Document Lp6m6JMjKwLjy6GJEoOqdLyxX

FILE NAME: Kaiser Gypsum (KG) DATE: 1974 DOC#: KG091 DOCUMENT DESCRIPTION: Journal Article - The Effects of the Inhalation of Asbestos in Rats Hr, J . ( 'a Herr (11)74)29, ST,2 THE EFFECTS OF THE INHALATION OF ASBESTOS IN RATS A. ( \ WAt i NKK, U. HKHHV, .). \V. S K I D MO H K and V. T i M B H K L L Fror ihn Aftdivnl UrHeoirh ('uuuri'x f^trnmocotiartix Unit, Uamfoufjh Huxfiiful, Fniorth, (Hmorgnu liccvtvt'tl /* I !#7*l, A coopind 2# Nnvcmliot* ID7 Summary.--Two experiments in which SPF Wistar 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 ail 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 mesotheliomata occurred, 4 of which were with crocidolite and 4 with Canadian chrysotile. Two of the mesotheliomata occurred with only one day's exposure to asbestos, There was a positive association between asbestosis and lung tumours. Waonkh, Berry and Timbrel! ( I reported the results of experiments in which rats were inoculated intrupicnmlh with samples of asbestos. In (he dis cussion it. was mentioned th a t tw o e x p e ri ments in which rats had been exposed to dust, clouds of the UlC'C reference samples had been carried out, .mil the results of these are now presented. .Some preliminary results of one of these experi ments were reported by W agner (1 !)72) la both experiments the rats were exposed to similar dust concentrations and the dose varied by exposing rats for various lengths of time. The main aim was to establish the relationship between the development, of malignant tumours in the lungs and th e dose and ty p e of asbestos dust but, additionally, the am ount of fibrosis was assessed ,WATi;iiiA),S AND MKTIIOtih Caesarean derived rats of the Wistar strain were, used which had been bred at (he tinU from SPF stocks giieit to us by the Imperial Chemical Industries. Pharma ceutical Division a t Alderlev Edge fhes-lnre in HHU and 10fi8 The asbestos samples used consisted of the 5 DlCC standard reference samples (Ttmbrcll, Gilson and Webster, 1068) which a ere prepared following recommendations of the DiOO. These, samples are of 3 am phi bolt* types--antoMtc. anthophyllitc and crocido lite--and i chrysotiles--a Canadian and a Rhodesia sample. The ruts were exposed in 1-4 m3 inhala tion chamlicrx {Timbrcll H /.. 1070) which contained 8 cages, each of which could hold 0 rats, or. for a short period with yoims; rats, here was room for a seventh. Fin (handlers were used, one for each type of asbestos The chambers were constructed so that the rats could he tended without the chambers being opened. The dust clouds were grunt ated using a specially devised dispenser (Timbre!!, Hu-tt and Skidmore. 1068). The clouds Mengenerated for 7 hours a day and 5 days per week. The respirable dust concentration' were meusuml using size selective gumtrie dust mimjdcs _[('nsM>lla Type 11-tAi The collected samples were evaluated nt the cud of each daily session, lit order t<> achieve (he imputed dosage, calculated the product of concentration and tin mirations occurring in the concentrationwere corrected on the following days. A', the end of exposure the rats weie left in tie clmmlirrs for a few days, to allow time fi>: UFKHt'TS OF INHALATION OF A.XliKSTOS IN HATS 252 their fur (<> become clear of asbestos bclote transferring thorn to a clean environment for the remaimlei of then lives. At transfer some rats, generally 2 01 ,'i of eueli sex, ere killed and (lie lung. reiinned for histological examination and determination of dust, content. As each type of asbestos Inis a consider able silicate content (about o(l%) the amount of dust in the lungs was determined by first assessing its silica content and referring this value to tha t of the respirable dust to which the mt had been exposed. This method was used in previous expeiintents (Morris et al., 1967), After exposure the rats uere caged m 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. Hixlillogical pr*titration. nhtinimj anil micrascri'jiic metiimln.-- Foi the, scheduled killings, animals were killed by chloroform anaesthesia and following exsanguinate! the thorax opened and the lungs removed. The left lung was air inflated and suspended in formalin Representative portions of the right lung \sere taken for electron microscopic examination and the remainder of the lung dilated with neutral buffered formalin. Slices were taken from both lungs for hl.xtologiea! examination. After the histological sections had been cut. the embedded tissue and the trimmings wore dewaxed and added to th remainder of the lungs which were used for Vihe chemical estimates. For other animals, at post mortem the lungs were dilated with neutral buffered formalin and after fixation were sliced sagittallv, routine sections being taken of the hole left lung and the upper and lower lobes of the right lung. In addition, any other suspicious lesions from the lungs ot other organs wore taken for histological examination. In all cases, sections were stained with Imematoxylin and eosin and the lung sections ere stained for elastic, retieulin and col lagen. Special stains were used in some cases as an aid to diagnosis of the tumours. Asues#men! of the sererili/ of i h M ' w ' s -- Sections of both lungs were examined wtf limit knowledge of the duration or type of asbestos exposme and with the animals in random order The sections were observed on a viewing screen of a Project inn Microscope 4013 15K using a X 7 objective. At tins magnification a large proportion of the lung rmild be assessed in a single field and, as it was not possible to observe asliestos