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FILE NAME Threshold Limit Values TLV DATE 1974 DOC TLV004 DOCUMENT DESCRIPTION Journal Article - The Effects of the Inhalation of Asbestos in Rats ~ t. eo . am ee Ee ce ee Br Cancer 1974 29 252 a i 7 ae from Meso ee 774409 09 SETKI SETKISETKI SETKI LULELS LULELS LULELS THE EFFECTS OF THE INHALATION OF ASBESTOS IN RATS C. WAGNER G. BERRY J. W. SKIDMORE AND V. TIMBRELL From the Medical Resourch Council's Purumoroninsis Unit Landough Hospital Penarth Glamorgan Received 5 September 1973 Acerpted 29 November 1973 Summary 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 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 11 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 WAGNER Bercy and Timbrell 1973 reported the results of experiments in which rats were inoculated intrapleurally with samples of asbestos In the dis- cussion it was mentioned that two experiments in which rats had been exposed to dust clouds of the UIC reference samples had been carried out and the results of these are now presented Some preliminary results of one of these experiments were reported by Wagner 1972 In 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 the dose and type of asbestos dust but additionally the amount of fibrosis was assessed MATERIALS AND MISTIROIDS Caesarean derived rats of the Wistar strain were used which had been bred at the Unit from SPP stocks given us by the Imperial Chemical Industries Pharma- ceutical Division at Alderley Edge Cheshire in 1964 and 1968 The asbestos samples used consisted of the 5 VICC standard reference samples Timbrell Gilson and Webster 1968 which were prepared following recommendations of The UICC These samples are of 3 amphibole types amosite anthophyllite and crocide and 2 chrysotiles Canadian and a Rhodesian sample The rats were exposed in 1-4 minhala- tion chambers Timbrell et al 1970 which contained 8 cages each of which could hold 6 rats or for a short period with young rats there was room for a seventh Five chambers were used one for each type of asbestos The chambers were constructed so that the rats could be tended without the chambers being opened The dust clouds were generated using a specially dovised dispenser Timbrell Hyett and Skidmore 1968 The clouds were generated for 7 hours a day and 5 days per week The respirable dust concentrations were measured using size selective grau samples metne dust The collected Cassella Type 1141 samples were evaluated at the end of each daily session In order to achieve the required dosage calculated a- the product of concentration and tim vanations oreurring in the concentrations were corrected on the following days A the end of exposure the rats were left in the chambers for a few days to allow time fic SUB ME q ne he OP wea: > EFFECTS OF INHALATION OF ASBESTOS IN RATS 253 their fur to become clear of asbestos belove transferring them to a clean environment for the remainder of then lives At transfer some were rats generally or 3 killed and the lungs of each sex removed for histological examination and determination of dust content As each type of asbestos lias a consider able siliente content about 50 the amount of dust in the lungs was determmed 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 al 1967 After exposure the rats threes or fours isolated in supplied with filtered air chambers were also in this were caged in a special unit The inhalation 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 examinto preparation staining and staing microscopie methods the 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 electron microscopic examination and the remainder of the hung 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 were sheed sagittally routine sections being taken of the whole left lung and the upper and lower lobes of the right lung In addition any other other suspicious lesions from the lungs or organs were taken for histological examination In all cases sections were stained with haematoxylin and eosin and the lung sections were stained for clastin reticulin and col- lagen Special stains were used in Some rases as an aid to diagnosis of the tumours Assessment of the severity of asbestosis Sections of both lungs were examined without knowledge of the duration or type of asbestos exposure and with the animals in random order The sections were observed on viewing screen of a Projectinn Microscope 4013 BK using a ^ objective At this magnification a large proportion of the lung eould be assessed in a single field and as it was not possible to observe asbestos fibres at this magnification the results were nof biased by knowing the type of asbestos to which the animal had been exposed Experiments In both experiments and for all doses there all 5 were groups which referetice samples were exposed to there were also exposed control groups which were not exposed Rats were allocated to treatments et raudom 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 munders of males and females Experiment There were 2 time inter- vals of exposure in the first rats were exposed for 3 months starting in May 1967 and in the second groups of rats were exposed for one day only in August 1967 In addition to the killings at the end of exposure in the month group there were also intermediate sacrifices at 5 8 and 10 weeks Experiment Experiment There were 3 time inter- vals of exposure months 12 months and 24 months The experiment started in January 1969 and after 6 months half of the rats were removed from the cabinets They were replaced and a year later these replacement animals were in turn removed They were replaced by animals to be used for special electron microscopy examinations which will not The