,..i.,.figa^iyir i'iW^<wrfferi^rTrt>*%MnrTTiiejadaiia^B^itaiaia6haMfeaa 4S4 Annals New York Academy of Sciences tion of 1.25 mg. of th is sample in 0.1 ml. saline. T h ere w ere no losses of animals within the first week, and this dose was repeated to a total of ten injections at weekly intervals during which seven animals died imme diately afte r the injections. In the 16th week, two died of "wet tail." Six su rv iv e a t p rese n t. 15 m o n th s a f t e r t h e i r first injection, h a v in g received a total of 12.5 mg. of soft chrysotile. Roentgenological exam ination of the chest does not show a tum or in any of these six survivors. G roup 7. F if t e e n h a m s te r s w e re given i n tr a tr a c h e a l in je c tio n s of 2.5 mg. of amosite Sample 7 in 0.1 ml. saline. One died immediately a f te r the first injection and another immediately after the third. Thirteen survived a course of ten injections, thereby receiving a total of 25 mg. each. Of these survivors, th r e e died between 147 and 16S days a f te r the first injec tion. Seven died of "wet tail" between 209 and 216 days, one at 442 days, one at 483 days, and one is still alive. No tum ors were found in those that died. Experiment C T hree groups of 60 ham sters each were started on weekly intratracheal injections of, respectively, amosite, soft chrysotile, or saline. A fourth group was set aside as an untreated control. The preparation of amosite used in this test was from the same batch used in all preceding tests. The chysotile was prepared from the same ore as that used in the preceding work, but was processed to an average fiber length of 30 microns. T w enty-one h a m s te r s survive 11 m onths a f t e r th e first injection of the amosite sample, of which they each received a total of 25 mg., delivered in ten weekly doses of 2.5 mg. apiece. Twenty-five ham sters survive six months after the first injection of the chrysotile sample, of which they each received a total of 6.25 mg., delivered in ten weekly doses th a t were reduced, because of losses, from 1.25 to 0.5 mg. apiece. No tu m o rs have been found in the respiratory tract of any animals that have thus far died in this experiment. In sum, no tum ors have been found in the respiratory tracts of ham sters that have thus far died :n any of the tr e a te d with, u-sfoe.-tos b y the intratracheal route. Each of the samples of asbestos induced pulmonary lesions resembling asbestosis, as will be described in another place. Large numbers of asbestos fibers were widely distributed through rungs, as was readily seen in the case of amosite or harsh chrysotile by closing the diaporagm of the microscope condenser in exam ining th e sections. This procedure seldom visualized fibers of soft chrysotile in tissues, but asbestos bodies were found with ordinary illumination in lungs treated with soft chrysotile, in addition to those treated with the other varieties of asbestos. There were numerous islands of adenomatoid tissue ar.d occasional small. -.lUV'1-- i HW" iv- Sciences There were no losses of repeated to a total of -ven animate died irr.me et died of "wet ta il." Six jection, having received gical examination of the urvivors. racheai injections of 2.5 ed im m ediately a f t e r the third. Thirteen survived .otal of 25 mg. each. Of lays after the first iniec!6 days, one a t 442 days, :ms w ere fo u n d in th o se 1on weekly intratracheal file, or saline. A f o u r t h : p re p a ra tio n of a m o site i all preceding tests. The it used in th e preceding h of 30 microns. r the first injection of otal of 25 mg., delivered re ham sters survive six i sample, of which they weekly doses that were apiece. Xo tum ors have aals th a t have thus f a r story tracts of hamsters Red w ith asb esto s by th e itetos induced p ulm onary !1in an o th er place. L arg e id through lungs, as was hrysotile by closing the lining the sections. This ile in tissues, but asbestos n lungs treated with soft ther varieties of asbestos. ?sue and occasional small. Smith ct nL: T ests for Carcinogenicity 485 calcified pleura! plaques. Fibrosis was a prominent feature, b u t it tended to be delicate and interstitial. In no instance did these ungs show thicx fibrotic lesions com parable to those described in the h am ste rs given single massive intrapleural injections. D iscussion The three preparations of asbestos studied each induced granulomatous inflammation, necrosis, calcification, and fibrosis afte r intrapleural in jection into hamsters. These reactions were most extensive on the side