fibres at this magnification, the results were not. biased by knowing the type of asliestos to which the animal had been exposed. h'.r/ieriinenE In Isitli experiments and for all doses there were groups which were exposed to all 5 reference samples: there were also control group which were not. exposed. Rats were allocated to treatments at random. At the start of the exposure the majority of the rats were between F> and 7 weeks old, a few being slightly older or younger, and there were approximately equal number of males and females. Experiment 1,--There were 2 time inter vals of exposure: in the first, rats were exposed for 3 months starting in Muv J!Mi7 and in the second, groups of rats were exposed foi one day only in August 1907. In addition to the killings at the end ot exposure, m the 3-month group there were also intermediate sacrifices at 5, 8 and 10 u eeks. Experiment 2 --There were 3 time inter vals of expostm-- (i months, 12 months and 24 mouths. The experiment started m dammiy and after (i months half of the rats were, removed from the cabinets They were replaced and a year later these replacement animals were m turn removed. They were replaced by animals (o be used for special electron microscopy examinations which will not he reported in this paper The remaining animals were removed m .January J071 after 2 years' exposure,-- fu tile (-month groups, in addition to the killings at the end of exposure. iats 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 about a year, and some were removed prematurely alter KH months For analysis these rats have been r>4 I. WACNFK. (!. HKKUV. W. XKIU.UOKK AXI) V. TfJIHliKU. It it! \{< 1 <fm `l m ouths h m onths 12 Jn o u iii.v 2 1 m oni hs .\(iH 4i> :2 24 21 T ahlk 1. A'lutin' o f Hals Hxjiosrd A iilh t ijilit U ito 4! Y2 24 Hi <'i t i f i i It iif 4U ;Y 2 24 2U 2ti ('h rv -a itile m 24 2:1 24 {H lio ilo s irtn ) ( 'tint m l 4 il 4K 5H '2a 1 27 > 4<S 2D J Tablk II. -Mmv timpimUl*' DhkLCoucevtmtivv (wf//w:!) finrf ( 'muitf/iCivf' Dose (Htr/jiii* lautrs) h of 1 <l*n 2 n v m i !< m onths 12 m o n th * 24 m onths A mo-mo 1 4 ') 12 4 11*2 ID 'S H i-fi mi olKMI R350 17 (am S3SM0 A iilh o {* h ^ Hilt* 12-X 1:i - r, t o (1 11-4 H i-fi hit f 24<> K :!40 17 MM <Y<x` l<lo!lM* i 2 r !2 **i mi 7 ! iim u nk ;>n;h> K 4:m | MMt xu m (lu ^ s o h li* M n m u im i ) U -7 l*2` \ M i-2 M-7 Ut- l <K 4`K ih >4 17 MM iW SOo < H i o u m ) 14 7 12- t 10-7 1(1 II Id l H3 ;m 7u H5iH) I7M M UHfiOO a included in the 12-month groups find hence The lesions consist initially o f n deposi the num Ikm uf nits in these groups is slightly tion <il asbestos fibres, alveolar macrophages higher, mul in the 24-month groups slightly and cell debris in the alveoli aiming directly fe lower, than planned. from the respirut oiv bronchioles. These In Tables II the mean respirable dust deposits become organized firstly by being concent iat ions and the euimilainc doses enmeshed in a thin leticulm network winch the produets of concentration and time, are coarsens with time and becomes replaced In shown. The one-day cx|M>suie. was 7 hours collagen (litres. The alveolar epithelium for all dusts For the other 4 time intervals reacts with replacement of the typo t celts o( exposiite. there were 'light vat iat ions in and becomes completely lined by grainil.u the niimhei of hums required to achieve pneiimocytes. In some of the aheoli the approximately equal doses hut the mean epithelium is shed into the hunina. m other times were 402. 788. 574 and 3287 hours there is a wailing of the alveoli by these respectively. The mean concentrations were cells. This usually occurs at the hifurcalioii- usually higher in K.xpenment I than Kxpcii- where groups of alveoli are closed off irniii menl 2. and the h-month group had an the Iuinma of the lespirntoi v bronchioles, a\einge dose of (Ml0;, o) that of the (i-moulh guiim the so-called pseiido-acmar ap- grout* I'easooable equality of (lose between pcaianee I I I the gumea-pigs tlicse small the dusts was achieved for all the lengths of cystic spaces contained asbestos (litre ami exposure, except for the one-day which was degenerating maeiophages, but m the rat- too short to allow a m adjustments numemus granular pociimocytcs were al-o picseut. The initial lesions were coniine'! in occasional discrete individual tespiiamn / iile r f iir liilio n fij hi.slohif/ii'ffl ftii(lni(/s bronchioles scattered tluoughnut the lime I'Ifl^sijicalion of ii'.brxloxis --The lesion- siilistanee Alter turthei exposure. more am: seen m the lungs of iats~e.\posed to nil types of asbestos weie similar to those described m guinea-pigs (Wagner lOtsi, lillio) '1 here were 2 main (liUcn-iices- tirstlv. asbestos liodies were never seen in the lung tissue of the iat although they aie frequently seen in the pleural graiiiilomata which follow the intm-pieinal inoculation ol amphibole lihres; secondly. there was a tar greater production of gratiulai pueumnevtes fix jm* llj alveolar epithelial cells in the ml more ivspmilmx brom hiolc' become mvnb.o! and all the M--oii,itoiv bmucliiolcs an-ur. from leiinioal Inoiii liioles become Ilurkem-, as (he lil n o t i s tissue net wink extends into 1lie wall ol tin1 lespuuloiv InoU'-luulc. ami this iiitelstilial leaetion sprt aiU down in!. the peripheral elements ol tin- pnni.m unit, u n o h m g llii' alveol.il flints ,nrn and filially the an sacs and alveoli Wilt pt *>gr< -sum IIn twin dual lesions lend ieoalesee lead 111I to tiie development ot . K HKK('T,S OK IN i L \ l , A T I O N ' OK A.Slili.STO.S I.V UATM 4 va ?v 4 > A ^ s rtA '; '; 4>r* j P ' & i ' i l s * r " P i? .