remaining be reported in this paper animals were removed in January 1971 the month after 2 groups years exposiett exposiett in addition to the killings at the end of exposure rats were also folled after 2 years 1.A 18 months after removal from exposure The numbers of rats are given in Table 1 In the month groups overcrowding amongst the males tended to ocem due to merease in size after about a year and some_were_removed some_were_removed some_were_removed prematurely_after_13 prematurely_after_13 prematurely_after_13 months For analysis these rats have been TITLHILH TITLIH LH TILH mons ^minut Bre EE A wee eb bt Ry a cia Oi A t ati h ed we Annee TIMBRELI 254 J. ( WAGNER 1. BERRY W. SKIDMORK AND Y. TIMBRELI ceapstine TABLE 1. Number of Bats Exposed Exposed i Chrysot Veattle Long ol exposure exposure exposure 1 1 day months 4 mathms aths 12 months 1 months Amosthe 49 52 + 2 21 Authuplis ibto 49 S2145 S2145 S2145 S2145 Crossdzelite 49 62 24 26 20 Canadian 49 oe OE < 23 24 Rhodissim Conbol $ 48 2457 58 2457 \ 2457 > 48 2457 3 Respirable mg e Table H. -Mean Dust Caucentration t- and Cumulative Dose mginemgine hours beysolile Chav Rhodesian Length of Chemtrofoslabe Chemtrofoslabe Cunedinn Rhodesian vt Exposure Ambosite Authophyllte iM) 12.5 ** 11-7 14.7 103 ay KF elaty 14.1 12.8 12.8 5038 12-1 4930 12.3 2 OAM 13.5 5240 10 2 82401 10-7 8500 why months months 12.4 1t-2 8350 10-9 8540 14.7 14.7 8430 19-9 17100 17100 6 months 11.4 17401 HG 17108 30.7 17100 17100 10-1 33800 months | 12 months 10-8 17000 10-3 33200 peed 33200 ORME, 24 months 10-6 335010 20.8 33700 Aye and hence The lesions consist initially of a deposi tg fibres alveolar macrophages included in the month groups number of rats in these groups is slightly tion of asbestos in the alveoli arising directly the and in the month groups slightly and cell debris ae bronchioles Thuse higher from the respiratory ed lower than planned become organized firstly by heming In Table 11 the mean respirable dust doses deenpmoesisthsed in a thin reticulin network network which concentrations and the cumulative ~ , and time are coarsens with time and becomes replaced by The alveoku epithelium the products of concentration was 7 hous collagen fibres 1 cell shown The day exposure intervals intervals reacts with replacement of the type for dusts For the other 4 time and becomes completely lined by granular of there were slight variations in in some of the alveoli the exposure number of hours required to achieve pneumocytes shed into the lumina in others the approximately equal doses but the mean 788. 1574 and 3237 hours epithelium is of the alveoli by these there is a walling occurs at the bifurcabifurtcatioines obinfurceatiosnes times were 402. The mean concentrations were were respectively cells This usually of alveoli are closed oft frian where groups usually higher in Experment I than Experi- the lumina of the respiratory bronchioles ment 2 and the month group hai an the called pseudo a^ dose of 60"of that of the month Ring In the gameit pages These small average Reasonable equality of dose between between peatan^'e continued asbestos hine and gthreoudpusts was achieveil for all the lengths of evstic spaces but in the rats macrophages except for the one which which was degenerating were also granular picumocytes etxooposshuorrte to allow any adjustments adjustments numerous present The initial lesions were contine individual individual respiratory to occasional diserete scattered throughout the Inig further Interprctation Interprctation Interprctation Interprctation of histological findings findings lesions bronchioles substance more and After further exposure exposure bronchioles become micab al Classification of ashostosis. The of sapuned to all types more respirartespiroatoryry respiratory brandinoles arism seen in the lungs and all the respiratore respiratore respiratore of asbestos were similar to those described from termmal broncinoles herome tha kons in guinea pigs Wagner 1943 1965 There the Gbrous tissue network extends into 2 mam differences firstly ashiesto as launchiole and wall of the respiratory respiratory w boedriees were never seen in the lung tissue the down into interstitial reaction spreads of the rat although they are frequently which follow this elements of the primary the peripheral duets athe seen in the pleural granulomata which unit involving the alveolas the pleural inoculation of amphibole and finally the aur says and alveoli Wui fibres secondly there was a far greater the individual lesions tend .. of pneumocytes ty progressionleading to the development of granular production cells in the rat coalesce 11 alveolar epithelial +Yi S wEi e cat e4vt, a oo ae e Le an ee ae be oom v OT PN ad -~ wed Fig 1 -Slight asbestosis Grade 4 Thickening of the walls of the alveoli arising slirectly the respiratory bronchioles with replacement of normal epitheliura by type 1 cells H & frons SO * {-. oo . | 4 e a ra : , Ps N ohh Fi ve Oe - 4 t tn, ty oe ye ce ay* j gs sg Bone os s * ga SN, ~ ; rs: : ^' 4 wt a * qtss Fu 2 Slight asbestosis Grade 4 Higher power showing numerous retr^eulecrocidilule bbmus in the alveolt of respiratory branchiale Asbestos hodien nie razely seen in the lungs of mits i . iljumination reducer to illustrate the libres H. 6 BIK mY comes en. ~~ ', am, amecr ~ - o. (meg. am Gite Taner USICH 7 i . _ raNDON ay org 04 PEs te Gtr neva, Cling? Wee Ore . > 54 ep S147 wee Sth, -& #297 a urte PAS 3 POS nes f . ees wee pr ReextAaya, be ow. 8 a reg 3 ad se Pte : ve & raNDON 3 Moslerate as^>rostosis Ginde li A lower power propretion dhistrating that the lesion fu diffuse Aggregations of type II bad although still mainly involving the sespantory Irinihiole now raNDON the walls H. & R. 80 les