Document 5b6q9bEmeKrGK3G6a6ZJ74LB8

".......... -- . M_ _OV -?C -14f Pneumoconiosis. Proceedings of the International Conference, id Johannesburg. 1969. MESOTHELIOMAS IN RATS FOLLOWING THE INTRA-PLEURAL INOCULATION OF ASBESTOS J. C. Wagner, G. Berry <m<; V. Timbreli. Pneumoconiosis Reset, ch Unit of the Medial Research Council: Penerth, Glcmor^r. Epidemiological studies have suggested that mesotiiciiomas of the pleura and peritoneum are teLiui to exposure to asbestos dust. Pleural mesotheliomas may be produced in rats following intra-pleural inoculation of asbestos dust and experiments in animals may help in the determination of the factors iuh'.'-'ocmg the occurrence of such tumours. In tl.L paper the results of three experiments in which asbestos was inoculated intraplcuraliy into rats will be given. Experiments ExraiU.i.ENT A: Comparison of Tyres on Asdestos Tilt treatments were amosite, ebr senile, crocide'.ite, oil-extracted crocibolitc and a control. There were 76 4.A.F. rats per treatment and the experi ment was duplicated with a similar number of Standard rats. The dost w. s 20 mg. of dust per rat and the experiment was st.-.ted at the end of 1062. Experiment B: Varying Dose Experiment This experiment involved only ohryori!e and crociilohic each it 5 doses, 0.5. 1, 2, a and S my. per i it with 12 rats per dose per dust. Injection was curing .March 1965. Experiment C: Comparison of Canadian Chrysoiu.es Samples of chrysotile from seven Canadian mines were used, the same chrysotile as i.n experiments A and 11 and a saline control. The co'c '.vas 20 mg. per rat. and there v.-cr; to rats per Canadian chrysolile, a: for the original sample of chruoule and 4S controls. Imection was during December 1966. All the experiments were survival experiments, i. e. the animals were only kill. . i: they aptci to be distressed. Experiments A and li are ccmoietc but an - o: the ran in Experiment C ware ii. 'l living at tile cad of January ic.-io so tlx -.t the rruits presented of this experiment arc not Nn.il. A necropsy examination was carried cut on each anim.'!. O: the ran .iioaulateJ with .'.Actios an appreciable proportion .vve'.nped mesotheliomas in all tile experiments put v. a have r.ca ..r fv.and 'his type of tumour wicn the control treatrtv.mi. Thus a ;.-.esotht!icr.ia can re said to be a direct result of iiioculaiic.a isith asbestos. Method of Analysis The treatment cur.vpjrhor.s to fc: rtwcti ?.:c bated on the incidence of mcsothciio.iU s. Tit: proportion of animals develop,:'.': ;uc;.n- theiiomas m?.y be used for comparisun v.-'ti-in an experiment but it has to be rcait'.d that this proportion is the result of tv/c- lectors, the first being the r.aiur?: mortality experienced by a group of animals v.iwtl.cr iiijeclcd with asbestos cr not, and the second the increased mortality due to the visit of developing a mcsoinalierr.:.. I: is tin.- latter component we are interested in and \vc would like to separate ir from '.ho iou;::r. With large groups of sricnais sued as experiment A this can r.c achict;-d h; life table methods and the rtsJ: of the ilcaii- tions depicted by a survival curry tliov'.tj the proportion 0? animals alive a: a n :i when the Rnimn's ".cry r::l; of c. -\'ph:;; a mesothelioma but the risk of m,uv.i do ,:n has been eliminated. If we hr.cw .i.e mathematical form of tins curve v. : estimate its parameter: and elteotiv.'.-. v; ;a a smoother version of the survival xu Til, would he partiruieriv useful tor vw: B and C where srnrli croups of j-.-'; v.rc used and it would be expected iLn: latcd survival curves would shew irregi.'arities. A 'node', relating rite induction r 'te tumour; with time was rivci by ; (1953) and developed bv A-mh > (1954; end Pike Tc'6: and In; l:.-. . both for analysing cpidv.miulccic i . animal experiments. It; ott: situ. :i. .. : : the age-.