Document JvrqqzO76rvBNZRrOqk6aq3ja

l i ;|i :|i . :1 | \. ; \: ;' I i. > j :i^ : | [: i : j. . Hr. ./, Cancer (1074) 29. EXHIBIT I j THE EFFECTS OF THE INHALATION OF ASBESTOS IN RATS J. C. WAGNER, G. BERRY, J. W. SKIDMORE am> V. TIMBREL!- From the Medical Itreearch Council'e l>neutnoconio/tie Cnit, IJnndough llorjiitol, I`(north, (Hantortjnn Kwivetl fi Scptcmltcr 1073. Accepted 20 Xovomlter 1073 Summary.--Two experiments In which SPF Wlstar rats were exposed by Inhalation to dust clouds of the UICC standard reference samples for periods of between one day and 2 years are described. All the samples of asbestos produced asbestosis which continued to progress after removal from exposure but only a little fibrosis was observed in control rats. Lung tumours, ranging in severity from adenomata to squamous carcinomata, were produced by all samples but in the controls there were only a few adenomata and none of the more serious tumours. Of the 20 tumours which metastasized, 16 occurred after exposure to one or other of the 2 chrysotile samples. In addition, a total of 1! mcsotheliomata occurred, 4 of which were with crocidollte and 4 with Canadian chrysotile. Two of the mcsotheliomata occurred with only one day's exposure to asbestos. There was a positive association between asbestosis and lung tumours. Wagner, Berry ami Timbreil {197X1) reported tho results of cxj>crimcnts in which rnts were inoculated intrapleuraliy with samples of asbestos. In the dis* eussion it was mentioned that two cxjicri* ments in which rnts had been cxj>osed to dust clouds of tho UICC reference samples had been carried out, and the results of these are now presented. Some preliminary results of one of these experi ments were rej>ortod by Wagner (1072). In both experiments tho rats were exposed to similar dust concentrations and the dose varied by exposing rnts for various lengths of time. The main aim was to establish the relationship between the development of malignant tumours in tho lungs and the dose and type of asbestos dust but, additionally, the amount of fibrosis was assessed. MATERIALS AND METHODS Caesarean derived rats of tho Wistar strain were used which had been bred at tho Unit from SPF stocks given to us by the Imperial Chemical Industries, Pharma, ceutical Division at Atdcrley Edge, Cheshire in 1054 and 1 DCS. The asliestos samples used consisted of tho 5 UICC standard reference samples (Timbreil, Gilson and Webster, 1908) which were prepared following recommendations of the UICC. These samples are of 3 amphibole types--amositc, nnthophyllitc and erocido* lite--and 2 chrysotilcs--a Canadian and a Rhodesian sample. Tho rats were exposed in 1*4 m* inhala tion chandlers (Timbreil et al., 1970) which contained 8 cages, each of which could hold 0 rats, or, for a short period with young rats, there was room for a seventh. Five chamliers were used, one for each type of asliestos. The chamliers were constructed so that the rats could be tended without the chandlers being ojiened. The dust clouds were generaied using a specially devised dispenser (Timbreil, Hyctt and Skidmore, I9G8). The clouds were generated for 7 hours a day and 5 days per week. Tho respirable dust concentrations were measured using size selective gravi metric dust samples (Cassella Type 114A). Tho collected samples wore evaluated at the end of each daily session. In order to achieve tho required dosage, calculated as tho product of concentration and time, variations occurring in the concentrations were corrected on the following days. At tho end of exposure the rats were loft in the chambers for a few days, to allow time for *ATS inhalation tween one usbestosis le fibrosis idenomata trols there .'0 tumours chrysotile were with j occurred >n between cd of -o ..mples -MS) which mlations of I amphibole nd crocidoliun and a m3 inhala: 070) which could hold with young ontli. Five .ch type of constructed without the ted using a broil, Hyett Joudx were 5 days per icentrations ticc graviypo 1I4A). aiuatcd at /n order to dcnlatcd as and time, icentrations : days. At .* Ier -i the >w for , i 1 I | * : their fur to become clear of asbestos before transferring them to a clean environment for the remainder of their lives. At transfer some rats, generally 2 or 3 of each sox, were killed and the lungs removed for histological examination and determination of dust content. As each type of asbestos has a consider able silicate content (about 50%) the amount of dust in the lungs was determined by first assessing its silica content and referring this value to that of the respirable dust to which the rat had been exposed. This method was used in previous experiments (Morris et at., 1967). ' After exposure the rats were caged in threes or fours isolated in a special unit supplied with filtered air. The inhalation chambers were also in this unit but in a separate room. They were fed on a pro prietary brand of autoclaved cubes, and water ad libitum. Except for the scheduled killings, each rat was allowed to live until it died or appeared to be distressed, and a full necropsy examination was carried out. Histological prejnrntion. staining and in icroscopic methods.--For t he scheduled killings, animals were killed by chloroform anaesthesia and following exsanguination the thorax opened and the lungs removed. The left lung was air inflated and suspended in formalin. Representative portions of the right lung were taken for electron microscopic examination and the remainder of the lung dilated with neutral buffered formalin. Slices were taken from both lungs for histological examination. After the histological sections had been cut, the embedded tissue and the trimmings were dewaxed and added to the remainder of the lungs which were used for the chemical estimates. For other animals, at post mortem the lungs were dilated with neutral buffered formalin and after fixation wore sliced sagittally, routino sections being taken of the whole left lung and the upper and lower lobes of the right lung. Iu addition, any other suspicious lesions from the lungs or other organs were taken for histological examination. In all cases, sections were stained with haematoxylin and eosin and the lung sections were stained for elastin, ruticulin and col lagen. Special stains wore used in some cases as an aid to diagnosis of the tumours. of the severity of nsbestitsis.