are seen in the lumina as well as investing amen pneumocz imam imam noend shy 8Sree Wipe Sg eiee say etoee, ? Fi dovere ashestosis Grade 8 There a generalized onderstafind libroNIS H. & E. fe a a 6g a + : a, oo . ^' : 4. ve EFFECTS OF INHALATION OF ASBESTOS IN RATS 257 257 257 diffuse interstitial fibrosis with gradual un crease in the density of the fibrous tissue ultimately resulting in the replacement of most of the long parenchyma by a dense collagen network surrounding distorted ar spaces many of which contain large clumps of granular pneumocytes which in some areas have lysed leaving foci of alveolar prote^>nosis The assessment of these lesions was based on 4 grades of minimal sfight moderate and severe Typical examples of slight moderate and severe asbestosis are shown in Fig 1-4 In addi tion when carrying out the assessinent if was found convenient to introduce the intermediate categories minimal slight moderate and moderate 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 different assigned reading random to within order and one category 90 were of the first Classification of tunwars 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 by Kuschner and Laskin 1970 and Shabad and Pyley 1970 Further these authors have described the development and morphological features of the tumours that we are reporting All these tumours were peripheral and appeared to arise from the region of the respiratory bonchioles in which the asbestos fibre had accumulated The origin of the adenomata appeared to be from accumulation of type 1 epithehal cells that proliferated in the alveoli of the respiratory bronchioles Fig 6 In many of the exposed animals these tumours were multiple and m ! mmmmber of animals particularly those with the more grades of asbestosis there seemed severe to be adenomata arising from numerous adjoining respiratory bronchioles giving an impressin af contiguous adenomata invading large areas of the hang } Harold Stewart personal communcation suggested that this type of lesion should be refered to uN adenomatosis In contrast to this a few control animals were seen to adenomata which were small have sulitary in size The adenocarcinomata were of the same type and origin as the alveolar adenocaremo mala described by Shabad and Py ov 1970 many of these tumoms were papillary papilary carenomata The squamous eat- cinomata appeared to originate from foci of squamous metaplasia becurring in the achestotic lesions in the respiratory bron- chioles Fig 7 8 As far as can be ascer- tained all 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 the Morphology of Experimental Respiratory Carcinogenesis in 1970 and the chapter by M. Stanton 1974 in the IARC Monograph on Pathology of Tumours in Laboratory Animes contains detailed descriptions of the tumours that we have illustrated The rats used in this experiment are from a caesarean derived harrier maintained colony and fortunately they have been kept free of rat bronchitis therefore the squamous metaplasia was not associated with bronchi- vetasis Metastases in the thoracic cavity to the chest wall diaphragm pericardium or the bronchial lymph glands were seen 14 animals the majority had lesions mvarling 3 of the sites and in only 2 animals were metastases observed in the tracheo bronchial glands In one animal secondary deposits were soon in sections from a kidney Eight adenocaremomata and 6 squamous tumours had metastasized RESULTS All except 2 of the rats in the groups with exposure of 12 months OF less survived for the whole of their planned exposure In the month 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 only & because of cannibalism Dust retention The mean weights of asbestos dust the lungs of animals killed at the J. MENER J. W. SKIDMORE AND V. TIMBRELL 8912BS 8912BS 8912B8S912BS nandes nandes nandes iram i Oy er.Non os os pneumocytes showing showing Type El pneumocytes Electron Electron micrography micrography micrography of an adenoma we a4, ne! a) t EFFECTS OF INHALATION INHALATION OF ASBESTOS IN RATS ey Se @. wna, hd < at 4 posta at " . aed * a4 aye, . y ats ae wee 1 9 wens to}. 2 o,f if BS tefae 2 * eo ST , * hd 4 .. ve 5 | & : wef 8 cA i | Byeees ae wt tt Pot Pag f som a Dat a eo a er re GRE Lata: Ly S! De ah we m.e an - . t yrs 1s: Mr MN a AES . ene so 4 ? v a eo 14 ae ~ * a ? othe : rd ed . . e*, URED an . - ey ee, a mas + eat pr. : sory @. Byes ~~ od . L 5 . . $ ? 259 : Fra oe < <. * at op -2 - Squamous metaplastat metaplastat metaplastat superatalposed on moderate asheshusts H & ot, Fie 8 Early squamous caremota an amenal with se voter aishes^ UNIS & E 300 + . : t ae . . a ay Weich WWeeiicchWheich Medi^as Medi^as ( kuraiy kuraiy kuraiy 260 C. WAGNER G. BERRY J. W. SKIDMORE AND V. TIMBRELL Length of exposure weeks 8 weeks 4 weeks months months 6 months 12 months 24 months G month after 18 month~-s Bon expisine expisine Table Menu dose mug hours 1880 2450 329 3030 8170 17100 33400 33400 Dust Retained in Lungs mg Chuysotske Chuysotske Chuysotske Amosite Authumphy llte Crondolte Canadian 1.0 01-9 2-60 3-7 7 8.3 16.8 " 3 1-0 2.8 3.5 4-4 9 6 13.8 2-5 Vl 1.6 2.1 3.0 4.5 9.3 14.9 1.2 11.1 (tek G5 9.6 0.4 0.8 03 0.0 Chrysotile Rhodesian 0.1 0.3 1-6 Od 7.4 1.4 0.6 ) - ems. rene No ee ae vn , Length of exposure exposure exposure 1 day 3 months anal 6 months 12 months 24 months TABLE Mean Survival after Amosite 804 771 763 692 807 AnthophylAnthophylitelite 806 823 686 759 778 Crucidolite 796 817 789 776 750 First Exposure days Chrysotile Canadian Chrysotile Chrysotile Rhodesian 763 753 790 689 778 857 766 826 585 758 Control 803 793 754 * Adjusted to be independent independent of sacrifices Fa s scheduled times are given in Table 111 More dust was usually found in males than in