in which the injection had been made, but transport of injected fibers was a t te - te d bv t h c- f re q u e n t f i r d i n g o f s im ila r reactions in the peric ard ia l cavity and occasionally in the contralateral pleural cavity, in eai.y spetim ens'int ceiiular response consisted principally of macrophages. Later, multinu- \ cleated giant cells, often filled with asbestos fibers, were numerous. Still later, fibrosis was the outstanding feature. Tissue reactions of this nature formed extensive adhesions which encased lobes of the lungs and some times the heart. Granules of calcium developed in these adhesions. The i thicker adhesions tended to have necrotic centers. By closing the diaphragm | of the microscope condenser, asbestos fibers were often readily visible in i large num bers in histological sections of pleural adhesions in hamster^ \ that had been injected with amosite or harsh chrysotile, but were rarely j visible in ham sters injected with the preparation of soft chrysotile. Multi| nucleated giant cells were much more numerous in pleural adhesions of j ham sters treated with amosite than in those treated with chrysotile. ] Pleural mesotheliomas were found at 244. 338 and 482 days a fte r in tra ! pleural injection of amosite. at 419 and 527 days a fte r injection of har^h S chrysotile. but not a f te r injection of soft chrysotile in groups of 15 h am ? sters given single injections of 25 mg. of preparations of these materials. { Amosite and harsh chrysotile fibers have less w ater associated with the { m olecule1- an d can be felt to be s ti lle r an d m ore spiky to th e touch w hen handled. Histologically, two of the tumors resembled epithelial types of mesotheliomas seen in man. and three resembled fibrous types.- Injection ' of asbestos into the pleural cavity, as done in this experimental worm j does not. of course, reproduce conditions of exposure in man. The experi i ments do show, however, that intrapleural injection in ham sters affords a J means for estimating the potential tumor-inducing action of asbestiform S minerals. Intratracheal injections, which more closely sim ulate the route of ex posure in man. have thus far failed to elicit tum ors in ham sters treated 1 w ith the three preparations of asbestos studied in the present work. A fter : intratracheal injections, fibers of asbestos were widely distributed through 1 out the lungs but no lesions resulted that were comparable in thickness to the fibrotic areas found after intrapleural injection of a massive dose of < i - '~ii*rTl>HrMlft,iiti i'iViitori11- ^ ^ w .i.., 4S6 Annals New York Aeademv of Sciences asbestos. In the four large tumors found in the pleural space, the precise point of origin could not be determined. The fifth tumor, however, was small and it could be seen to have arisen in the thick fibrous wall of a pleural adhesion. Similarly, a sarcoma was found in a thick fibrous wall that surrounded a deposit of soft chrysotile in the subcutaneous tissue of the axilla in another ham ster in this work. These observations suggest that asbestos may posses no prim ary carcinogenicity, but that tumors may arise from the reactive tissue engendered by it. W arburg has detailed evidence supporting a theory that carcinogens act by i n t e r f e r i n g w ith cellular r e s p ir a tio n .1' T he pleural adh e sio n s seen in early specimens in the present work were highly vascular, but in the later specim ens v ascular channels w ere choked off by th e development of dense fibrous tissue. Respiration of many cells in densely fibrotic pleural ad hesions was manifestly impaired as evidenced by the many central foci of necrosis described in these lesions. It may thus be that tum ors associated w ith asbestos arise from cells whose respiration is impaired by en tra p ment in dense fibrous tissue. In our early case of a fibrous type of meso thelioma. the tum or was. as mentioned above, seen to arise from such dense fibrous tissue. As mentioned in descriptions of other specimens from these experiments, somewhat cuboidal cells were often seen lying in islands or lining clefts in fibrous pleural adhesions. These cells resembled mesotheiial ceils of the pleura. E n trap m e n t of cells preserving these characteristics in fibrous tissue affords a possible theoretical explanation for the develop ment of epithelial types of mesotheliomas. exposed to as- have been de em asbestos in this may be a : animais thus t a r studied. A g a in s t th is concept sta n d s tn e r.nc .reinoma is not unusually frequent in the