-- Sections of both Jungs were examined without knowledge of the duration or type of asbestos exposure and with the animals in random order. The sections were observed on a viewing screen of a Projcctina Microscope 4013 BK using a X7 objective. At this magnification a large proportion of the lung could be assessed in a single field and. as it was not possible to observe asbestos fibres at this magnification, the results were not biased by knowing the type of asbestos to which the animal had been exposed. Experiments In both experiments and for all doses there were groups which were exposed to all 5 reference samples; there were also control groups which wore not exposed. Rats were allocated to treatments at random. At the start of the exposure the majority of the rats were between 5 and 7 weeks old. a few being slightly older or younger, and there were approximately equal numbers of males and females. Experiment I.--There were 2 time inter vals of exposure; in the first, rats Here exposed for 3 months starting in May 1967 ami in the second, groups of rats were exposed for one day only iu August 1967. In addition to the killings at the end of exposure, in tiio 3-month group there were also intermediate sacrifices at 5. 8 and 10 weeks. Experiment 2.--There were 3 time inter vals of exposure--6 months, 12 months and 24 months. The experiment started in January I960 and after 6 mouths half of the rats were removed from the cabinets. They were replaced and a year later these replacement animals were in turn removed. They wore replaced by animals to Ik* used for speeial electron microscopy examinations which will not be reported in this paper. The remaining animals were removed in January 1971 after 2 years' exposure. In the (I-month groups, in addition to the killings at the end of exposure, rats were also killed after 2 years, i.e. 18 months after removal from exposure. The numbers of rats are given in Table I. In the 24-month groups, overcrowding amongst the males tended to occur due to increase in size after al>out a year, and some were removed prematurely after 13$ months. For analysis these rats have been i .. t t .1. (\ WAtiNKK. ItKKUY, .1. W. sKIlhMOBK AM> V. TJMBKKU. "f I <lny :i month* (> month* 1 2 mouth* 1'4 month." TaIU.K I.--S anther of HaU Exposed Amiw-ito 40 .72 24 27 21 Aoiliojthylhio -40 V* .'4 2S 1!) tVooidoliu' 10 '' 24 2t> 20 C}iry*otiI( (Cmmiimn) 40 24 2:i 24 Cliry*otilr (IthoilCMIUl) Control 49 4S V* -,s its 1 -7 4S i l^'liyth of r\po*urt 1 ituy 0 mouth* <! month* 12 month* 24 month* Tahi.k 11.--Menu Rtspindde f)n.sf ('onecntrulion {ntj'tu n} and ('nniitialivr liose (m/jj/n3 hours) .Amo.-iio 14 1 12-4 11-2 IO-8 lo ti 00 utitU) s.".( 170(10 aaaoo Anthnph.vllitr 12* ta;> 11>- 9 it*4 lod 90 :.mo s.vw it tun aa7oo Crttcitlohio 12 .*> 12 u jo-7 iott io-a SS r.oao s4ao jTonu 00200 Chrv>mi tin (('nnuilmtij 0'7 12 1 10-2 tO-7 10- J OS mao *240 17 Jt*t) 00200 Chrv SOtilf ( UIuhU-miui) 14-7 j2a 10-7 10-9 JO' 1 loa ,*.070 x.-.no 17)00 aaouo included in the 12-month groups and hence the nmnhet of rats in these groups is slightly higher, and in the 24-month groups slightly lower, than planned. In Table 11 the mean respirable dust concentrations and the cumulative doses, tho products of concentration and time, arc shown. The one-day exposure was 7 hours for all dusts. For the other 4 time intervals of exposure, there were slight variations in the muulier of hours required to achieve approximately equal doses hut the mean times were 402. "88, 1"74 and 3237 hours .respectively. The menu concentrations were usually higher in Experiment 1 than K.vj>eriment 2, and the 3-month group had an average dose of (>0% of that of the 0-nionth group. Reasonable equality of dose Indween the dusts was achieved for all the lengths of exposure, except for the one-day which was too short to allow any adjustments. /!i/er;>re/fgio>} tf histological ft mlitigs Classificution of ashcstat<i,--The lesions seen in the Jungs of rats exjKised to all ty}K*s of asl>estos were similar to those descried in guinea-pigs (Wagner, 1003, 100,7). There were 2 main differences; firstly, asU'stos ixxlies were never seen in the lung tissue of the rat although they ore frequently seen in the pleural grnnulomata which follow the intra-pleural inoculation of amphibole fibres; secondly, there was a far greater production of granular pncmnocytes (typo If) alveolar epithelial cells in the rat. The lesions consist initially of a deposi tion of nsliestos fibres, alveolar macrophages ami cell debris in the alveoli arising directly from the respiratory bronchioles. These deposits liecome organized firstly by being enmeshed in a thin reticulin network which coarsens with time and Incomes replaced by collagen fibres. 'Hie alveolar epithelium reacts with replacement of the type ( cells, and ln-comes completely lined l>y granular pnemnoeytes. In some of the alveoli the epithelium is shed into the Ittmina. in others there is u walling (if the alveoli by these cells. This usually occurs at the bifurcations whew groups of alveoli arc closed off from the luniina of the respiratory bronchioles, giving the so-called pseudo-acinar appearanee. In the guinea-pigs these small cystic spaces contained asl>estos fibre and degenerating mueroplmges, but in the rats nmnemtts granular pnemnoeytes were also present. The initial lesions were confined to occasional discrete individual respirutory bronchioles scattered throughout the lung substance. After further exposure, more ana more respiratory bronchioles become involved and all the respiratory bronchioles arising from terminal bronchioles l*come thickened as the fibrous tissue network extends into the wall of the respiratory bronchiole, and this interstitial reaction spreads down into the peripheral elements of the primary unit, involving the alveolar ducts, atria and finally the air sacs and alveoli. With progression, the individual lesions tend to coalesce, leading to the development of a EFFECTS OF INHALATION* OF ASBESTOS IN* HATS loposiullage's irectly T!