females and on average the female lungs contained only 70 as much dust as the male lungs The values in Table 111 are the averages of the male and female means For the 3 amphiboles there was a similar pattern with an almost pro- portional increase of lung dust with dose The 2 chrysotiles were similar to one another but much less dust was found than with the amphiboles also the chrysotile figures did not show the same clear increase with dose The main features are summarized in Fig 9. The dust in the lungs of the animals which had 6 months exposure had been partially eliminated 18 months after removal from exposure The proportions eliminated were %% for amosite 73 for crocidolite but only 41 for anthophyllite ever the lower elimination of Howantho phyllite was not significantly different from the aunosite and crocidofite figures Suvriral The mean lengths of survival from the day the rats were first exposed are t- given in Table IV The survival times have been estimated so as to be inde- pendent of the sacrifices The short survival of the month group exposed om to Canadian chrysotile was largely due to 8 rats dying before Day 400 in the ~ other four month groups only 2 rats y died before Day 400 These early deaths were not due to exposure since ^ died due to an infection in one cage and 2 * were falled in fight Discounting these ~ 8 deaths the mean survival was 698 o n days which was still the lowest of the month groups When the mean was taken over all the lengths of exposure the Canadian chrysotile groups showed a least survival but only a month less than the control groups The amosite | the and anthophyllite groups had mean sur- vivals only a few days less than controls while the crocidolito and Rho- desian chrysotile 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 wee overall survival of the animals This is in marked contrast to our intrapleural moculation experiments in which injection Weight of dust in lungs ing 15 4 EFFECTS EFECTS EFECTS EFFECTEFSFECTS INHALATION OF ASBESTOS ASBESTOS IN RATS 201 10 $ 7 Arphiboles ar m See Mer removal from exposur ' Chrysotiles ee Tag 0 r OM 3 G Y% Fa T ee 12 I T T re wee ee T T Lg 2400 i. Time months , wae 10000 20000 Cumulative dose mg hours 30000 PoPo Mean weight of dust my hinigs of rats aulatuant aulatuant aulatuant to diesse apud tatar of asbestos reduces the expectation of life by several months Wagner al 1973 the 3- and month micans there was an merease of asbestosis with exposure for all the dusts Also following 6 months rn Asbestosis Asbestosis exposure there was progression during the following 18 months without exposure exposure showed 4, for all the asbestos types but these tats The amount of asbestosis WILS SSPSSed did not fire all the rats killed at scheduled times as badly as those which continued exposure There were signi in Experiment 2 and after 8 werks and ficant differences between the asbestOS months exposure in Experiment 1 types / < 0.01 annosite invariably There were 5 or 6 rats po treatment for the least asbestosis gave each throughout antho exposure except that there were phyllite phyllite and Canadian chrysotjle showed only 3 after 8 weeks Overall the 2 most asbestosis after 6 months sexes had similar amounts of asbestosis or longer exposure crocidulite and Rhodesian ebry- and they have therefore been combined sotile were intermediate give the mean asbestosis scores in Table V which are summarized in Fig 10 Except for somie mconsistenty between The mean asbestosis scores wluch were allowed to hye lives are given in Table VI of the rats out ther Those rats cn w Ce tag SaoE a apy eae tae wet Daw ce Rega re wai TejonTejon Media MediaMedia Media oe Livrais Livrais LivrLiavirsais 262 C. WAGNER C. BERRY J. W. SKIDMORE AND V. TIMBRELL Asbestosis grade Ag Moderate 7 8 grein Slight ai = rr) ceeet oy Exposed to wae -" weet After removal from exposure asbestos 4 7 er Minimal = Nil ~ t 0 Controls be ny eet ee 3 6 i iL , Li 10000 Ce eee 12 a x ii 20000 -4 - .? ey ue 24 Time month a a t 30000 Cumulative dose mg mhours Sag 1 10 Ashestosis in sacrificed rafs in relation to dose and timu Length of BS pea Raa he months months months 12 months TABLE V Ammosites 2u 2-3 22 tt Menu Ashestosis Scores Arthophyllte ay 3 2 hoard Crondolte 8 8 G 3 of Sacrificed Chrysottle Canadian 2 4 ta 4.3 Rats Chrysotile Chrysotile Rhodesuae 2.7 77-19 2.6 3 -- outro Ie't " 4 je. 18 months 24 mouths mouths months after 18 months % 3 2 2 ae 4.8 7 0 3 3-8 ard ts ge n + nun exposure * wl 21 mmubual 4 shght Gemocrate Gemocrate 8 vere scheduled for 24 months have been dividert months exposure of Rhodesian chry- tile The rats which died before com into those that died before completion of exposure and those that survived for jileting 21 months exposure had mon asbestosis than those sacrificed afta :: a period of exposure The amount anthr of asbestosis found in the rats exposed months exposure for amosite for one day was no more than that found phyllite and Rhodesian chrysotile This the case for crocidolite and ^fin control rats Comparing Tables V was Hot and Vi for the rats which completed their Canadian chrysotile for which those tats bad occurred be that died during exposure had shorter exposure progression and death with mean survivals than for the other shirts tween the end of exposure the Meaned over all dusts those rats that all dusts the single exception being 400 law Qu lar 01 bet ty ith Thi Tu clu ier 110 ee of THE I the DEERREY shoy Late lung cond abser UNH SUPE to hi Schen withi 2072 IR eee ae Pet y th + s te Se _e SHE stale h h 111itsel 11+ rinte EPPECTS OF INHALATION OF ASBESTOS IN RATS Table Length Length of .-- Mean Asbestosis Scores of Survivors Mean Survival in Months NHL Taly Talay 3 dwuths fi months 12 montlis montlis Up to 250 tubuths tubuths Ainosala 1.3 9.9 3 4.8 4. 