`T,'r V .- 7 > . / -"iw- *. W i '- Y . .'"* V <7*i*v . . -X* 'sM& A/ I ^ . f y $ .F r > u p -, - * f& i. . _ ** k--_ % 4 ' :is4 ` M iI V. yf;\ H o 1 - - S h ^ h t isb(`Nt>Hjs ((iriuft* 4) Thcl*iMig ol th<* wails o {la* tiivooh wn-mt* <lfi ***ttv iron th n H*s|>ii'ftt4fy ImmchmU'-, w ith t a p h u v m a n t ut n u im alap iftio tm in b\ i \ p t I (*<{K U ' K KO `i . % v w ^ . :m yf\% ' V i . X s'VV* %f $1 * \vv >ik i U & .Sf' m JT K \ %V Pt m* t j ^ * r * 'A fYt t ' a J S t i c h t m -l x - ao x i- , (<_jada 4) U tutta pou.* 4 \ i m i g m u t i n mi's l a h a a n V a i o c i i l o l n - n) m t h o utvff*li <t a r a ^ p i r a t o r v tuonala)*. A s t a ^ j o s b o d i n x ms* mais. -< o * t h- i n n ^ - ol m l Ulummuium `odtwod U vtUwirata Uta 1bn*s H tV K y .*>00. K K F E t T K U K ! X11A I . A T H l.\' i l l ' A S l i K s T O S l \ H A T S diffuse interstitial fibrosis with giaduul in crease in the density of the fibrous tissue, ultimately resulting in the re|)fileenient of most of the. lung parenchyma by a dense collagen netwoik sitnounding distoited air spuccx many of which lontaiu huge clumps of guuudai ptiuuimicytcs which. m some amis, have lysed leaving foei of hpo-alveoku proteinosis. The. assessment of these lesions as based on 4 grades of fibrosis--minimal, slight, moderate and severe. Typical ex amples of slight, moderate and severe asbestosis are shown in lug. 1-4. In addi tion, when carrying out tlx* assessment it was found convenient to introduce the intermediate categories minimal/slight, slight/ moderate and moderate,/severe. The 7 re sulting categories were scored 2-8 and the normal lung, in which there was no sign of asbestosis, was scored 1, and the assessments were averaged for each group of rats. The repeatability of this form of assessment was tested on 154 sections, reassessed in n different random order and 90% were assigned to within one category of the first reading. CloMiifiatittitt o f (iwtoit)x--The tumours found in the lungs of rats al'tci exposuie to the various types of asbestos dusts were peripheral adenomata, widespread adeno matosis, adenoeareiiiomata and squamous carcinomata The ratify of pulmonary t u mours in rats has been stressed in the reviews by Ku.sehner and Luskin (1970) and Slnihad and Pylev (11)70) Further, these authors have described the lies elopinent, and iimiphoiogiciil features of the turnouts that we are reporting All these tumours were |ieripheral and appeared to ai ise from the legion of the respiratory bronchioles m which the asbestos fibre had accumulated The origin of the adenomata appealed to he iroin accumulation of type II epithelial cells that prohfeiated m the alveoli of the inspiratory bronchioles (Fig 5 (i) til many of the exposed annuals these turnouts wvic multiple, and in a number o( animals, particularly those with the mole seven* crudes of asbestosis, there seemed to he mluiomata arising Ijom numerous adjoining respiratory bronchioles, giving an impression if contiguous adenomata invading large ireas of the lung. I)r Haiohl Stewart, (personal eommumeathm) suggested that ties type of lesion should be veferred to ns adenomatosis In contrast to this, a lew control animals note seen to have solitary adenomata which were small in sr/.e. The uleiioearemomala were of the same type and origin as the " alveolar udenucareinomala " described by ttlutliail and Pylev (1970); many of these tumouis were pupillary adenoma-carcinomata. The s<|tiamous oareinomata appeared to originate iroin foci of ,s<|uamoii.s metaplasia occurring in the ashestotie l<*.sions in the respiratory bron chioles (Fig. 7, 8) As far as can be ascer tained, all these tumours were peripheral in origin and not bronchia! papillomata. The classification of these tumours was discussed in some, detail in Session VI at the tiatlinhurg Conference on the Morphology of Experimental Respiratory Carcinogenesis in 1970; and the chapter by M. F Stanton (1974) in tile IA RC Monograph on Pot/ioloijy of Tumour in. Luborufory A mills contains detailed descriptions of the tumours that we have illustrated. The rats used in this experiment are fiom a caesarean derived, harrier maintained colony anil 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, pericaidium or the traehco-broiiclual lymph glands were seen in 1-i animals; the majority had lesions invading 3 of the sites and in only 2 animals wire metastases observed in the tracheo bronchial glands. In one annual secondary deposits were seen in sections from a kidney Fight adeiiycarciiiomata and (i squamous tumours had metastasized RESULTS All except 2 of the ra ts in the groups with e.xposuro of 12 months or less survived for the whole qf their planned exposure In the 24-month gioup then* was appreciable mortality before the end ol exposure and only oil",,, survived for the full period. O u t of 1013 ra ts it was impossible to obtain adequate histological material in only S because of cannibalism b u st rbcnVii) The mean in the lungs weights of of animals asbestos dust killed at the 2<i() C C. WACNIiR, C. UKRUV. .1. W. SKIDMOK!'. AND V. T t MR R K U . I/CMtel h o f .> u 'r c ks Hw ok* H) uci'k s .1 m o n th * d months I2 months 24 m onth> c> m o n t h s IX m o n t h s r) T aiU.K i l l . -- Du.st Retained in Lungs (mg) M.-iui Utim* m a / i M h.mt'K Amosiit* Atillt. pliviS it <* ( V.KM.lohte f \ .-.of A.* (Vnmniuui) 18X11 l-ll 1-3 1-1 0-1 2450 < Mi l-ll (i- 1 3290 2-0 2-8 21 0-5 50311 .1-7 3-5 3 - (I 0-6 847 4-7 4-4 4 -5 0-4 I7MMI n-:i !