-pt-ific death rate v! r.r . ' with a mviotheiioma a: titr.e 1 . - tion the model is m " ck(t -- w)"'1 for 1 4 v where c, k and to are cot: ' theoretical approaches lend "j : it may oc consider? d as the ex11 o.ive val.ic clis.riu>>.to.i > ir 216 OHER 0004364 4 Mesotheliomas in Rats *17 number of cells are considered at risk of malignancy and a cancer cell is formed when the first such cell succumbs. Secondly the model would hold if a cancer cell was the end result of h successive cellular changes. With both approaches the constant c is related 10 the dose and vi is the induction or latent period. In our analysis we will also describe the age-specific natural death rate as cxp(a + bt) which gives a satisfactory fit to our control groups and has been used in epidemiological studies. Results Experiment A. The percentages of rats developing mesotheliomas an* given in Table 1. We note that the SPF and Standard rats crocidolite. For each dust the pattern was similar, consisting of an initial period during which nc mesotheliomas were found foliowed by a rapid onset of cases. However, the length of the initial period, i.e. the induction period, was dependent on the dust. For Standard rats similar results were obtained but the dific-ance between chrysotile and crocidolite wa:. smaller. The model given earlier has been fitted to this experiment and generally gave good fits. The constant fc had values near to 3 and may be taken as 3 without any loss of precision (this value has been assumed to hold for experiments B and C also). As an Table i: Percentage of Rats Developisto Meso thelioma--Experiment A SPF Amosite.............................. ChrysotiL........................ . 64 Crocidolite........................ Oil-extracted Crocidolite . 59 Standard 31 69 68 64 gave similar results, that amosite gave fewest mesotheliomas and that the oil-extracted crocidolite gave similar values to the natural crocidolite. Cal rule'ion of the survival curve eliminating natural death, i.e. considering only Fig. 2. Expcri'-tnt A: Comparison of observed and expected distribution of deaths far croct- dolite m Standard rats. The expected deaths are calculated from the model ziv ri ui the text with 2 --10-44, b " o 005$6, c = I-J4 N IO-`,k = 3, w -- 332. Fig. 1. Experiment A: Survival of S.P.r. rats with mesotheliomas after dim mating crlec. of mortality due to other causes. mesotheliomas as causing death, gives Fig. 1 for SPF rats. For convenience of presentation the oil-extracted crocidolite has been excluded since its results were so similar to the natural illustration of the fit of the model, Fig. 2 shows the cumulative number of deaths with and without mesotheliomas for c'.e.c:\oii:e in Standard rats; there is close ferment between the observed and expected d,:'ributions. Experiment B. Averaged over doses, 36'' and 19% of rats developed mesotheliomas for chrysotile and crocidolite respectively. For each dust the constant w was assumed independent of dose. This is required on theoretical grounds but with such a smell experiment the data cannot be used to cheek this assumption very rigorously. There is a relationship between the estimate of ike i.onsta-.n c aid dose. With only 12 rats per d-se the scatter is l*.6e but it has been shewn that the relationship may be taken as li.-.mr, i.e. at any age the risk of developing 3 mesothelioma is proportions' to amour.: of dus: iniecred. The induction period was t:e days longer for crocidoiitc than chry-otiie. HER 0004365 2lS J. C. Vagner, G. Berry and V. Timbrclt Fig. 3 shows the observed and expected numbers of mesotheliomas. If we extrapolate the results of this experiment to a dose of 20 mg. we find that for both dusts this would lead to a higher rate of mesotheliomas than actually occurred in experiment A, the difference being significant for chrysotilc but not for crocidolite. of c but an average cobalt content, docs net fit into the same trend as the other mines. In fact the correlation is significant only if mine C is excluded. The correlation between e and chromium docs not suffer from this dis advantage since mine C has a low chromium content. The relationship between c and Table 2: Values of c and Brucite Content Experiment C Mint B D F E A H C e x /0* 9-1 8-7 8-5 69 54 4-3 2*0 Brucite Content 20% Trace Trace Trace to 5% Trace to 5% Trace Absent Fig. 3. Experiment B: Observed and expected numbers of mesotheliomas (out of 12 rats) on the basis that the risk of developing a mesothelioma at any age is proportional to amount