26 25 24 23 23 Anthojdyflite Anthojdyflite 1-3 3-2 4.2 4-4 64 24 27 20 25 22 Croculudito 1.2 + 3.2 5-6 4.2 28 27 24 25 14 Chrysatile Camedian +2 33 3-7 5.1 5-1 95 20 20 25 16 Chrysotik Chrysotik Khork Khork stu^ 1-4 2.8 4.2 28.1 61 23 28 21 27 22 24 months 63 28 7.0 28 6-8 29 vs 6.8 28 * uil 2 minimal 4 slight 8 moilerate 8 severe 263 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 VI there is again less asbestosis for amosite than the other dusts although the difference is not as large as in Table V. For rats which completed their exposure the difference between amosite and the other 4 asbestos types types had a mean of 0-7 for rats sacrificed and 0-5 for survivors The results in Table VI do not support the findings in Table V that anthophyllite and Canadian chrysotile produce more asbestosis than erocidolite and Rhodesian chrysotile and we conclude therefore that there were no important differences in the amount asbestosis produced by these + samples samples Tumours of the lung Lung tumours were observed in 247 of the rats exposed to asbestos The total naabers of each kind for each dust are shown in Table VII where those rats with more than one tumour of the hung classification is by the more severe condition No tumours of the lung were ^,lserved within 300 days of the start of xposure and 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 AQ control rats out of 84 survivors in Experiment 1 with adenomata but in Experi- Experiment 2 there were no lung tumours out of 42 control rats There were slightly more male than female rats with tumours -128 compared with but the only 2 tumour typos for which there was any major difference between the sexes were adenocarcinoma and squamous carcin- oma Out of 50 adenocarcinomata 35 occurred in males whereas 30 of the 40 squamous carcinomata were in tomales Metastases occurred in 20 nits 10 of each Sex There were also mex0- theliomata Table VT ) in males Two of the mosotheliomata occurred with only one day's exposure 1 with 3 months none with 6 months 6 with 12 months and 2 with 24 months The m080- 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 1 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 coresponding controls there was clearly no evidence that these adenomata were a sure to asbestos consequence There were of expo- expo- ^ more serious tumours 2 of these were meso- theliomata one with amosite after 715 days and the other with erocidolite after 531 days There were also 3 adeno- carcinomata one with crocidalite after * * a 3 _ a i. = z : ae iH of 3 cA ney . . Welch Welch Welch Welch Marioli Marioli Marioli Marioli Marioli Qutay Qutay QutayQutay Qutay Qutay __ ag ae: aint Rh 11 Tes Se we oA eee ee 264 C. WAGNER C. BERKY J. W. SKIDMORE AND V. TIMBRELL TABLE Number of Animals with Lung Tumours or Mesotheliomata Type of lung tumour BA awe ecane comnenvenmmemennee No with No. No. of No. with gece ome Adenos Squamous 800 won rats at Jung miremomat entomomat htt Exposure tumour Adenoma Adenomatosis risk Exposure Exposure ht A mostle 3 QUO ( 1 day 45 QUO " months 37 7i QUO h 6 months 19 2 A 1 12 months 25 1G A 3 24 months 21 13 3 I 5 Total 146 38 19 i 1 iF) F 6 " } 6 0 Gi 1 = 0 -0 2 Anthophyllite 44 * 26302Z 00-002 1 day 26302Z 00-002 months 37 6 months 18 7552 26302Z 0-02 12 months 28 7552 26302Z 0 -0 2 24 months 18 7552 26302Z Total 145 50 26302Z 12 Cracidolite 43 DITZER 502546 CANIKI 1 day CANIKI 3 monthmosnths 36 14 502546 CANIKI 6 months months 7 DITZER 502546 26 DITZER 502546 CANIKI 12 months 502546 CANIKI 24 months DITZER CANIKI Total 141 55 502546 ***** 0 ***** 0 *** ' ( I ***** G * } 8 v a tt oe 1 } 2 me 0 1 1 { i a a 0 " G2 2 7 2 u 9 2 4 Chrysolile Chrysolile at. Canution { a a 1 day # ' th i) $ 0 U 3 months 35358 18 N^ ! 0 0 5 N^ 2 3 6 months 35358 1 - 3 6 1 1 2 } 12 months 35358 MW 2 35358 24 months it 11 3 4 a 20 Postal 137 43 Chipotle Chipotle Wholesany 1 =" 595 488559 i 50 " day 978-3 WM 595 ( ft months months 488559 595 1 50 months months 488559 978-3 2 74 4 ts 2 * a . 12 months 488559 978-3 - 2 24 months 488559 59784-3 139 10 19 9 11 4 U Total 144 } Cantial Cantial u ( 4 + play 44 { u months months 40 I J } a 6:21 mouths % tl { } Tocat 126 v 7 tebisfases Rats winch survived survived at least 300 days after start Numbers in brackets me those with tebisftaebsisfeasses exposure exposure ) = } = = a = 807 days another with sotile after 719 days Rhodesian chry and one which metastasized with Canadian chrysotile after 838 days interpreting interpreting Table VII and Fig 1 has to be borne in mind that there was a greater tendency for rats to develop adenomata m Expernment | than in 2 as shown in the controls Experiment of an- Therefore the higher proportion mals with adenomata after 3 months than after 6 months exposine exposure an tefact We do not iksnopwrobwahbyly adenomata occurred in the 3 MONTHS AMOSITE EFFECTS OF INILALATION OF ASHESTOR IN RATS 265 6 MONTHS 12 MONTHS 24 MONTHS J 300 730 ANTHOPHYLLITE 1000 500 500 750 1000 500 750 1000 a 4 750 500 750 1000 500 750 1000 CROCIDOLITE X 300 750 500 1750 1000 500 750 1000 500 750 1000 3} 500 750 eo 1000 fiAdenocarcinoma Adenoma Adenomaadenomatosis Other couses Fig 11 Disiributions of survival times m days after first exposure controls in one experiment and not in the other but as the finding is significant P = 00 in its own right and is supported by the results from the exposed nuimals it is unlikely to be due to chance There was a higher incidence of tumours with 12 months exposure than with 6 months but little difference between the 12 and 24 months exposure Half of the 8 mesotheliomata in Experiment 2 occurred with Canadian chrysotile so that in total crocidolite utul Canadian chrysotule produced + meso- theliomata each Of the 20 tumours which metastasized 16 were after expo- sure to chrysoti1le0 with the Rhodesian sample and 6 with the Canadian Three others were with crocidolite and one with anthophyllite Two of the meso- theliomata in the month groups occur- red within 400 days after first exposure one with crocidolite after 399 days and one with Canadian chrysotile after 355 days the only rat which failed to survive survive for its scheduled 12 months exposure f, $ 4D 7 os . 