> t> `1-3 0-8 3340 HI-8 13 14 9 (Ml 1 :> 2-0 1-3 (Ml Clii-yriolilo (RhinloHmn) o-1 0-3 0-5 0-7 n-4 1-4 0-6 0-1 iit-ngth of nxpnsmv I day 3 month* U month* 12 m o n t h s 24 montha T iiii.b 1\ \ - M ean Su rvival* after First FxpoHure (days) AmosiK* Anthophylftto Crondohto i lt r y s otdo { ( 'rmt l mt t ) ('lii-yxotilc (Hhilomaa) (3>l pot 8<H SCO 77! 823 '(ill G8(i S>2 750 807 778 705 7(13 753 803 817 790 857 703 788 00!) 700 1| 77(5 778 820 ;) 754 750 585 758 JI * Adjusted to ton mdu}x*ndt'nf nf sumheu*. scheduled finu*s are given in Table i l l Metre d u s t was usually found in males th a n in females and on average tin* female lungs contained only 70% as much dust as the male lungs. Tim values in Table 111 are th e averages o f th e male and female means. For th e It amphiboles th e re was a similar pattern, with an almost propovtiema! increase o f lung dust with dose The 2 chrysotiles were similar to one another but much less dust was found than with the amphiboles, also the chrysotilc figure-, did not show the same clear increase with dose. The main features are s u m m n r a e d in Fig. 0. The dust in the lungs of the animals which hud 0 months' exposure had been partially eliminated IS mouths after removal from exposure. Thu proportions eliminated were 7-1% tin amos'ite, 73% foi croeidolite but otll\ 41" ,', lor a n th o p ln Ihte Ifowevoi. the lower elimination of aitihophyllite was not signilieantlv different Irom the amosite and croeidolite figures. S u m rat The mean lengths of survival from th e day the rats were iirst exposed are given in Table if '. T he survival times have been estimated so as to be inde pendent of th e sacrifices. The short survival of the 24-month group exposed to Canadian chrysotilc was largely due to 8 rate dying before Day 400 (in the other four 24-month groups only 2 rats died before Day 400). These early deaths were, not due. to exposure, since a died due to an infection in one cage and 2 were killed in a light. Discounting these X floaths, th e mean survival was OiiS days, whieh was still th e lowest of the 24-mont-h groups. W hen the mean was taken over all the lengths of exposure, the Canadian ohrysotile groups showed least survival, but only a month less th a n th e control groups. The amosite and .mthophvllitc groups had mean sur vivals only a few days less than (lie" controls, while the croeidolite and Rlio desian chrysolite groups had longer sin vivais of 2 and 3 weeks respectively, lienee, there is very little indication tlmt the exposure bad any effect on tino \c n d l survival of th e animals. Thi? is in m arked co n tra st to our intraplem.il inoculation experim ents in which injection K K K K ( T.'\ U K I X It A t.A TIU .V U K \ s l t K s T O s | \ H,\T,S gUf Weight of dust in lungs (rng) 1m., (1 JU. "1--------- *--------- ------- Time (months) 10000 20000 Cumulative dose (m g/m 5hours) '10000 \ l< *a u W f t j x h t ti t l n - d mi i u n i i w <j! 'M m 1f Lit UM f*|n>(` unii 1nil* <l a sb e s t os r e d u c e s the e x p c i (,)tion of life l>\ several months fW.igner >1 n/,, I !)73) Asbf'slftxi.s Thu am ount of asliestosis was assessed tut all the m ts killed at scheduled times in H x| H'rifih -i 11 2 and alte r x weeks and .1 months' e \|io s iu e in ex p e rim e n t I There u e ie a or (i rats pet tre a tm e n t tor I'.teh exposure, except that th e ie were only II aftei X weeks t)vt*mll the 2 sexes had similar am o u n ts of asbestosis .uni they have', therefore, been eomhiued to give the mean asbestosis scores in Table V, which .ire summarized in Kig, It), r.xeepl for some inconsistency hot wet'll the '!- and (i-inontli me.ms, there was an increase ot asliestosis with exposure for all the dusts. Also, following ti m o n th s' exposure, there was progression during tile following IS months without exposure tor all the asbestos types, Imt these vats did not fare as badly as those which continued expoMiie There were xigniliennt dilterenees bet ween the asbestos types ( / ' y ll ill | aniosite iu \a r ia b h gave the east asliestosis throughout; a n llu e phyllite and Canadian ehiwsotile showed most asliestosis after (i m o n th s' or longer exposure, eroeidolite and Rhodesian chrysotile were intermediate. The mean asliestosis semes ol the rats which were allowed to live out their lives are given in 'fable VI Those uits 2 i> 2 .1. ('. WAO,VER. U. JlEKUY, J . W , SK1DMOHK A N IJ V. TI.MliRICU * Aebe(otis grade Moderai Slight Minimal Nil 36 12 I---------- f----- -- ,----------1----1----- 1 1 1 0 10000 20000 l 1 3000Q 24 Tim e (moniht) 1 Cumulative dose (m g/m 3hours) \'u 10 Asbo-toni* ut cniicod imK in ivIhI kiii h lose a n d (mu . r AWU5 Y.-- Moan Ashcrtoxi* Hooro** of Sacrificed Mats <*f \mnsht Aiith}>hvlht ( `hry w oii ' (i'anadmn) C h n > o t *lc* (Rhodesian) iVmu X u ., U J u 2-f> M UK lilt li 4 ' f. - tt m* iu h'x 2 1 4-2 12 m o n lh " 4 11 > i I s m<>niIt" 4 month'' 4 a ( t (i m on th'' :t .1*0 {nfti't IK mmi! b* iion-rxjM>miio) 2 o *2 ti 2*7 1 f 2 *s 2' 7 :j`0 ) 4 2 0 {0 2 h i 2 4 :i 4 *i 4 ;* I . -) 4 S (i-o .v<s i-s *>. T !r * I ml, 2 im m m .il, 4 : liiilil. 0 mmlmaO'. K scheduled for L'-t m onths have been ilividwt' It m ouths' exposure of Rhodesian e h n - ' info (hose lfi.it (lieti before completion tile T he ra ts which died beiore com of exposure ,ui<l those fbal .sitrviveii lor piotine L' I m ouths' exposure hud moo ^ a period ol n o n -ex posare. 