lungs of men with the ucec r.y silica .1 Wagner, however, has reported a mesothelioma :L` irtrap.ir'jrai in :eetion of silica. :i provides an rr. observa rions described in the present paper on tissue reactions precei ' appearance of such tum ors in ham sters it can be seen th a t they followed exteus.". e f.orotic reactions. In the present work, the fibrotic reactions were iu i-e e d by asbestos. It is possible that mesotheliomas may arise in ham sters from other m aterials th a t induce equivalent pleural fibre sis. The findings thus leave unanswered the question of whether asbestos fibers to -ses prim ary carcinogenicity. Because of a recent report: of an increased frequence of carcinom as in Smi the gastrointestina' asbestos, we added ments reported her In these animals th invaded the panerea lymphomas and chi lesions in hamsters tumors of hamsters was an anaplastic i is being studied fu asbestos fibers in ti The question of ' carcinogen present ported in an accomi Gross and histol injections of asbest adhesions ensued. I hamsters that had that had received a No tumors have given repeated intr; monary fibrosis occ not comparable in t injections. The experiments Hters. They raise a carcinogenic action epiphcriomena o f fib Roentgenological experiments was m In-partment of Ita> N. .1. Their collabo I .Sm i t h , W m . K. u{ nccnpatioiia Vi 20.T ( IP i. :u itnoi t, i .7., . hi a pla sia. .1. A ! VVM.M it. .1. C. Mu |ili iira hy I Mu IMI. *.*** -m i.y nwgfcr* Sciences Smith f t fil.: Tests for Carcinogenicity 487 leural space, the precise tb tjrr.or, however, was th ick fibrous wai! of a I in a thick fibrous wail ; subcutaneous tissue of se observations suggest ty, but th at tum ors mav the gastrointestinal tra c t of an occupational group of men exposed to asbestos, we added a.-bestos to th e d iet of ham sters in one of th e ex p eri m ents reported here, and m aintained them on such feed thoughout life. In th ese anim als th e re w ere occasional g a s tric ulcers, or.e lym phom a th a t invaded th e p a n c re a s, and a cho'.an g io c arcin o m a of th e liv er. G a stric ulcers, lymphom as and cholangiocarcinom a have been described as spontaneous lesions in h a m ste rs by F o rtn e r f t ol. - in valuable p ap ers on sp o n tan eo u s tum ors of ham sters. In one of our ham sters th at was fed asbestos, there ory th a t carcinogens act . w as an anaplastic neoplasm in the m esentery n ear the colon. This lesion eural adhesions seen in is being studied fu rth e r by a recently developed method fo r detection of ascuiar, but in the later asb esto s fib'-:-- in t i - s u t ' le developm ent of dense , The question of w hether asbestos fibers may potentiate the action of a sely fibrotic pleural ad- ! carcinogen present in cig arette sm oke was explored in experim ents re the m any central foci of ' p orted in an acco m p an y in g p a p e r.11 i th a t tum ors associated i Co n c l u s io n is im paired by entrap- ; a fibrous type of meso- ! Gross and histological changes following single m assive intrapleural to arise from such dense j injections of asbestos into ham sters have been described. Thick fibrotic er specim ens from these i adhesions ensued. L ater, pleural m esotheliom as w ere found in occasional seen lying in islands o r ! ham sters th a t had received b rittle varieties of asbestos but not in those Is resem b led m e so th e lial j th a t had received a s o ft v a rie ty . ug these characteristics j No tum ors have as yet appeared in the resp irato ry tra c ts of h am sters an atio n fo r the develop- J given repeated in tratrach eal injections of these varieties of asbestos. P ul m onary fibrosis occurred following in tratrach eal injections, b u t th is was ja n im a ls exposed to as- not com parable in thickness to th e fibrotic lesions induced by intrapleural no cases have been de- j injections. resu lt from asbestos in j The experim ents show th a t asbestos can induce m esotheliom as in ham ?ts th a t th is m ay be a > sters. They raise a question as to w hether these tum ors signify a prim ary je lu n g s o f an im als th u s carcinogenic action of asbestos fibers or are secondary and nonspecific ;g th a t carcinom a is not I epiphenom er.a of fibrosis. >rosis induced by silica.1 ' A cknow ledgm ent a rat after intrapleural Roentgenological exam ination of the chests of selected ham sters in these m y m eans provides an tors. From observations preceding appearance of llowed extensive fibrotic experim ents w as m ade by F ra n k lin Reed. M.D. and Bruce B u rtan . M.D.. D epartm ent of Radiology. M orristow n M em orial H ospital, M orristow n. < X. J. T heir collaboration is appreciated. j References :tions w ere induced by arise in ham sters from rosis. The findings thus >s fib e rs p osses p rim a ry luency of carcinom as in i : ; i 1. S m it h , W m. E. 1952. Survey of some cu rren t B ritish and E uropean studies of occupational tum or problems. A. M. A. Arch. Indust. Hvg. Occup. Med. 5: 212-2*13 (R efer to pp. 249-254. 