48) ial vat % { re - : } see jega _ : a : - er wees pein tn waich waich waich, Medini Medini Medini MediniMedini Medini litaj litaj litaj litaj litaj ae 6 1. W. SKIDMORE AND V. TIMBRELL 266 J. WAGNEK G. BERRY f least 600 days the mean survival times Asbestosis a Asbestosis and bong lumow's of those with and without lung tumours An analysis was carried out to deter- similar There were 17 lung these mine whether there was any relationship were very in 201 rats only ti of Te between the grade of asbestosis and the Since the tumours occurred in 157 rats without asbestosis in the 44 rats presence of lung tumours survival it 3.8 while 11 occurred asbestosis gra~ e depends on with minimal or slight asbestosis 25 0.001 to standardize to a constant difference was necessary time This was achieved by a highly significant eet survival coefficients of calculating the regression for at sites other than lung grade on survival time asbestosis exposed for Tumours A total of 412 tumours other than the rats without lung taunmdouwristh survival of or mesothelioma of tumours 3 months or more lung tome observed least 400 days after first exposure were pleura or peritoneum adenomata at adenomata between The majority of these were Differences in these coefficients adenomata of asbestos and the 4 lengths of the breast or pituitary find- the <types not significant and the which were common mortem of of exposure were of 0-00304 -0.00065 ings Both Both these adenomata ococccurredurroc eur edd ings Both frequntlyfreqfureeqnutenltyly atdenhomeatsa e tfuemmoaulress e t in pooled coufficient used The frequently as grade units per day was then times The other gradle of each rat was mates numbers of these tumours benign and malignant asbestosis it benign using this slope to an arbitrary other tumours are adjusted mean asbestosis for each type of asbestos adjusted survival The tumours for for each of Table VIH . FoForr noneooff tthhee were were then calculated shown in Table grades for those with and without Sile the 20 groups groups tumours lu 15 of these groups the the VH VH Number of Tumours at Sile TABLE than the Lung higher hung asbestosis grade grade was Other Benign mean Tumas animals with lung tinnours in the other Malig only the opposite occurred but Paraut- nant 5 groups The of Other tumKnits Other slightly so in 4 cases accuracy asbestosis asbestosis asbestosis Breast os estimate of the difference in Arnestler 4 22 40 * 201 uf 15 ",, a bnetween those with and those without without a of Autisglas tumour is dependent on the number | which varies Chrysatile 23 Mis 7 animals in each category 37 " 3 3 amudian and weighting each group Chiy 1919 Rexdeset between between groups a mean 2 13 to take account of this gave Hence overall | Comiral 20 34 of 0-71 0-13 slifference with lung tumours haul the difference between the animals -20-001 -20-001 ) more asbestosis tumour types was the control and the asbestos treated significantly significantly P than those without Differences between between the significant In Table IX more detail ail but the dusts were not significant authophyllite 0 25 of the sites of the tumours with given combined The largest wile range in means authophyllite erocidohte 0-66 vsotile 0-42 chrysotile types of asbestos the treated and com differences between Canachan chrysotile 0-85 and Rhodesian for tumours of the ovary amosite are insufficient trol rats were in controls and 1-22 shows that there 40 in treated and none on 11 to reach any firm cunclusions of male genito unnary organs data tumours in controls However this question for only one day The groups exposed exposed of a relation- in treated and none neither difference was significant malignant provide supporting evidence and asbestosis lung tiranours A few rats bad multiple 2 of the rats with mesothelioma ship between between little asbestosis in these Lamours also had a lung carcmoma There was very Table VI and restricting survived atten- for at of the pleura carcinoma and one rat with a squamous groups tion to animals which OW om, he . ' ' y + : . . ; f 5 4 | j i i 4 RIPECTS OF INILALATION OF ASBESTOS IN BATS . TABLE IX Sites of Tumours Other than Lang aati SitefTumman type Ashestas Ashestas Deated A Ehtetgeus Malignant Control Bengar Madiggnant Digestion organs and peritonema peritonema 4 3 pm Bono and skun I 3 3 1 ( Rd Breast 100 " Ovary 3 " ny 1 ~. Other Other female t fj genitosuunnan^ genitosuunnan^ tagitis > * " Male genitorumiary genitorumiary genitorumiary Organis { 8 { 1 Louaera } 173 i 31 Thymetna Thymetna j 0 1 LymphomuuLymphomuu leukatenant * Others Others .3 ad ! a ~ Tby rond mediastinumA Sup arde raced auftemil 4 salivary glassl tlacoel oe + weayer Weth catel wih ods ah wll ugest con Gary Gary ~ and sas, 11 weyed, Aignant Chom epnpecbbys 1 of the lung had a mesothelioma tunica vaginalis Mesotheliomata of the vagin alis were seen in this and one other rat there is no evidence to suggest an associattion with exposure to asbestos Also in some cases there were secondaries for example a synovioma had spread to the hung In all such cases tumours have heen classified by the primary site and in the few cases of multiple malignant tumours there was no difficulty in recognizing