'Dio amount a.xbostosiK than those sacrificed allei ft o f asbestosis (hand io (Ite. ruts ox postsI m onths' exposure for amosito, india for one dn_\ was no more lltttii (hub found phvllile and lUiodesiitn ehrysofilc. Tinin control rats, Com paring Tables V was not th e ease for croeidolile ana a n d VI for (lie n its which completed their C anadian ehrvsotile. for which tho-e Mi exposure, progression had occurred be litai. died during exposure, had sli<>i'<t tween tile end o f exposure a n d d eath with mean survivals than for the other d.1-1- 1 all dusts, the single exception being the Moaned over all dusts, those rats lli.it n ! I <w )r ill t < In an t,, , h en ! no of. Tu, the nun via, l.lts lum. ( oia Ol>M l-Xp( son to I) -eile wit j) KKKKITN OK l.VIfAl.ATION OK A.SIUCSTOS I S H A T S 2>:t i* T a h i a -; W . -- M m n Axhcstosw S a m s * o f Survivor$ (Mean S m viva l in Slonihx) MNJIOSltl * (7irv-.,>l it,- (`hi >><lif Am,,Mi,- Amlrnpliyllai- t'luiailiiiito ('immlmii) (Ktm, 1mm) si \ 1 liny i moiilhs l-.-l (.'(,) -'!) (2') l-.'l (it., :< 2 (27) 1-2 (2(i) :i-1 (27) 1-2 (2.7) .4 It (.) 1 4 (24) 2-S (28) <\month* 2-2 (4) 4-2 (2) :ia (24) :i-7 r2") 4-2 (22) IIHHlths 4-S CXI) (-'A) .(> (2.->| r,-t (27) ti- 1 (27) U|> to 'I (Ml (21) a 4 (22) 4 2 (14) 1 (HI) ( 1 (22) mmitlH * 24 months (i-.'l (2S) 7-0 (28) 0-0 {>!>) fl-S (2) * )! ml, i: mmimtit, 4: mIj^IW, ih uvkU'VaU',, S-. ft'\riv. 4 t i i >tn { i>>1 4 Mi" 1 i ud (lied during exposure hud slightly more asbestosis than would be expected from the sacrifice ruts, consistent with the more severely affected animals having the shorter survivals. However, the effect was very slight and, as observed earlier, the exposure did no t affect survival to my extent. In Table V I there is again less asbestosis for am osite th a n th e other dusts although the difference is not as large ns in T able Sr. F o r r a ts which completed their exposure, the difference between amosite and the other 4 asbestos ! \ jk;s had a mean o f 0-7 for ruts sacrificed and 0-5 for survivors. T he results in Table VI do not su p p o rt the findings in Table V th a t anthophyllite and Canadian chrvaotilc produce more asbestosis than <rucidoHte and Rhodesian chrysotile, and uo conclude, therefore, t h a t there were no im portant differences in the a m o u n t of asbestosis produced by these 4 samples Tumours o f the Iv ikj Lung tum ours were observed in 247 of die rats exposed to asbestos. T he total numbers o f each kind for each d u s t are lien in Table V II, where for--th e s e ruts with more th a n one tu m o u r of the hme, classification is b y th e more severe (umlition. N o tu m o u rs o f the lung were oWrved within 300 days of the start of 'vposure a n d therefore only r a ts which arrived tin's initial period are considered io have been a t risk. A p a r t from the clssluled killings, only 13 ra ts died within the first 300 days. T here were 7 An control rats out of 84 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 -- 128 com pared w ith 110-- but, th e only 2 tumour types for which there was any major difference between the sixes were adenocarcinoma and squamous carcin oma. O u t of 70 adenoeareim m m ta, 35 occurred in males whereas 30 of the 40 squamous carcinomata were in females Mctastases occurred in 20 rats, to of each sex. There were also t! mesotheliomala (Table VII), 7 in males. Two of the mesothelionuita occurred with only one d a y 's exposure, 1 w ith 3 m o n th s ', none with G m o n th s', 0 witli 12 m o n th s ' ami 2 with 24 months'. The meso thelioma which occurred with 3 months' exposure to crocidolitc was a peritoneal tumour; tiie otIters were all of pleural origin. The distribution of the lung tumours with time after first exposure are shown in Fig. 11 for all dusts a n d all lengths of exposure ex c e p t one, day. In the. 5 groups exposed to asbestos for one day there were 14 a d e n o m a ta 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 mesotheliomata, one with amosite after 715 days and the other with crocidolitc after 551 days. There were also 3 adenoearcinonuita, one witli crocidolitc after 24 ,T, 0 . WAG IS' K It, (1. JIICHKVj \V. HKIDM ORK A N D V. T I M I t l t E h h Tam .K ICvjkwurt' Atnostfd 1 llltv 3 month (>month. 12 months 24 months Total V iJ.-- X vm b rr of A nim als No. ol cuts 1 rwk* No. with lung himom r------ --- 45 :i n 37 10 7 IS 2 i 25 JO r> 21 13 a Mil 38 1!) with L u n g T u m o u rs or J / fsolhpliomaki Tvj><` o f lo n g tumour AOono* onrcmonmt Squamous crtrcuiomnf No, woh moo* thohooiu 0 0 It O 0 t 4 i 1 3 8 5 0 i 0 0 0 0 0 0 0 0 it 1 A iilhn/ihi/iliti1 1 thvy 44 2 2 0 :i months a? 0 f> 0 f> months IS fi It 1 12 month* 2S 20 <4 0 24 month IK Jfi 2 5 Total 145 no 22 12 0 0 0 0 0 <1 1 1 0 4(1) 1 1 3 0 ) 8 (1) 8 2 ( *rorniabfr 1 day 41! 0 r> 0 3 motii h :u> 14 10 2 f> month IS 4 2 2 12 moil)ha 20 IS 0 4 24 mrmth* IS in 4 .1 Total 141 ,K> 20 in 1 1 0 1! 2 (1) 7 (1) 0 I I U) 1 0 0 0 o 2 (1) 0 9(2) 4 ChnjfOftU' {('anafh'tft) 1 tiuV 42 I 0 `l months 114 IS ir. 0 month 17 c> 2 12 m o nlh s 21 11 i 24 months 21 10 2 Tolal 137 4'** 20 a 1 U) 0 0 0 3 0 0 2 0 1 0 it m 1 (1) :i 3 i id 4 (2) i K 11 (3) 0 CD 4 ( `hujfiohlr {Uho>h v/mj I .lav 45 .1 4 3 moiith.s 'Ui ir. 11 0 moot 1>n 10 8 2 12 m onths 27 hi 2 24 m om h.s 17 11 0 T..ll 144 .V) Ml 0 1 0 II 2 It (U 0 (I H It (1) 0 0 4 7 121 (4) t) 1 5 (2) 5 u 10 10 (0) l i (4) 0 f'onhoi 1 (lu\ 44 4 4 a U `5 moot hs 10 .1 0 0 0 24 mmillw 42 0 0 0 0 Ti.i.il 12i* 7 " 0 0 0 0 0 u 0 0 0 0 { < a U w I n c h * m r w w < l af I*'** 3 0 0 <iu\ * Jif I*/ Onrl o' *,\|Mi'-iur' l NnnO *M in Siiai h*i s n i r th* >*>' ns \\ \\ m t i*u ,W4`". Nil" d a \ s , ano th e r wit.li Rhodesian ehrvsotiic afte r 7 Ih d ay s ami one wliielt metastasized with (Canadian chrysotiie after K3K days. In interpreting Tatile \ 'lt and Kig H , it has to he h o m e in mind (hat ( h e r e was a greater tendency for rats to develop a d e n o m a ta in Rxperimont. 