Document dQXyxGvenovoBRmm04mmVajkR

I TfmrTrTs- MOV ~~F In: Pneumoconiosis. Proceedings of the International Conference, Johannesburg. 1969. MESOTHELIOMAS IN RATS : FOLLOWING THE INTRA-PLEURAL INOCULATION OF ASBESTOS J. C. Wagner, G. Berry <i V. Timbsell Pneumoconiosis Resce.ch Unit of the Medical Research Council, Penarth, Glamorg-sn Epidemiological studies have suggested that mesosheitomas of the pleura and peritoneum are teh.wi to exposure to asbestos dust. Pleural mesotheliomas may be produced in rats following intra-pleural iuoailation of asbestos dust and experiments in animals may help in the determination of the factors ir.`t,.,i'ncing the occurrence of such tumours. In this paper the results of three experiments in v/hich asbestos was inoculated intra pleural;}' into rats will be given. Experiments ExraniAtasT A: Comparison of Types of Asbestos Tilt treatments were nmosite, cbrysotile, crocitl-jHte, oil-exiracred crocidolite and a .uint control. There were p6 il.P.F. rats per treatment and the experi ment \ "as duplicated with a similar number of Standard rats. The dose w. a 20 mg. of dust per rat and die experiment was started at the end of 1062. Experiment B: Varying Dose Experiment This experiment involved only chrysotile and crocidolite each at y doses, o.j. 1, 2, 4 and S rnsr. per it1 with 12 rats per dose per dust. Injection was curing .March 1965. Experiment C: Comparison of Canadian Chrysotiees Samples of chrysotile from seven Canadian mines were used, the same chrysotile as in experiments A and B and a salir.c control. The cose was 20 mg. per ret, and there were :6 rats per Canadian chrysotile, 5: for the original sample of chrytotde and 4S controls. Injection was during December 1966. . All the experiments werr survival experiments, i.c. the animals were only kill.:--. if they api-v.-.red to be distressed. Experiments A and 11 are ccmaicte.bat 20^ o; t!ie rats in Experiment C were &i.'l living a: the end of January texa so that the re-uhs presented of this experiment arc not final. A necropsy examination was carried one on each animal. O: the rats inoeuiateu with Asbestos an appreciable proportion developed mesotheliomas m all tile experiments put v.e have never found `his type of tumour with the control treatment:. Thus a tr.esotheiicnia can be said to be a direct result of inocutatic.i with asbestos. Method of Analysis The treatment comparisons to bs given arc based on the incidence of mesotheliomas. The proportion of animals developing :ue-;otheiiomas may be used for comparison within an experiment but it has to be realised that this proportion is the result of two factors, the first being the natural mortality experienced by a group of animals whether injected with asbestos or r.ot, and the second the increased mortality due to. the risk of developing a mesothelioma. It is the latter component we are interested in and we would like to separate it fretrt the former. With large groups of animals such rs in experiment A this can be achieved by life table methods and the result of the calcula tions depicted by a survival curve shr.vv.g the proportion of animals alive at a f.ivvn time when the ani:ria;$ were rick of clevc'-.igmg a mesothelioma but the risk of natural uc tth has been eliminated. If wc knew ii.e mathematical form of this curve vr.j veil estimate its parameters and dtectivvly a;:o a smoother version of the survival av.v. This would he particularly useful for cxpcr-Vvuts B and C where small groups of j.ru!; vrerc used and it would be expected lint :!: c.v:ulatcd survival curves would show is.-:.-: irregii'atities. A model relating the inductlct! r>:e tumour; with time was given by :.Tr; (1953) and developed by A-mks.y r'; I(1954) and Pike 'ic56! 'and In; hrc-i . .:! both for analysing cpidcmiolcgtc.d c . -:d animal experiments. In our situ.:;:.-::. the age-specific death rate of an:'-:: ' -V * with a mesothelioma-a: time 1 J'.r tion the model is m *= ck(t--w)--* for t > vr where c, k and v are cc.r.s: r . theoretical approaches lead tti :l:-t : it mav be considered as the -h.