` I. willed I'd by H'lium colls, tutilur li the others these ations ' from hioles, ap. small ' and ' rats also .'dined atory lung re and olved rising koned ' into . and i into inmry atria With ml of 1 4 I 1 Kin. 2.--Slight aitlN'HloHix (Oniric 4), Wiihor |x>\vrr rimunne immcrmi* n'fructilo cmeiriolito fibre* in dn* ufvetili of a n**|>irwtory bronchiole. Adjesto* IhhIio* nro rarely *et m die tunspc of rat*. Illumination reduced to illustrate tb<* libn**. H. \ E. < Hi. 2f.fi J. (\ W.\(iN'KK <: HKIlliV. J. W. SKIDMOItK AX!) V. TIMJSKKI.L Fm. X--Mtwiomto a*tac*t<w<iM.. (G--nvlo --ft). A lower power projection iliuM rati'ni g SthSat'%the , Wio*r,,t although hi ill ninthly involving thi* respiratory bronchiole in now diffuse. Aggregation* of typo II pnemnoeyto* nro seen in the himina n we!) an investing tho walls. H. & K. x 80. EFFECTS OF INHALATION OF ASBESTOS IN RATS 257 diffuse interstitial fibrosis with gradual in crease in the density of the fibrous tissue, ultimately resulting in the replacement of most of the lung parenchyma by a dense collagen network surrounding distorted air spaces, many of which contain large clumps of granular pncumocytcs which, in some areas, have lysed leaving foci of lipo-alveolar proteinosis. The assessment of these lesions was based on 4 grades of fibrosis--minimal, slight, moderate and severe. Typical ex amples of slight, moderate ami severe asbestosis arc shown in Fig. 1-4. In addi tion, when carrying out the assessment it was found convenient to introduce the intermediate categories minimal/slight, slight/ moderate and moderate/severc. The 7 re sulting categories were scored 2-8 and the normal lung, in which there was no sign of asbestosis, was scored l, and the assessments were averaged for each group of rats. The rejicatability of this form of assessment was tested on 154 sections, reassessed in a different random order and 90% were assigned to within one category of the first reading. Classification of tumours.--The tumours found in the lungs of rats after exposure to the various types of asbestos dusts were peripheral adenomata, widespread adeno matosis, adenocarcinomata and squamous carcinomata. The rarity of pulmonary tu mours in rats has been stressed in the reviews bv Kusclmer ami Laskin (1970) and Shabad and Pvlev (1970). Furtiior, these authors have described the development and morphological features of the tumours that we arc reporting. All these tumours were peripheral and appeared to arise from the region of the respiratory bronchioles in which the asbestos fibre had accumulated. The origin of the adenomata appeared to be from accumulation of type II epithelial cells that proliferate in the alveoli of the respiratory bronchioles (Fig. 5, 0). In many of the exposed animals these tumours were multiple, ami in a number of animals, particularly those with the more severe grades of asbestosis, there seemed to be adenomata arising from numerous adjoining respiratory bronchioles, giving an impression of contiguous adenomata invading largo areas of the lung. Dr Harold Stewart (personal communication) suggested that this type of lesion should be referred to as adenomatosis. In contrast to this, a few control animals were seen to have solitary adenomata which were small in size. The adenocarcinomata were of the same typo and origin as the " alveolar adenocarcino mata " described by Shabad and Pylov (1970); many of these tumours were papillary adenoma-carcinomata. The squamous car cinomata appeared to originate from foci of squamous metaplasia occurring in the asbcstotic lesions in the respiratory bron chioles (Fig. 7. 8). As far as can be ascer tained. ail these tumours were peripheral in origin and not bronchial papillomata. The classification of these tumours was discussed in some detail in Session VI at the Gatlinburg Conference on tlm Morphology of Experimental Respiratory Carcinogenesis in 1970: and the chapter by M. F. Stanton (1974) in the IARC Monograph on Pathology of Tumours in Laboratory Animal# contains detailed descriptions of the tumours that we have illustrated. The rats used in this experiment are from a caesarean derived, barrier maintained colony and fortunately they have been kept free of rat bronchitis; therefore the squamous metaplasia was not associated with bronchi ectasis. Metastases in the thoracic cavity to the chest wall, diaphragm, pericardium or the trachoo-bronchial lymph glands were seen in 14 animals: the majority had lesions invading 3 of the sites and in only 2 animals were metastases observed in the tracheo bronchial glands. In one animal secondary deposits were seen iu sections from a kidney. Eight adenocarcinomata and 6 squamous tumours had metastasized. RESULTS All except 2 of the rats in the groups with exposure of 12 months or loss survived for the whole of their planned exposure. In the 24-mouth group there was appreciable mortality before the end of exposure and only 53% survived for the full period. Out of 1013 rats it was impossible to obtain adequate histological material in only 8 because of cannibalism. Dust retention The mean weights of asbestos dust in the lungs of animals killed at the .1. (*. U\\(J\K1{. (i. MKKHV. J. U'. .sKIDMOKK AXI) V. TI.MUKKU. EFFECTS OF INHALATION' OF ASBESTOS IN' RATS 250 hr.**H 7,__.Sqimmoiw metaplasia *ti|>ortiupo.<ocl *m m-Klerate usbestosis. II. A K. X 2<>0. I fn>. 8.__Kurlv squamous ouroiuomu in an animal with seven* nslx'stnsi*. II. A E. X SOO. 2 i 2fi0 J. WAfJXKR. 0. HKRRV. J. \V, SKIUMOUK ANt> V. TIMUKKU, length of exjxwiro 8 week* 8 weeks 10 week* 3 month* 0 month* 12 month* 24 month* 0 month* (after IK month* nc>n-exjx>*ure) TaM.K HI.