1 than in Kxpeniiien!. 2, as shown in the controls, Theretoiv, the higher proportion of imr m uh with adenomata after 3 months' exposure th a n a fte r (i m onths' exposim* is probably in .triefaet. W'O do not know w h\ a d enom ata occurred in the 3 months AMOSTC KPt'ROTS ()!' IN H A L A T I O N OI-' A.SIUNTOS IN' KATS SOO V i 1000 anthophyuite i--i r^i 500 0 00 otoaooure JX 500 *750 CHRYSOTIU Canadian 500 7S0 KXJO CHRYSOTIE Rhodeiton JX 500 .750 00 Sqwonvxn corctnoma SB Adeoocartmoma S3 Adenoma/ odenomaov Q Other caves Im;. 11.- <*) mu vu uH imc^ ti (lav** alte* hts u\j>o*uit controls in one experim ent and n o t in tlic other b u t as the finding is significan! U` -- U-Qti) in its own right and is s u p ported by the results from th e exposed animals, it is unlikely to be due to chance. There was a higher incidence of tumours with 12 m o n th s' exposure than nth G m o n th s' b u t little difference between the 12 and 24 m o n th s' exposure. Half of the 8 m esotheiiom ata in K\|;riiucnt 2 occurred with Canadian dirjsotilo, so th a t in total crooidolito and 2 (6 i. C . \ V A ( ! N K K , (. K K H V , 4 . \V. S K I D M O H K A X I ) V . TI.M IJ R K b b Asbestosis ft ml hint/ Iunion is Aii analysis was curried out in d eter mine whether there was any relationship between the grade of asbestosis and the presence of lung tumours. Since the as best os is grade depends on survival, it was necessary to standardize to a, c o n sta n t survival time.. 'Phis was achieved bv calculating the regression coefficients of asbestosis grade on survival time for rats without lung tumours, exposed for 3 months or more and with survival of at least 400 days after first exposure. Differences in these coefficients between the 5 typos of asbestos and the 4 lengths of exposure were not significant and the pooled coefficient o f 0-00304 rj-: O-UdObJ grade units per day was used. Tho asbestosis grade of each rat was then adjusted using this slope to an arbitrary sunival. The adjusted mean asbestosis grades were then calculated for each of th e 2u groups, for those with and wit Smut lung tum ours In 1.1 of these groups the mean asbestosis grade was higher in the animals with lung tumours: in the other .1 groups the opposite occurred b u t only slightly so in 4 cases. The accuracy of an estimate of the dilferenee in asbestosis between those with and those without a tu m o u r is d ependent on the num ber of animals in each category which varies between groups, and weighting each group to take account of this gave a mean dilferenee o f 0-71 ; (HU, Hence overall Hie animals with lung tu m o u rs had significantly (P < O-OO!) more asbestosis than those without Differences between the dusts were not significant but the wide range m means - an tlm p ln Hite o 2.1, C anadian ehiysotile o-42, eroeidolite U-UH, amosite O-S.l and liliodesian eluysotile 1-22 - slo w s tha t there are insufficient d a ta to reach an y firm onebisions on this (piestion. The groups exposed for only one day provide .supporting evidence of a relation ship between lung tumours and asbestosis T hen' was ver\ tittle asbestosis in these groups (Tabic VI) and icsti letiiig a tte n tion to animals which su rv o e d for at least <>00 days, the mean survival times of those with and without lung tumours were very .similar. There were 17 lung tu m o u rs in 201 ra ts; only (i of these occurred in 157 ra ts w ithout asbestosis (3-8%) while 11 occurred in the 44 rats with m inimal or slight asbestosis (2.1%), a highly significant difference (i*<0-00l) Turnout's at sties other than lung A total of 412 tumours, other than lung tum ours 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. Both of these adenomata occurred 4 tim es as frequently in females as in males. The numbers of these tumours, other benign tumours and malignant tu m o u rs for each type of asbestos art- shown in Table V III. For none of the T aui.k V il 1. - N umber o f Tum ours af Ail< Other than the. i.wn<J Bcntj'n hfiimtiD t , Maine IMmt lUitil f'w try Other ttimmu- AmoMl' 'I'Z 40 4 S An! hoplu 1/ih* M AS 4 *2 Ai 4 Ill rhiwtHif `2.\ 2<> 7 1 ((`murium) `Im 'id Hi `{7 9 H (HuhI M.tH} (Smlml 20 M *> 13 tu m o u r type *s was th e dificreneo between the control and the asbestos treated significant. In Table IX more detail igiven o f the sites o f th e tumour* with all t \ pex of asbestos combined The huge-t differences between the treated and eon tro! ra ts were for tu m o u r s o f the. ov.ui, to in tre a te d and none in controls, and tu m o u rs of ni.de genito-urinarv organs, 11 in Irouted a n d none in controls. However, neither dilioronco was significant A few ra ts had multiple, maligua'i! tu m o u rs. 2 of Die rats with mesothelioma of th e plcuia also had a lung carcinoma and one r a t with a squamous carcinoma * lil-'F KC T.S <>!' I X I I A I ,A T H i \ u|,' n S H K . S T O S !.X U A T S -(7 ) * * I 11 t I i s ,->i in 11 e h" \ 1 V\ f<*il At.