the distinct types iz one was not a secondary of the other DISCUSSION Our finding that the asbestosis produced by exposure progressed after cessa- tim of exposure is a agreement with human experience but contrasts with the early inhalation experiments reported by Vorwald Durkan and Pratt 1951 which progression did not occur Wagner 1963 reported more asbestosis with amosite than with chrysotile m gunea rats and monkeys but our xperiments show that of the DICC -tandard reference samples amosite is the Past fibrogenic in rats Gross et al 1967 found lung cancers ne of 72 rats which survived 16 months exposure to chrysotile dust at a mean concentration of 86 mg for 30 hours week They considered that contamirun the asbestos by trace metals from the worn hammer of the mill used to produce the respirable fibre could have been a factor in the causation of the tumous However our results now show that there is no need to invoke such a hypothesis to explain the high rate of lung tumours The amount of chrysotile retained in The lungs did not show any clear increase with than dose in rats exposed for longer 3 mouths In two carber expezi ments Wagner and Skidmore 1965 Morris et al 1967 a higher airborne dust concentration was used to give a cumulative lose m 6 weeks similar to that given in the present experiment experiment over 3 months The weight of asbestos found in the lungs of rats exposed to amphibole was 3 times greater than in those exposed to chrysotile In the present experiments the ratio was 6 to t after 3 months but increased with continuing exposure as the weight of amphibole in the lungs continued to increase did not but the onount of chrysotile The previous experiments had shown that the rate of clmination of dust from the lungs was much greater for chrysotile than for the amphiboles The present results may be explained on this basis the weight of chrysotile having reached equilibrium level ie the rate of elimination equalling the rate of retention There are a number of features of the results presented above which we found wajchwajchwajch wajch tebe Madini alte MadiniMadin MadinMadini Madini Madini 268 C. WAGNER G. BERRY W. SKIDMORE SKIDMORE AND V. TIMBRELL surprising First an Experiment 1 mesotheliomuta occurred with the two one of sites other than the lung is equivocal Although an association with gastro- day exposure compared with only on with the 3 months exposure which had a dosage Norr than 30 Limes greater the incidence of mesothelionuda wars intestinal tumours has been found ept- demologically it is not yet regarded as plearly established Nelikoff Hammond and Churg 1972 Newhouse 1974 and proportional proportional the inoculation dose as is mebrated for experiments Wagner et of luster lung cancer magnitude risk Our than the excess experiments pro- al 1973 then the probability of such an extreme result occuring by chance would be about in 1000 vile no support la such an assoemtim The experimental work of Graham and Grahani 1967 suggested that intra- me Secondly there was no evidence of peritoneal injection of tremolite asbestos either less carcinogeneity or less ashestosis could produce ovarian tumours but the in the groups exposed to chrysotile than follow of women asbestos workers those exposed to the amphiboles even reported by Newhouse at 1972 pro- though the amounts of dust in the lungs dured no definite conclusions on this Were so different In particndar the question because of the rarity of the UIC Canadian chrysotile produced as tumour Our experiments do give some mesotheliomata as the UICU croci- support an association between asbestom many dolite The 2 UICC samples of chrysotile exposure and ovarian tumours as well produced 12 of the 14 tumours with as tumours of the male genito autastases However much less dust system Mthough neither was significant was retained in the lungs of rats exposed this could be because of the relatively to chrysotile than amphiboles Fig 19 small size of the control group To Moreover after intrapleural inoculation overcome this we have included the the risk of a mesothelioma occurring control rats from some of our ollur with U'100 crocidolite is 3 times the risk experiments thus mereasing the noti with chrysotile Wagner al 1973 treated group to 403 nits of the sam - Therefore allowing for the greater reten- strain This larger group contained 2 of crociolite after inhalation we malignant tumours of the ovary and ^ tion the risk with aroa- tumours of the urinary tract in might have expected dobte to have been of the order of 20 males In over 700 rats exposed to ashestos there were 30 ovarian ovarian tumours times that of chrysotile Two of the mesotheliomata occurred 7 of which were malignant and 11 within 400 days of the start of exposure timioues of the urmary tract in This may be compared with our injection males Hence based on the larger set controls the association between ashes experiments in which only 20 out of 803 of and ovarian tumours is occurred within 400 days Wagner and tos exposure Berry 1969 Wagner et al 1973 Also weak and significant whereas there is the earliest mesothelioma occurred after no support for association with tumour- 355 days and we observed only 3 within this period in our injection experiments The positive association between as- bestosis and lung tumours which we have established in the animals is in agreement with epidemiological findings e.g. Minister of Labour and National Service 1949 Knox et al 1968 Elmes and Simpson 1971 The failme to establish any association between asbestos exposure exposure and tunwurs of the male armary system The UCC chrysofile samples are her than the chrysofile which has been use an industry in the past However the is a trend for industry