-- Dual Hetnined in Lungs (mg) Moan dose mg/m* hour* IKK0 24f>0 3200 5050 8470 I7IOO 33400 -- Amosite to 0-0 20 3-7 4-7 8-3 10-8 1 3 Anthophyllite 1 -3 1 -0 2'8 38 4-4 00 13-8 20 Crocidolito II 10 2- J 30 4-8 03 140 t-2 Chryxutile (Canadian) 0-1 <M 0-5 uo 0-4 U-K 0-3 00 Chryxotile (lUiodesian) 0-1 0-3 0-8 0-7 0-4 1-4 00 01 Is-ngth of i'x|x>*im* l ilny :i month* 0 month* 12 month* 24 month* TaIU.B IV.--Mean Suridval* after First Exposure (days) Amosite 804 771 703 <>02 807 Anthophyllite 80(1 823 080 780 778 Orocidolite 708 817 788 770 780 C'hrvsotilo (Canadian) 703 700 0G0 778 885 Chrysolite (Hhndpsian) Cimt rol 783 803 Hf>7 703 700 I| 820 f 754 788 J Adjusted to J>o independent of sacrifice*. scheduled times are given in Table III. More dust was usually found in males than in females and on average the female lungs contained only 70% as much dust ns the male lungs. The values in Table III are the averages of the mule and female means. For the 3 amphiboles there was a similar pattern, with an almost projKjrtiomd increase of lung dust with dose. The 3 ehrvsotilcs were similar to one another but much less dust was found than with the nmphiboles; also the chrvsotilc figures did not show tho same clear increase with dose. Tho main features are summarized in Fig. D. The dust in the lungs of the animals which had C months' exposure had been partially eliminated 18 months after removal from exposure. The proportions eliminated were 74% for amosite, 73% for erocidolite but only 41% for anthophyllite. How ever, the lower elimination of anthophyllito was not significantly different from the amosite and erocidolite figures. Survival The mean lengths of survival from the day the rats were first exposed are given in Table IV. The survival times have been estimated so as to be inde pendent of the sacrifices. The short survival of the 34-month group exposed to Canadian ehrysotile was largely due to 8 rats dying before Day 400 (in the other four 34-month groups only 3 rats died before Day 400). These early deaths were not due to exposure, since 5 died due to an infection in one cage and 2 were killed in a fight. Discounting these 8 deaths, the mean survival was G98 days, which was still the lowest of the 24-month groups. When the mean was taken over all the lengths of exposure, the Canadian ehrysotile groups showed least survival, but only a month less than the control groups. The amosite and anthophyllite groups had mean sur vivals only a few days less than the controls, while the erocidolite and Rho desian chrvsotilc groups had longer sur vivals of 2 and 3 weeks respectively. Hence, there is very little indication that the exposure had any effect on the overall survival of the animals. This is in marked contrast to our intrapleural inoculation experiments in which injection rysotilo 'xlosiim) 0] 0-3 0-5 0-7 D-4 1-4 '>(5 0. 1 Weight of dust in lungs (mg) EFFECTS OF INHALATION OK ASHKSTOS IN RATS 2>l ntrol '03 :03 M4 sf re 11 ...lies io incle short `X posed -'ly clue (in the 2 rats deaths 5 died and 2 ig these as m of the au was .posurc, showed ith less amosite an sur- an the <1 Rho- :er surctive/y. lication on the . This ipleural ije \ | Ki<;. 9. -- M'-iu* weight of <lu*t in lung* of rata in mint inn to <hwo mu I linn*. of asbestos retluees tile expectation of life by several months (Wagner H ni, The amount of asbestosis was assessed for ail the rats killed at scheduled times in Experiment 2 and after S weeks' and 2 months* exposure in Experiment 1. There were a or <> rats per treatment for eaeli exposure, except that there were only 3 after S weeks. Overall the 2 sexes had similar amounts of asbestosis and they have, therefore, been combined to give the mean asbestosis scores in Table V, which are summarized in Kig. 10. Except for some inconsistency between the 3- and 0-month means, there was an increase of asbestosis with exposure for all the dusts. Also, following 0 months' exposure, then* was progression during the following IS mouths without exposure for all the asbestos types, but these rats did not fan* as badly as those which continued exposure. There were signiUcanf diilerenees between the asbestos types (/* < tH)l): amosite invariably gave the least asbestosis throughout; authophyllite and Canadian ehryxotile showed most asbestosis after t> months1 or longer exposure; eroeidolite and Rhodesian ehrysotile were intermediate. 77te mean asbestosis scores of the rats which were allowed to live out their lives are given in Table NT, Those rats 2f>2 .1. V. WADXKK. (*. ItKKIiV. J. W. NKlDMOitK AND V. TlMltUKU. A*b**to*ia grtde Moderate - Slight -4 Minimal -4 NU J r o 36 12 24 Time (month*) V 1 *l ` l 10000 30000 30000 Cumulative dose fmg/m3 hours) Ktfi. 10.--Af>!x*t<<iK in xneriiiooU mix in Tvlntinn to iloso ntnl time. l*-rij;t)i of fxjKJsuro K wool** 3 month* 0 mouth* 12 month* 18 month* 24 month* 0 month* (nftcr 18 month* mwexpoHUtv) Taui.k V .-- d/mn Ashcstos'is Scores* of Sucrtfici (1 Rats Amosito A)iili<>i>liyllit> Crocidotito Clinxitilo C'hrywilito (('itmulmn) (Khndcsiiiii) 20 2 0 2-5 2-7 o, > 3-2 40 5-2 __ __ 4.1 0-2 3*2 5-0 2 0 20 2-7 2-8 2-7 30 20 no 2-fl 4*3 4-3 4-3 __ _ __ 4-8 GO .`8 :i 7 5- 5 3-7 I; nil, 2: minimal, 4: *h>:ht, 0: tmxlemto, 8: xovorp. x'otitrul 1-3 1-3 1-2 1-2 1 -8 scheduled for 24 months have been divided into those that died before completion of exposure and those that survived for a j>eriod of non-exposure. The amount of nsbestosis found in tiic ruts exposed for one day was no more than that found in control rats. Comparing Tables V and VI for the rats which completed their exposure, progression had occurred be tween the end of exposure and death with all dusts, the single exception being the W months' exposure of Rhodesian chrvso tile. The rats which died before com pleting 24 months' exposure hud more asbestos!