-a ill l- 4 h nl! i-i -t I HU v.trv. * Sll'l ns, U ign.uU l-liom l in' 'in ' UU'lll. T a m .k IX. S i leu o f T n m a u n O l l i e r ll i n t t 1.HHff A-Jx'Hi iis ( t( .tlrtl <*imu 11<1 Ni?f/Tum<itu tyj* Dii'iX'-hvf nii'ftiix mid iM'ntohtMiin Houu itnil skill Hivjut Ovary f*nah* /fNMtwummy i Mith* `''iHtMmnury ut ImiiU'iiuuiil Thymoma Lymptumva loukawiiM -I ;{ 1(Hi :i J 17:i 7 a 1hyroul M) Mnitonnnt :t 4 0 i h S I 1 X .1 m. <haMilium xaln ary 1hvruai 1 o 'Jii o 11 u i 0 I Mi|>tuh*nai Muh^nanl ) { <1 A ii 0 1 ti of the lung lm<! a mesothelioma, tunica vaginalis. Mesothcliomata o f the vagin alis were seen in this and one oilier r a t; there is no evidence to suggest an associa tion with exposure to asbestos. Also, in some cases there were secondaries, for example, a synovioma had spread to the lung. In all such eases tu m o u rs have been classified by th e prim ary site and in (lie few eases of multiple m alignant tumours there was no difficulty in recog nizing the distinct types, t>. one was not a secondary of the other. DISCUSSION Our finding that the nsbestosis pro d u c e d by exposure progressed after cessa tion of exposure, is in ngicem ent with human experience b u t c ontra sts wills the early inhalation experim ents reported hv Vonvakl, D urkati a n d P r a tt (Ih o i) m which progression did n o t occur W.itruer (1903) reported more nsbestosis with nmosite than with chrysotile m uiuea-pigs, rats and monkeys but our f\|ii'iiments show t h a t of th e U lt't' -tandard reference samples nmosite is the least iibrogemc in rats. (iross et al. (1907) found lung cancers <:i '.'.l of 72 rats which survived Hi m o n th s ' <\|ii>Mire to elnysoiile thw t at a mean diluent rat ion o( 30 msr/nr' for 30 hours v week They considered th ,d contami: itinn of the asbestos by trace metals from the worn hammer o f the mill used to produce the respirable fibre could have been a factor in the causation of the tumours. However, our results now show t h a t there is no need to invoice such a hypothesis to explain the high rate of lung t umoui s The amount of eluysotjlc retained m the lungs did not show any clear increase with dose in rats exposed for longer than 3 months. In two earlier expetiinents (W agner and Skidmore. 199.7, Morris el al., 1997) a higher airborne dust concent ration was used to give a cumulative dose in 0 weeks similar to that, given m the pic,sent experiment over 3 months The weight of asbestos found in the lungs of lats exposed to ampliibole was 3 times greater th an in those exposes I to chrysotile. In the present experiments the ratio was 9 to I after 3 months, hut increased with continuing exposure as the weight of ampliibole in the. lungs continued to increase, but the amount of chrysotile did not The previous experiments had shown that the rate o( elimination ol dust from the lungs was much greater for chrysotile than for the amphiboles. The present results may he explained on this basis, the weight of ehiysotile having readied equtlibt him level, be. the rate of elimination ecpi,tiling the late of retent ion. There arc a num ber of features of the results presented above whi<h we found J 29X .1. C. WVINK! ! . liKHKV. .1. \V. SKIDMUKK AND V. TIMUHKU. surprising. First, m l-Apciimciit I two of sites rilhcr than the lung t.s equivocal. IlIrM li hrlioinat.t ul-l tilled with the one All hough an association with gastro 5 fill v d a \ ]o'iin t* (otiipared with <>nl\ one intestinal tu m o u is has been iound epi- * f '.III with (iic 5 m o n th s' t*x|o-.m<*, which had ,1 demiologieally. d is not yet icgarded as ] W hi. dosage m u l e Ilian all times greater. eleaih established (Xelikolb Hammond 4 II' t In* incidence ol mesiil liHiomala was and ( l u n g , 1972, New house, 1974), and i proport iunal to dose, as is indicated for is o| lower m agnitude than tile excess l 1lie | d l 1! 1 till inoi nlation o p c u m c n l s (W a rn e r <1 lung (.nicer risk Our experiments pro K 'siil til., I(17;!), then the probalnlit v ol such vide no siippoit for such an association \ an extremis result occurring by chance The expei mental woik o f i.'ruham and would he about 2 in a Inon dralnon (1997) suggested th a t intra- i Secondly, there was no evidence of pentonoal inject inn of tremolite asbestos res,,, tills (Is H ie 1 cither less eareinogenieit \ or less asbestosis could pioduee ovarian tumours, but the lif in the groups exposed to ebrysolde than follow-up of women asbestos workers i b those exposed to the amphibolos, even reported by New house ft a! (1972) pro though the am ounts of dust in the lungs duced no definite conclusions on this w ho polls, were so different In particular, the question because ol tin 1 m rity uf the H- U l d d Canadian ehrysotile pioduced as tnnioiu Our experim ents do give some i ' Wo n t m any m e s o th d io m a ta us the t Ti ( `(' eroei- .support to an association between asbestos * t o I) dolite. T he 2 U ! ( V samples of ehrvsotile exposure and ovarian tum ours as well Lam. produced 12 of the 14 tum ours with as tum ours of the male genito-urmary class). inetastuses. However, much less dust svstem Although neither was significant ` grate, was retained in the lungs ol ta ts exposed this <mild be because of the rolativelv Walt. to ehivsotile than umphiholes (Fig. ID) small si'/e ol the control group. To I urn i I Moreover, after intrapleural inoculation oveieome this, we have included the 1` V p e i the risk o f a mesothelioma occurring ro n d o ! rats Iron) some of our oilier <otiip . wills UICC ctoeidolit.e is 3 times the risk experiments, tints increasing the non I with ehivsotile (W agner <1 nl., 1973). d e n te d group to 4(13 ra ts of t,he sann Theiefore, allowing for the greater reten strain This larger group contained - tion of croeidoiite after inhalation, we might have expected the risk with 'Toeidohte to have been of tie' ordei ol 20 malignant tum ours of the ovary anil .5 tu m o u is of the genito-urm ary tract in males. In over TOO ra ts exposed to 1.1 Ml s. U-di f V, *> Kl t i times t h a t of <In vsotile asbestos there weie In ovad.m tumouis. 