injected. Experiment C. At this stage 69% of the rats injected with the original chrysotile have developed mesotheliomas and between 19 and 56% for the 7 samples from different Canadian mills. Again to was assumed constant for the different forms of chrysotile and in fact the best value was almost identical with that of experiment B. Comparing the treatments in terms of the parameter c, this pirameter was significantly large: for the original chrysotile than the Canadian samples and the value fitted in with what would be extrapolated from experiment B. In Table 2 arc shown the values of c, and the brucite, cobalt and chromium contents of the dusts. There is correlation between cobalt and c, but mine C, which has the lowest value chromium is shown in Table 3. The correla tion is high (0.91) and significanr (p <0.01). Nickel, scandium and iron have also Ven looked at. Nickel gives a similar patt xr. to cobait except that mine C is even more diver gent from the relationship shown by the ether 6 mines. The relationships of c with scandium and iron showed nothing of interest. Mine C is in western Canada, the other 6 in a relatively small area of eastern Canada. Table 3: Intp-a-Pleural Inoculation Or Canadian Chrysotu.es Values of e (Carcinogenicit) and Chemical Properties Brucite Alin* * x 10' Content Original Sample B D F E 17-7 9-r 8-7 8-5 69 A 5-4 H 43 C 2*0 -- 20% Trace Trace Trace to 5% Trace to 5% Trace Absent Cobalt {ppm) __ ZIO 78 78 57 3 43 60 Chromium (ppm) 7S0 930 730 440 520 480 120 Discussion All these experiments were started before the U.I.C.C. Reference Samples became available but the seven Canadian dusts used in experiment C came from 7 of the 8 mines from which material was supplied to form the Canadian chrysotile sample. Also, the sample of chrysotile used in all three experiments was a super-fine grade from cue 0? these HEB 0004366 Mesotheliomas in Rats *19 mines ()>). The difference observed between the original chrysotilc and the sample from mine D in experiment C is therefore of intercsr s>.nd couid be due to different milling procetiurcs or since the samples were obtained several years apart due to coming from a different part of the mine. It is also of interest to note that the mesothelioma rate due to the original chrysolite in experiment B and C is greater than in experiment A. This could be due to a change in the susceptibility of the rats or a change in the dust during storage. An injection experiment was started in 1967 using the U.I.C.C. Reference Sampics, repealing the separate Canadian samples and including a pure brecite treatment, this last treatm ait being of interest in view of the results of experiment C. The procedure of inrra-pleural inoculation is unrealistic when compared with human experience and results obtained from inhalation experiments would be more infor mative. Chrvsotiie has been shown to be a biological! / active dust but epidemiological studies suggest that crocidolite is more dangerous. This may be because the spirallike shape of chrvsotiie fibres inhibits their inhalation. In 1967 rats were exposed in chambers to dust clouds of the reference samples and 3 months ago another inhalation experiment was started. Summary' Rats have been inoculated intrapleurally with sampics of asbestos. With all types of asbestos an appreciable proportion of animals developed mesotheliomas. There was no difference in effect between the natural and oil-extracted forms of crocidolite. Amosita produced fewer mesotheliomas than cid chrysotilc and crocidolite, this being a result of a longer induction period. When different doses were applied the risk of developing a mesothelioma at a given age could be taken as proportional to the dose. Samples of chrvsotiie from seven different Canadian mines all produced mesotheliomas. YTe are ;ratrful for the heip we have received from our colleagues, Mr. O. Is. Munday, Mr. W. H. Roberts and Mr. J. U7. Skidmore. The trace clement results were determined by Mr. A. Morgan and his co-workers. References Armitase, P. and Doll, R. (1954): Brio J. Cancer, S. t. Aordlir.s, C O. (t953): Brit. J. Cancer, 7, C. Pike, M. C (:95C): Biometrics, 22, tea. 0004367