* than those sacrificed after 24 months' exposure for amosite, anthophyllite and Rhodesian ehrvsotiie. This was not the case for crocidolite and Canadian ehrysotile, for which those rats timt died during exposure had shorter mean survivals than for the other dusts. Moaned over all dusts, those rats that died usbe: the mon short was the any less i dust' large comj betw types and Tabl< Tublt ehrvs crocii we c no ir of ns) Tunic Li the tu numbf shown ruts v lung, condif obser expos survh to ha sched withii O " (month*) Control 1-3 l -3 1-2 1-2 is a chryso on* eommd more after 24 , anthoilo. This dito and lioso rats l shorter ier its. rut* Jiat EFFECTS OF INHALATION' OF ASBESTOS IN RATS Table NT.--Mean Asbestosis Scores* of Survivors (Mean Survival in Months) Length of exposure Amositc Chrysotil<> Chrvsotilo Anthophyllito CrocidoJife (Canadian) (Rhodesian) 1 flay 3 months Q months 12 months to 24 months 24 months 1-3 (26) 2-9 (28) 3-3 (24) 4-8 (23) 8-0 (23) 0-3 (28) 1-3 (20) 32 (27) 4-2 (20) 0-0 (23) 0-4 (22) 70 (28) 1-2 (20) 3-1 (27) 3-2 (24) 5 0 (23) 4-2 (14) 0-0 (29) 1-2 (25) 3 3 (20) 3-7 (20) 5-1 (25) 5-1 (10) -- 1-4 (23) 2-8 (28) 4-2 (23) 6-1 (27) 01 (22) 0-8 (28) * I: nil, 2: minimal, 4: alight, 0: moderate, 8: severe. 2(13 died during exposure had slightly more asbestosis than would be expected from the sacrifice rats, consistent with the more severely affected animals having the shorter survivals. However, the effect was very slight and, as observed earlier, the exposure did not affect survival to any extent. In Table NT there is again loss asbestosis for amositc than the other dusts although the difference is not as large as in Table V. For rats which completed their exposure, the difference between amositc and the other 4 asbestos types had a mean of 0*7 for rats sacrificed and 0*5 for survivors. The results in Table NT do not support the findings in Table V that antliophyllitc and Canadian clirvsotile produce more asbestosis than crocidolite and Rhodesian chrysotile, ami wc conclude, tiiercfore, that there were no important differences in the amount of asbestosis produced by these 4 samples. Tumours of the lung Lung tumours were observed in 247 of the rats exposed to asbestos. The total numbers of each kind for each dust are shown in Table VII, where for those rats with more than one tumour of the lung, classification is by the more severe condition. Xo tumours of the lung were observed within 300 days of the start of exposure ami therefore only rats which survived this initial period are considered to have been at risk. Apart from the scheduled killings, only 13 rats died within the first 300 days. There were 7 control rats out of 34 survivors in Experi ment 1 with adenomata, but in Experi ment 2 there were no lung tumours out of 42 control rats. There were slightly more male than female rats with tumours --12S compared with 110--but the only 2 tumour types for which there was any major difference between the sexes were adenocarcinoma and squamous carcin oma. Out of 30 adenocarcinomata, 3.7 occurred in males whereas 30 of the 40 squamous carcinomata wore in females. Motastases occurred in 20 rats, 10 of each sex. There were also 11 meso* tholiomata (Table NTI), 7 in males. Two of the mesotheliomata occurred with only one day's exposure, 1 with 3 months', none with 0 months', 0 with 12 months' and 2 with 24 months'. The meso thelioma which occurred with 3 months' exposure to crocidolite was a peritoneal tumour; the others were all of pleural origin. The distribution of the lung tumours with time after first exposure are shown in Fig. 11 for all dusts and all lengths of exposure except one day. In the 5 groups exposed to asbestos for one day there were 14 adenomata and, compared with the 4 in the corresponding controls, there was clearly no evidence that these adenomata were a consequence of expo sure to asbestos. There were 5 more serious tumours; 2 of these were meso theliomata, one with amositc after 71.7 days and the other with crocidolite after 531 days. There were also 3 adenocarcinomata, one with crocidolite after * t - 1 i. S t. J. ('. W.AGNKK. (!. 15KKKV. J. \\\ NKID.MOKK AND V. Ti.MBUKU T.utu: VII.- A*umber of Animal* with Luny T amours or .Mc8<>th(liomntn hx|x<iiro . 1 1" OA1 tt No. til rat* nt risk* No. with lung tumour Adenoma Tvtx* of lung tumour -A .. -- Ailctio* Adenomntosi* enretnomnf --- ^ StjimmouH curciiiomuf No. with meso- thelioma 1 dnv 4.3 3 3 0 0 . 3 Illulit 1| :i7 10 / 30 tt months IJ months IK J5 J to I s 0 4 1 1 4 mouth* 21 13 3 1 3 Tutu! 14 ti 3H Hi K 3 01 0 0 0 o 01 . 1 nlho/ihi/lli/e 1 dnv 3 months tt months 12 months -4 months Total 44 37 IK 2S IK 14r> o 0 20 in .*0 1 0 3 >> >. 0 0 1 0 5 12 0 0 1 4 (I) 3 8 (1) 0 0 1 1 0 8 0 0 0 1 1 2 Crocitioli/e 1 dav 3 months tt months 111 mnutlis 24 tnonths '1 otal 43 30 IK 20 IK 141 0 14 4 IK 13 55 5 10 > r, 4 20 0 > 2 4 r 13 1 1 0 3 2 (1) T <1) 0 1 (1) 0 0 2 (1) 9 (2) J l o 0 0 4 ChnmtiU (Ctiuttiluiit) 1 tlay 3 months <> months 12 months 24 months Total 42 34 17 23 21 137 i IK 5 11 to 45 0 ir> > I > 20 0 1 (1) 0 0 0> 3 0 0o 10 3 0 fl) > (1) 3 3 1 (1) 4 (2) I 8 11 (3) (1(3) 4 (lilitxlmian) l tiny .1 months 0 month* 12 months 24 mouth* Total ContrtJ 1 dnv 11 months 0-24 month* Total 43 30 19 27 17 144 44 40 42 120 5 10 8 10 11 ;'>!) 4 3 0 7 4 II > 2 0 19 4 3 0 7 0 o 3 4 1 10 0 0 0 0 Hut* which survived at least .'JOO day* uftcr start of exposure, t Number* in bracket* utv those with metastasis. 1 3 (1) 3 (I) 7 (2) 6 (2) 19 (0) 0 0 0 0 0 0 0 0 (4) 5 n (4) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 S07 (lavs, anotlicr with Rhodesian clirvsotile after 719 days and one which metastasized with Canadian ehrysotile after 838 days. In interpreting Table VII and Fig. 11, it lias to be borne in mind that there was a greater tendency for rats to develop adenomata in Experiment I than in Experiment 2, as shown in tiie controls. Therefore, the higher proportion of ani mals with adenomata after 3 months' exposure than after G months' exposure is probably an artefact. We do not know why adenomata occurred in the No. with fni'so- 'Hcliomu 0 0 0 0 han in mtrols. of aninonths' vposure lo "Ot ii: y 3 MONTHS AMOSITE EFFECTS OF INHALATION* OF ASBESTOS IN' RATS 205 6 MONTHS 12 MONTHS 24 MONTHS MO 7M WOO ANTHOPHYUITE rin! ^f, )00 7M WOO MO 7M WOO MO 7M WOO JOO 7)0 CROCIOOL1TE COO MO 7M WOO no WOO -CL )00 7M WOO CHRYSOTILE Cooodton n mo n m no woo *4- MO 750 WOO mo no woo js MO 7)0 WOO CHRYSOTILE Rhodeiion -4 Mi MO 7M WOO n l . JOO 750 WOO MO 7M WOO n MO JM WOO j3 MO 7M WOO MO 7M WOO MHI u MO 7M WOO V Squomcu) caranorng Adenocarcinoma E Adenoma/ odenomatoui Other cavtet Fto. U.