'd'-lss Two of the m esotheliomata occurred 7 ot which v e i c m alignant, and II K within 4(K) da.vs of the sta ll of exposutc This nun- be compared with our injection tumours of the genito-urinary tract m males Hence, based oil the. huger ' t nil jH < in , < lit \ experim ents in which only 20 out. of X03 occurred within 400 days (Wagner and lh>rrv, 11)159; W agner cl nl , 1973) Also, the earliest mesothelioma occurred after llaa days and we observed only 3 within this period in our injection experiments. T he positive association between axhcstosis and lung tu m o u is which we have established in (lie anim als i,-> in agreement, u ith epidemiological lindmgs (e .j . Minister ol Labour and N ational Service, 1949, of controls, the association between ad tos e x p o sm e and ovarian tumours iweak and non-signilieant, whereas then- e liosuppoit for an association with tumourtil the male genito-urm ary system 'flic U K V c h n s o t i l e samples are lin>-r than the e h r w ,t i l e whieli lias been im i in industry in the past. However, d u e ls a trend for iudusti.v to use liner din sot lie (W right, 1999) and so the e\|n-u mental results mav be more relevant 9. ^ u K\'.\, I {*H>*\ , 1 tits id, K M II \ , i.|Mf fti m I, *rr;'HI.' W ~t. i ' s * f D s, Ml\/M, , ( l `l I'l. K nox el ftI , 190S; Limes and Simpson, 1971) The failure to establish any association the c u n e ilt situation than to the |a-f We are investigating the effects of iiili.i! i lion ol chi vsotile m mote detail in .it. f ! f t , / , m ivi M i -s ; IhM, between asbestos exposure and tum ours expeiim cnt mvoK mg U K V ( 'anaili i:i k k k k rs o r i m i u . v n o x o r a m j k s t o s in i: \ t s .Mi'. chis sol ile .i Alalie 7 su m | ile front ,i U IV. ( I % 7 ) i 'nmpm ml Ott-s( UvMi n i t o n m ( micidiali mute ,m<! (he Mipclhnc sample which proved (lie most e.uemogcinc of 'd H im^ M U m < ,(titl S I an* Iti ht I u h o h 'd f< > / m d I / t j / u u t u t l ivi U a v t t H O s f o j c l I V r g a m m i }t 2n,~t IhlJ s X the materials \\ hi* 11 sse inoculated jntrnplcui'iilly i\\ ;inri </ o / , 1!I7,'!) The experim ents ssc tepori have given Xl'VVllHt n (lit i'.'i P m , \J U { M>74) (*a!ii`ci \ s b iM n - , T\fl< 111( f11 si t \ , o f . \ s h i \ l ( / \ Uytn, 2 P .n n n y s k t, -I. ( ' n h m i , S' W mkofs in hh>t(ji(fi n l O rin i f 1972 Tif ffi f<Jf a m i I'isults which m s< vci.it inspects <or iosjhm<! !o tliosc found m m.m T hus, this expenmenful mctlioil is rstaidM icd u s a valid tool foi the investigation of the biological (`fleets of asbestos .1 1 ' SS f { I \ i<( ' Kt V nw f H PlpJflMi t 'ntts, V o Hp In (uittf \ i w m u s i \ M L , H m m v , \ ) , U ' v n v i u t , .1, ( \ A* Ti H itK , M K A M i oh "( fltt* Mortality nf Kmiml A M mM on \ V n f k l - s /V, J a i d J/r<h , 29. i M S i { i k o i r , .1 , if \ M \ i n \ n l\ c a ( 1 9 7 3 ) PdlCIIHiJti li l t ' l l y mi' ,\nm-vi!i ( `itt m., J \slw--dns. .l/c/ot t u t a , ({ l i f t, 2 5 , 5M We lire grateful to all our colleagues ^ t u i \ n , 1. M A h m . l . Lo -am s in liiti hunt's X (I'JTiii Mmplwln^uta! IrnltusA b\ P nhcyclu ' who over a num ber of \eni\s were res ponsible for the (fails a tte n tio n neressm y m carrying out the experiments. We would also like to aeknou ledge our (hanks to Dr .Harold Stew art of the National i'nicer Institute' who advised us on the classification of turnouts We are also giateful to our former colleague. Dr A. Walter, who had classified the mm-lung tumours occurring m some of our earlier experiments which we mentioned for ouuparisoii H V<>n-* lit \ f O / d t o l o r / i / o f I frt'i l H H U ii u l It i Ot Uf Of t) ( I ( f t I 111)1/1 III s i ' 1*1 *M ( `u i i f , O u t l m b u r ^ . \ 'A M, M a \ , h7D I7<1. P X V H o s h n m , M h tiu m m . J r mai .1 W {) itili la^n Jr, n . S A to m ic K im'il <'uiiimtssKu( Symposium S e i t z s X o } ) | , 2 S ' w t o n ', M }' ( I 9 7 i ) h i ( `itlhofoifis <>} T u r n o u t * m L u h o m to n j l / o m n h \XI I T t i m u t i f s at {Hi* } i a l l `. m 2 1 A R < ' N'miit <><' Pit PI ten fimi'* No. 9. Lv oit Itii< t tiitt it oh i Sj^rnt-v i'i / H " scuroh ( 'aittMW hi J>IC}NUMtH>n fJ IMIt liKi 1 , C h r s o v , .] (' s\ Wl-HMKH, 1 1 9UK) h i l ' i 1 Shut<ku<! Ih'itM'tMH'r Iti A' '! 'Ins I i d d ( ' u n a >, 5 , PM T i M i t u r u . , V , I h u i . A S\r A ^ u u * M ` ut . .1 \V ( MHiH] \ Simp !*1 ] ) i -.|m h m i I"i 11 i it'tu'tai tifM Otj'.i 1 IdUfl*- Itimi m i litii'K M it c S.u u ph ^ u* Ssht'-^ltts d u n //////, 11. 274 KKfKKKWKs i'1'ms, l1. C A .Snft'Hos, M ,f <' lli)71) hf-mini am \Voikn\x m litjfiwb A Mmiahtv hUn HI* H t . ./ iml Med . ZH, T t u i m m i ., \ ' , S hiovoitiA .1 \ \ \ t H vktT, A W \\ WhuM'.li, .1 0 (9 7 P ) K \| k u h ` r h a n i h o r s Pm CnhnUtKm 'Xpi'nmnnt s u n ii SiimAiiit} Kt'liM non Samiihs t( A-'U*.* **f` iIn* imt'r milDMi.il I ' liK/it A u . u u d ( `inti-} ( I ' H ' I ' 1 /rasoi `1 A''lM,-*lMstS b H \H SSf, J . (X* <fi.SHA M. ( 1 100 7 i O v i l i UUt ( Mucnn S " ,, 1, 31 i am i AsPs-sto'* l . t i t ' i t H i ' . 1 , 1 1.7. S'tntu u .i * .A .) \ )t ttk t \ T Si A i ' u v f j . P. i ' hf'uvs, P , o k T u n v ii .i )S T P , T m k t u , lh H., M<>.7i) K' \ p t `I HUtMli .1 St *ltii - * K s' K M u , M A U VMS' \ * M V i h . H w ) K x p t ' i t f M i I1u il s I m l l / 1/ 1/ A, i * m* nhvt A>b>*siosis 'I'h* f )( v <{i*pm<'i i f oi Utmi.' U .) r. -nmh v-tiv->i*>sis K\jk' i nml ` t u r n m K a f *. w n U 1'll hl lU IM I V l )t'j K>M<s oj \iuiiiiiX H t .J m d Mvd , 20. < l Us s stiifin \S , ;u:i AMn-stns 1)u si .l i ( h curii ffflh. S\' V'.Xf'M, J A s h o s l o s lOnsl Anti X P a d St i . 132. IPO. K m o s , J . K , U ts iM i'N , S 1). 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