262-2631. 2. S elik o ff. I. J.. J. Ch i sc. & E. C. H ammond. 1964. Asbestos exposure and neoplasia. J. Am. Med. Assn. 1SS: 22-26. 3. W agner. J. C. 1962. E xperim ental production o f mesothelial tum ours of the pleura by im plantation of dusts in laboratory anim als. N atu re 196: 1S0-1S1. ` ` ..................................... * 'I 1"HU WWRPWPWI. PP* i \ Mnuti itt 4SS Annals Xew York Academy of Sciences I j 4. Smith. W m . E.. L. Miller. J. Churg & I. J. S elik o ff. 1964. P leural reaction and mesothelioma in ham sters injected w ith asbestos. Proc. Am. A ssn. Cane. Res. 5: 59. t i I TESTS FOR EF CARCIXOGE 5. Yorwalp. A. J.. T. M. Durkax & P. C. Pratt. 1951. E xperim ental studies of asbestos:?. A. M. A. Arch. Indust. Hyg. Occup. Med. 3 : 1-43. j j Lionas 6. B ehrens, \V. 1951. ber experim entelle A sbestosis. Schw eiz. Z. Path. 14: 2T5-297. j \ Health Res,\ 7. X orpmaxn. M. & A. Sorge 1941. L ungenkrebs durch A sbeststaub im T ier- ; versuch. Z. Krebsforsch. 51: 16S-1S2. ' S. Ly n c h . K. M.. F. A. McIver & J . R. Ca in . 19-57. P ulm onary tum ors in , mice exposed to asbestos dust. A. M. A. A rch. Ir.dust. H ealth 15: 207-214. > In a preced in g pa 9. W agner, J. C. 196-3. Asbestosis in experim ental anim als. B rit. J. Indust. Med. 20: 1-12. 10. Sugiura, K.. W m. E. S m ith & D. A. S underland. 1956. E xperim ental i asso cia te d w ith an i> theliom as in m an, b production of carcinoma in rhesus monkeys. Cancer Res. 16: 951-955. ourselves, have faik 11. B alm)Liact, M. S. & W. A. Gantt. 196-5. P re p aratio n of asbestos fibers for experimental use. This A m ai. 12. S m ith . W m . E. 1956. E xperim ental Studies. I n Pulm onary C arcinom a: > anim als, except afte to find m alig n an t tt Pathogenesis, Diagnosis and T reatm ent. E d g ar M ayer &. H. C. M aier, : inhalation exposure ii Eds. : 54u. Xew York U niversity Press. Xew Y'ork, X. Y. 13. S m ith . W m. E., L. Millet:. J. Churg & I. J . S elikoff. 1965. Mesotheliomas [ pothesis th a t pulmo in ham sters following intrapleural injection of asbestos. J. Mr. Sinai Hosp. not be attributable t li 32: 1-8. hut rath er to a pron 14. S elikoff. I. J., I. G. Tchertkoff & E. H. Robitzek. 1948. In itia l pneumothorax. A new safe induction technique. Q uart. Bull. Sea View Hosp. : carcinogen (s > presei 10: 93-100. seemed possible that 15. H errold. K. M. & L. J. Du n h a m . 1962. Induction o f carcinom a and papil volved. E xperim ents lSii loma of the tracheobronchial mucosa of the Simian ham ster by intratracheal instillation of b en zo (a) pyrene. J . X'at. Cane. In st. 28: 467--492. Benzo [a j pyrene w 16. B erkley, C.tJ . Churg, W m . E. S mith & I. J . S elikoff. 1965. The detection ' c ig a re tte smoke.-' Se i and localization of m ineral fibers in tissue. This A nnal. rold an d D u n h a m ' hi 17. Chup.g, J., S. Mglten & S. Rosen. 1965. Histological characteristics of methothehoma associated with asbestos. This A m ai. and carcinom as in 18. W arburg, O. 1956. On the origin of cancer cells. Science 123: -399--314. benzo[a ] py re ne susp 19. F ortner. J. G., A. G. Mahy & G. R. Schp.odt. 1961. T ra n sp lan tab le tum ors of the Syrian (golden) ham ster. P art I: Tumors of the alim entary tract, endocrine g.ar.ds and melanomas. Cane. Res. 21< 2 ) : 161-198. Two varieties of : c h ry so tile p ro v id ed L. 20. F Op.tnep., J. G-, A. G. Mahy cc R. S. Cotran. 1961. T ransplantable tum ors icai School. T his m a` of the Syr .an (golden) ham ster. P art II: Tumors of the hematopoietic 15 m icro n s. X o car.ce tissues, g en.tourinary organs, m am m arv glands and sarcomas. Cane. Res. i i 21(2): 199-234. " ' 21. Mille?.. L., W m . E. S mith & S. W. B erliner. 1965. T ests for enect o f as in je c tio n s o f th is rr.a" tio n v. as a sam p le of bestos on fcer.zo (a) pyrene carcinogenesis in the resp ira to ry trac t. This Armai. ra to ry ' and found to laboratory, m esothei: injection of th is prep yet been found a fte r into ham sters.1 Male golden S yria: Colony, N'ev/field, X. -J ` This work was -up;. 07681 from the Xat.or Milhken Feur.datior-