--Distribution of survival tinu-s in days after tirst vx{io*un\ controls in one experiment and not iti the other but as tiie finding is significant (P = 0-0(5) in its own right and is sup ported by the results from the exposed animals, it is unlikely to be due to chance. There was a higher incidence of tumours with 12 months' exposure than with (> months' but little difference between the 12 and 24 months' exposure. Half of the* 8 mesotheliomata in Kxperiment 2 occurred with Canadian chrysotile, so that in total eroeidolite ami Canadian chrysotile produced 4 meso theliomata eaeii. Of the 20 tumours which metastasized. Id were after expo sure to a chrysotile (10 witli the Rhodesian sample and 0 with the Canadian). Throe others were with eroeidolite and one with anthophyllite. Two of the meso theliomata in the 12-month groups occur red within 400 days after first exposure, one with eroeidolite after 399 days and one with Canadian chrysotile after 355 days (the only rat which failed to survive lor its scheduled 12 months' exposure). 2(>() j. (*. \va<;nkk. w:i:ky. .j. \v. skidmokk and v. timhhkll Ashestosis and lung tumours An analysis was curried out to detcr- minc whether there was any relationship between the grade of ashestosis ami tho preseneo of lung tumours. Since the ashestosis grade depends on survival, it was necessary to standardize to a constant survival time, 'fins was achieved by calculating the regression coefficients of ashestosis grade on survival time for ruts without lung tumours, exposed for 3 months or more and with survival of at least 400 days after first exposuro. Differences in tlie.se coefficients between the "> types of asbestos and the 4 lengths of exposure were not significant and tho pooled coefficient of 0-00304 0*000(15 grade units per day was used. Tho ashestosis grade of each rat wus then adjusted using this slope to an arbitrary survival. The adjusted mean ashestosis grades were then calculated for each of tho -0 groups, for those with and without lung tumours. In 1.5 of those groups the mean ashestosis grade was higher in the animals with lung tumours; in the other 5 groups the opposite occurred hut only slightly so in 4 cases. The accuracy of an estimate of the difference in ashestosis between those with and those without a tumour is dejK'tulent on the number of animals in each category which varies between groups, and weighting each group to take account of this gave A mean difference of 0-71 ~ 0*13. Hence overall the animals with hmg tumours had significantly (/* < 0*00)) more ashesfosj* than those without. Differences between the dusts were not significant but the wide range in means--anthophyllite 0*25, Canadian ehrvsotile 0*42, crocidolite 0*06, umosite 0-S5 and ilhodesian ehrvsotile 1*22--.shows that there are insufficient data to reach any firm conclusions on this question. The groups exposed for only one day provide supjjorting evidence of a relation ship between lung tumours and ashestosis. There was very little ashestosis in these groups (Table VI) and restricting atten tion to animals which survived for at least <>oo days, the mean survival times of those with and without lung tumours wore very similar. Thoro were 17 lung tumours in 201 rats; only 6 of these occurred in 157 rats without ashestosis (3*s%) while 11 occurred in the 44 rats with minimal or slight ashestosis (25%), a highly significant difference (/><0*001). Tumours at sites other than tuny A total of 412 tumours, other than lung tumours or mesothelioma of the pleura or peritoneum, were observed. The majority of these were adenomata of the breast or pituitary adenomata, which were common post-mortem find ings. Doth of these adenomata occurred 4 times as frequently in females as in mules. The numbers of these tumours, oilier benign tumours and malignant tumours for each type of asbestos are shown iii Table VIII. For none of the Taw.k VIII.--Xumber of Tumours at Sites Other than the Lung Amovin' AmhopUyllito Crocidolite Chrvxolilo (Canadian) CUrvxutilo (KlnnJeMun) Com rut Hrtiijin tumour* .------------- ---------------- Mali* Pituit limit lJrvaxt ary Othor tlltllOU >. 40 4 8 To 38 4 15 '10 :U 4 10 13 20 7 7 15 37 9 3 JO 34 * 13 tumour types was the difference between the control and tho asbestos treated significant. In Table IX more detail is given of the sites of tho tumours with all ty|>cs of asbestos combined. The largest differences between the treated and con trol rats were for tumours of the ovary, 10 in treated and none in controls, and tumours of male genitourinary organs, 11 in treated and none in controls. However, neither difference was significant. A few rats had multiple malignant tumours: 2 of the rats with mesothelioma of the pleura also had a hmg carcinoma and one rat with a squamous carcinoma 'Jil times tumours 17 lung of these isbestosis .* 44 rats s (25%), <0-001). her than i of the >bscrvcd. lonomata enomata, cm findoccurred les as in tumours, nalignant t*stos arc i ' the f/t at Sites Mnfitfimnt r tumours 8 13 10 13 between treated detail is i with all ie largest and con ic ovary, rols, and rgans, 11 ifowever, nalignant rtholioma ur mia u nna EFFECTS OF INHALATION OF ASBESTOS IN HATS 267 Table IX.--Sites of Tumours Other than Lung A.sboHtoa treated cvntrol Sito/Tumour typo Digestive organs and peritoneum Done and skiit Breast Ovary Other female jienito-urinarv organs Male gonito-urmary organs Intracranial Thvmoma Lvmphomo/leuknemia Others Benign Malignant 4 3 too 3 2 3 173 ? -- a thyroid adrenal (l) 3 4 0 ? 