to use finer chay sotile Wright 1969 and so the expen mental results may be more relevant the current situation than to the past We are investigating the effects of mihal tion of chrysotile in more detail m as experiment involving ICC Caquadern chry Came win th plen resul Pesys Hus abs " the I Wy who pons 2t wouh to 1 Cann Aussi grater Walte furan, exper comp baw~ WGants " SHEN 46173 oy Vagina ! iNeed fest dat ties 41 tls tls Pateae escus, Equith 21 Gy A Moe, S MINISTEE 19100 P's fee^'y HMSO Movta. { Saluti ~eeae pclheryusrotaillely Wagner Canadian sample Thus investigation EFFECTS EFFECTS EFFECTS OF INHALATION INHALATION OF ASBESTOS IN RATS 209 chrysotile grade 7 sample from a GW mune and the 1967 Cumpurison of Dust Retepijon which superfine sample Speentie Patlungen Phu and Retepijon 21 proved the most The fadeddet Particles anal Standard Rats the materials which extremogenic of Davies Davies Befund Vapours 11 Ed CX CX we moculated intra Pergamon NEWHOUSE M. P. 205 al 1973 The experiments we in the hilfects stos of Asbestos 1954 Camera Lining Workers Pestik Pestik Tudustry Au Biolagrat results report live given which in seyrtal REP J. Asbestos Jann 23 Orbster Boggov sker 07. Hilson V 1972 Tundalf Tundalf respond respond to those respects for Wagne8 FYRC Serenituie Serenituie Serenituie Publicatiotis and found in 18 Thus - fl In mand Publicatiotis Publicatiotis No. this experimental experimental method method is XwHotst , 1. Brady as valid tool for the established established Terok M 2 G. WALSER 1972 A Study ( & the of of Female Ashratus Ashratus Workers of the Mortality biological effects of asbestos 29 131 Workers ButBut But Mril SERGE d Hontex^ E. ( 1972 Careining niity of UMR Anosite We Anche cuth HHh 25 1st Ashustus Ashustus are grateful to all our SHABAD M & Prity colleagues who over a number Lesions De Kat L. 1970 Marpatologiral Marpatologiral daily ponsible for the of years years attention attention atention attention were res - necessary Hudeorarbon Hudeorarbon Respantory ' Fangs Turucial in Polyes Polyes elic In Morphology Morphology of Esperonautal Esperonautal Esperonautal necessary attention in Respantory any May Ses, Pro necessary carrying out the experiments We Hung 13:16 13:16 May 1970 134 ' Cont Gatho- would also like to acknowledge acknowledge : Harin Jr and Netteshemy W. Deatherage to Dr Stewart our thanks Us Energy Deatherage Ji Us Atomic Harold acknowledge Stewart of the National National National Settes No. Crammission Sympost 1927 1927 Cancer Institute who advised STANTON M. 1974 1974 lu Tumours us on the Laboratory ob Pathology of in classification of tumours We are also Rut Laboratory Part eeeersits 2 Vol Vol 1 Tunus id the grateful to our former No. EARC Lyons Sextilie PublicationS Walter colleague who had classified the Dr. A Cancer International Ageney for Research Lui preparatio lung tianours Thankura V. Hersos C. Winspeak occurring in some of our earlier UNCC Standard Reference Winspeak , 1965 earlier 3 experiments which 111 mentioned for Int 1 Cunter 406 Reference Samples ut Ashestos comparison Tramurit V Hunt & W A SKIMORT W 1968 A Sumple Sumple Dispenser Dispenser for Clauds Generating Dust from Standard Rderin^'e Rderin^'e Asbestos Jun Rderin^'e Samples of oe REFERENCES REFERENCES Pratareid occap Hgg 11 273 TOMES TOMES P. it Pratareid V SKIDMORE J. W Herr A 11 eams. Workers in BeSltfausptson MJ ( 1971 fisulation W^ NERJ. Nor 1970 Exposure Exposure Chambers mnd Med 28 226 3. Mortality 1940 66 Bir Inhalation Refermer Experiments Experiments with Standard . a GRAHAM ) & Guinay R. me 1967 Ocarian Career national Samples of Vition Aganst Ashestras of the Cancer UTC Luter Scrovol and Asbestos Blour Res H15 H15 VORWALD 213 Scrovol T iss Dr Trevnar Trevnar i VORWALD A J. DuPLAN oe Kaschak M BABY M A.P. Tua KER R. R. 1931 Experimental DuPLAN T \ & Pryer C utal Ashestosis The 1967 Esperan Esperan Development Development of bung Studines Studines ol V I Archs ont Hgg 3. MACNIR Astarsprests 41200 121 Fon Bxpetituental Aluvantule Aluvantule Aluvantule Rats with Asbestos Pulmonary Pulmonary Deposit ol Ammads " 21163 Ashustosis Ashustosis in Bxpetituental Bxpetituental Bxpetituental 15.343 15.343 Dust luchs CHEE 11th 11th Br agred MEGE 20 Expostire ksox F Holmes S. Dott R. ibu D Wysur d C. 1965 The Sequelae of Ashistos Ashistos Dust Jun F Expostire tu tel Sep 1168 Mortality from Lang Lang Cancer and Other WAXER J. 1972 The Significanet 132 G91 Cruises Auning Workers an Asbestos m Pissipe In Recent Results of Asbestos Pucture B Fund Med 25 293 Textile Vol 39. Cunca Problems in Other ac the Research Research SCHENER & LASKIN Ipsthebal Tumors and Tipi4a9t70 Pulmonary Cancer and Lymphomes Lymphomes and Talmas New Epolemioloqa Epolemioloqa KI E Grandmaz~-n Verlag the Kat In Morphology Pridiferation 37 York Springer Verlag 1 qurtory Morphology of Carcinogenesis Carcinogenesis Proc Proc Papo Papo oncob^> Con Gatim WAGNER WAGNER J. & Benin 1969 Mesoth hans 13 16 May 1974 B Xetteshicum " Kats Folloy ing Inoculation Inoculation honks G Hunna Jr and W Xetteshicum Xetteshicum Hi Cumer 23 367 with Asbestos US Atomic Energy Deatherage di Warner ( Sapa No 21 Energy Commission Symposti^ IL 1 203 , Badoy G. Mesotheliomans & Tamree V 1973 me Rats Rats Podboxong Inoculation with MINISTER O Annad AND NATIONAL SERVICE Whestos and Other Materials fge F 4949 Annad Annad Report Report of the 28.17 28.17 Capter Tuspretta Lectories for the Year 1947 Chef fud of Wasser J. NIGHTMOR HMSO 7621 London Dust Depositions J. W. 19651965 Asbestos Meis C RODERPS RODERPS WH SHYERION NY Depositions and Retention in Rads beat os, 132.77 doen SKILLMORE ! W. WasxUK SHYERION bog RE WPHOPE W Asbestos A Crag, 1969 Im Ber Asbestos and Health Health m Pespr D 100 167