8 8 I 1 8 3 mi'dia.stinutn salivan- gland thvrmd Benign I 0 20 0 0 0 34 O -- 1 suprarenal Malignant 3 1 0 4 0 0 l 0 of the lung had a mesothelioma tunieaHjfrom the worn hammer of the mill used vaginalis. Mesothcliomata of the vngiti-;*to produce the respirable fibre could alis were seen in this and one other rat; have been a factor in the causation of there is no evidence to suggest an associa the tumours. However, our results now tion with exposure to asbestos. Also, show that there is no need to invoke in some cases there were secondaries, for such a hypothesis to explain the high example, a synovioma had spread to the rate of lung tumours. lung. In all such cases tumours have The amount of clirysotile retained in been classified by the primary site and the lungs did not show any dear increase in the few cases of multiple malignant with dose in rats exposed for longer tumours there was no difficulty in recog than 3 mouths. In two earlier experi nizing tiie distinct types, i.e. one was ments (Wagner and Skidmore, 1905; not a secondary of the other. Morris H al., 1907) a higher airborne dust concentration was used to give a cumulative dose in 0 weeks similar to DISCUSSION that given in the present experiment Our finding that the asbestosis pro over 3 mouths. The weight of asbestos duced by exposure progressed after cessa found in the lungs of rats exposed to tion of exposure is in agreement with atnphibole was 3 times greater than in human experience but contrasts with those exposed to clirysotile. In the the early inhalation experiments reported present experiments the ratio was 0 to 1 by Vorwald, Durban and Pratt (1951) after 3 months, but increased with in which progression did not occur. continuing exposure as the weight of Wagner (1963) reported more asbestosis amphibole in the lungs continued to with amositc than with clirysotile in increase, hut the amount of clirysotile guinea-pigs, rats and monkeys but our did not. The previous oxjx'riments had experiments show that of the UK.'C shown that the rate of elimination of standard reference samples amosite is the dust from the lungs was much greater least fibrogenie in rats. for clirysotile than for the amphiboles. Gross et al. (1907) found lung cancers The present results may he explained on in 25 of 72 rats which survived 10 months' this basis, the weight of clirysotile having exposure to clirysotile dust at a mean readied equilibrium level, i.e. the rate of concentration of 80 mg/m3 for 30 horn's elimination equalling the rate of retention. a week. They considered that contami There are a number of features of the nation of the asbestos by trace metals results presented above which wo found 268 (\ WAC.NKU. HKKKY. J. W. SKID.MOKK AND V. TI.MItKKU, surprising. First, in K.xj>criment 1 two mesotheliomata occurred with the otic day exposuro compared with only one with the 3 months' exposure, which hud a dosage more than AO times greater. If the incidence of mesothvliomuta was proportional to dose, as is indicated for the inoculation experiments (Wagner d al., 1973), then the probability of such an extreme result occurring by chance would be about 2 in a 1000. Secondly, there was no evidence of either less carcinogenicity or less asbestosis in tho groups exposed to chrvsotile than those exposed to the amplnboles, even though the amounts of dust in the lungs were so different. In particular, the IT1CC Canadian chrvsotile produced as many mesotheliomata as the I'ICC erocidolite. The 2 UICC samples of chrvsotile produced 12 of the 14 tumours with metastases. However, much less dust was retained in the lungs of rats exposed to chrvsotile than nmphiboles (Fig. 10), Moreover, after intrapleural inoculation the risk of a mesothelioma occurring with VK'C eroeidolite is 3 times the risk with chrvsotile (Wagner et al., 1073). Therefore, allowing for the greater reten tion of eroeidolite after inhalation, we might have expected the risk with eroci* dolite to have been of the order of 20 times that of chrysotilc. Two of the mesotheliomata occurred within 400 days of the start of exposure. This may be compared with our injection exi>eriments in which only 20 out. of 803 occurred within 400 days (Wagner and Herrv, 1900; Wagner et al., 1973). Also, t he earliest mesothelioma occurred after 335 days and we observed only 3 within this ]K>riod in our injection experiments. The positive association between ns* bestosis and lung tumours which we have established in the animals is in agreement with epidemiological findings (c.g. Minister of Labour and National Service, 1949; Knox et al.> 1068; Elmos and Simpson, 1971). The failure to establish any association between asbestos exposure and tumours of sites other than the lung is equivocal. Although an association with gastro intestinal tumours has been found epidemiologieally, it is not yet regarded as clearly established (SclikofF, Hammond and Churg, 1972; Xewlumse, 1974), and is of lower magnitude than the excess lung cancer risk. Our experiments pro vide no support for such an association. The experimental work of Graham and Graham (1907) suggested that intra- peritoneul injection of tremolitc asbestos could produce ovarian tumours, but the follow-up of women asbestos workers reported by Xewhouse et al. (1972) pro duced no definite conclusions on tins question because of the rarity of the tumour. Our experiments do give some support to an association between asbestos exposure and ovarian tumours as well as tumours of the male genito-urinary system. Although neither was significant, this could be because of the relatively small size of the control group. To overcome this, wo have included the control rats from some of our other exjK'rimonts, thus increasing the nontreuted group to 403 rats of the same strain. This larger group contained 2 malignant tumours of the ovary and 3 tumours of the genito-urinary tract in males. In over 700 rats exposed to asbestos there were 10 ovarian tumours, 7 of which were malignant, and H tumours of the genito-urinary tract in males. Hence, based on the larger set of controls, the association between asbes tos exposure and ovarian tumours is weak and non-significant, whereas there is no support for an association with tumours of the male genito-urinary system. The VICO chrvsotile samples arc finer than the chrvsotile winch has been used in industry in the past. However, there is a trend for industry to use finer chry sotilc (Wright, 1969) and so the experi mental results may be more relevant to the current situation than to the past. We are investigating the effects of inhala tion of chrvsotile in more detail in an experiment involving UICC Canadian ;tuVocuI. gastroaul epi;rded as arunond *4), and * excess ats pro>ciation. un and intra asbestos but the workers proon this of the ve some asbestos as well urinary n;<:"ant, * ely up. To ded the ir other he nonhe same ained 2 v and 5 tract in used to amours, and 1L tract in rgor set it asbesaours is there is tumours i. are finer en used t, there ier eliry experi- vnnt to lie past. >f inhaiail ' an a. an EFFECTS OF INHALATION OF ASBESTOS IN HATS 200 ehrysotile, a grade 7 sample from a Canadian mine and the superfine sample which proved the most carcinogenic of the materials which we inoculated intra pleural!}' (Wagner et al., 1973). The experiments we report have given results which in several respects cor respond to those found in man. Thus, this experimental method is established as a valid tool for the investigation of the biological effects of asbestos. We arc grateful to all our colleagues who over a number of years were res ponsible for the daily attention necessary in carrying out the experiments. Wc would also like to acknowledge our thanks to Dr Harold Stewart of the National Cancer Institute who advised us on the classification of tumours. Wo arc also grateful to our former colleague, Dr A. Walter, who bail classified the non-lung tumours occurring in some of our earlier experiments which wc mentioned for comparison. REFERENCES Ei.mkm, l'. ('. & Simpson. M. J. C. (1971) Institution Worker* in IMfu.it. 3. Mortality 1940-00. Hr. J. ind. Med.. 28. 220. Oiiajiam, .J. A (cKaiiam, R. (1907) Ovarian Cancer and Asbestos, Eneir. lira., 1, tin. Guoss, l'.. pk Tkkvu.i.k, R. T. P., Tm.KKti. K. B.. Kasi iiak. M. & BauvaK. M. A. (10(i7) Kss|x*rinu'iital AslH'xtosis, The Development of Luna Cancer in Rut* with Pulmonary Deposit* of (,`hrvsotile Aslx*stos Dust. Archa enrir. lilth, 15. 343. Knox. J. K.. Holmks. S.. Dm.t.. H. A llnx. l. D. {IiiiS> Mortality from filing Cancer and Other Causes Among Workers in an Asfx'.stos Textile Factory. Hr../. iml. Med,, 25, 2113. KrxrftyKK. M. Lankin, *S. (1970) Pulmonary Epithelial Tumors ami Tumor-like Proliferation in the Rat. In Morpholixjy of Ex/ieriincnlal Hcapirntorif Carcinogeneaia. Pmo. Conf. Gatlinburn 13-id May 11)70. Ed. P. Xetteshemi, M. (!. IIiiiiiui. -Ir. ami J. W. Dcnthrmgo. .Jr. V.X. Atomic Enemy Commission, Sym|xtsium Series, So. 21. p. 203. Mivistku ok Laiiocii and National Skhv`k (1040) diimmi Ucftort of the Chief Iiia/iectiir of Factories for the Year 1047 (Cmd. 7021). lemdoti: HMSO. Mounts, T. G., Rohkkts. \V. If.. Str.vKTt>.v, H. E., SKiPMotut, J. \V,, Waunku. .J. C. & Cook. G. W. (19(57) Comparison of Dust Retention in S|H'citic Pathogen Free and Standard Rats. In Jnhnlcd Particle* amt Vajyonra II. Ed. C. N. Davies. Oxford: lVrjjamon. p. 203. Xkwhoi sk. M. I.. (1974) Cancer Anions; Workers in flu* Asbestos Textile Industry. In Hiolo'jicai Effect* of A*bcMto*. Lyon. 2-5 Octolx*r 1972. 1-M. P. Hogovski, J. C. Gilson, V. TimbreJJ and J. C. Wagner. (IARC Scientific Publications, Xo. 8). In print. XRwttorsE. M. L.. Bkuuv, G.. Waonkr. J. C. A Ti'ttOK, M. E. (1972) A Study of tin* Mortality of Female Asfx'stos Workers. Hr. J. imi. Med., 29. 134. SmaKwr, I. J.. Hammond. E. C. A Cornu. J. (1972) Carcinouonicity of Amnsito Asbestos. .4rr/i* rnrir. Illtli, 25, 183. ShauaD, L. M. A Pyi.ev, L. X. (1970) Morphological Lesions in Rat Luntrs Induced by Polycyclic ilvilrncnrhons. In Morphology of Experimental ll.r.piratofj Cnreinogntraia. Proc. Conf.. Gatlin- bum. J3-J0 May. 1970. Kd. P. Xettcsheim, M. U. Hiinua. .Ir. ami J. W. Denthemue Jr. l*.S. Atomic Enemy Commission, Symjiosium Series Xo. 21. p. 227. Stanton. XI. F. (1974) In Pathology of Tumour* in {.aboratary Animal#, Vo| I. Tumour* of tin* Rat. Part 2. I.A.R.C. Scientific Publications. Xo. 0. Lynn: International Agency for Research Cancer. In preparation. TlMIUtKU.. V.. (Jii.son, J. (\ & Wkustku. I. (1908) U1CC Stamlard Reference Samples of Aslx'stos. hit. J. Cancer. 3. 40(1. 'VlUttUKU., V.. Hvktt, A. W. A Skiomoue. J. W. (BIOS) A Simple l)i*|x*nsor for Generating Dust Clouds from Standard Reference Samples of Asls'stos, .1 an. occup. Hug.. It. 273. TlMIIUKM.. V., SKIOMOItK. J. W.. Hybtt. A. W. Waunkk, J. C. (1970) Exposure Cltatnls-rs for Inhalation Kx|x>ritm'ntx with Standard Jb-forvurc* Sampli's of Aslx'stos of tho Intor- national I'uion Against Cancor (CICC). Arroaol Sri.. 1. 215. VuuWAi.n, A. J.. DruK.vN, T. M. A Phatt. P. C. (1951) Ex|x>rimontnl Stmli'-s of A-shoxtosis. Arr/ia ind, llgej.. 3. 1. Waunkii, J. (*. (1903) Aslx'stosis in Ex(x*rimi*ntal Animals. Hr. J. itnl. .Mod., 20. 1. Waunkii. J. C. (1905) Tilt* Si-quolm* of Kxposun* to A>lx*stos Dust. Ann. E.Y. Acad. Sci.. 132. 091. Waunkii. .f. C. (1972) Tin* Sii:m(iratu*i* of A'fx'stos in Tissm*. In Hcccnt Health* in Cancer llcarnrch, I 'al. 39. Current Problem* in the Epidcmiolixi;/ of Cancer ami Ed. E. Grumlmaim and II. Tulimus. X-av York: Spruitfvr-Vorlait. 1>. 37. Waunkii, J. C. A Bkuuv. G. (1909) Mvsothrliomnx in Rats Foilmvinit Inoculation with Aslx'stos. Ur. J. Cancer, 23. 507. Waunkk. .J. Bkhuy. G. A Timiiuku., V. (1973) Mesotheliomas in Rats Following Imx'ulation with Aslx-stos and Other Materials. Hr. J. Cancer, 28. 173. Waunku. J. C. A Skiumoiik, J. W. (1905) Aslx*stos Dust De|xisition and Ih'teutiim itt Hut*. Ann. ,Y. F. A<od. Sci., 132, 77. Wkiuht, (. W. (1909) Asbestos and Health ill 1909. Am. Her, reap. />*., 100, 407. l i i `s j i 'S i