-.l extreme value distribution r.rM._ > r 216 ASARCO ELP 0003553 Mesotheliomas in Rats 217 number of cells arc considered at risk of malignancy ar.d a cancer eel! is formed when the first such cell succumbs. Secondly the model would hold if a cancer cell was the end result of k successive cellular changes. With both approaches the constant c is related to the dose and w is the induction or latent period. In our analysis we will also describe the age-spceific natural death rate as cxp( + be) 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 are given in Table 1. We note that the SPF and Standard rats crocidolitc. For each dust the pattern was similar, consisting of an initial period during which no mesotheliomas were found followed by a rapid onset of eases. However, the length of the initial period, i.e. the induction period, was dependent on the , dust. For Standard rats similar results were obtained buc' the diuc-cnce between chrysotile and crocidolite was smaller. The model given earlier has been fitted to this experiment and generally gave good fits. The constant k had values near to 3 and may be talien 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 Developing Meso thelioma--Expcjumest A SPF Amosite.............................. CKrysocil- ..... 64 Crocidolitc . . . . . Oil-extracted Crocidolite . 59 Standard 31 69 6S 64 gave similar results, that amosire gave fewest mesotheliomas and that the oil-extracted crocidolite gave similar values to the natural crocidolitc. Calculation of the survival curve eliminating natural death, i.e. considering only Fig. a. Experiment A: Comparison ofobserved and expected distribution of deaths for croridoliic in Standard rats. The expected deaths are calculated from the model giv- n in the tt-:;: with 2 = --10-44, b *= o-oo;S6, c = 1-54 x I0"`, fc 3, w = 332. Fig. t. Experiment A: Survival of S.P.F. rats with mesotheliomas after eliminating crTec. of mortality due to other causes. mesotheliomas as causing death, gives Fig. 1 for SPF rats. For convenience of presentation the oil-extracted crocidolitc lus 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 ciocMolite in Standard rats; there is close agreement between the observed and expected attribu tions. Experiment B. Averaged over doses, 36 and 19% of rats developed mesotheliomas for chrysotile and crocidolite resnccrively. For each dust the constant w was assumed independent of dose. This is requited on theoretical grounds but with such a small experiment the data cannot be used to check this assumptior very rigorously. There is a relationship between the estimate cf the i.onstant c a/tri dose. With only 12 rats per dose the scatter is l*.5e but it has been shown that the relationship may be taken as lintar, i.e. at any age the risk of developing a mesothelioma is proportions' to amount cf dust injected. The induction period was :po days longer for crocidolitc than chrysotile. ASARCO ELP 0003554 2x8 J. C. VCagner, 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 chrysotile but not for crocidolitc. of e but an average cobalt content, docs not fit into the same trend as the other mines. In fact the correlation is significant only if mine C is excluded. The correlation between c 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 e and Brucite Content Experiment C Mine B D F E A H C cx/O* 9-1 8-7 8-s 6-9 5-4 4*3 2*0 Brucite Content 20% Trace Trace Trace to s% Trace to s% Trace Absent chromium is shown in Table 3. The correla tion is high (0.91) and significant" (p <0.01). Nickel, scandium and iron have also been looked at. Nickel gives a similar pattern to cobait except that mine C is even more diver gent from the relationship shown by the other 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 reladvcly small area of eastern Canada. Table 3: Intra-Pleural Inoculation of Canadian Chrvsotiles Values of e (Carcinooenicity) and Chemical Properties Fig. 3. Experiment B: Observed and expected numbers of mesotheliomas (out of ti 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 vs 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 e, this parameter was significantly larger 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 ar.d chromium contents of the dusts. There is correlation between cobalt and c, but mine C, which has the lowest value Brucite Aline ex lo' Content Original Sample B D F E 17-7 9*1 8-7 8*5 6-9 A 5-4 . H 4`3 C 2*0 _ 20% Trace Trace Trace to 5% Trace w s% Trace Absent Cobalt (ppm) no 78 78 57 3 43 60 Chromium (ppm) - 7S0 930 730 440 520 480 120 . Discussion AH 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 one of these ASARCO ELP 0003555 Mesotheliomas in Rats 219 nu'ncs (1>). The difference observed between the original chrysotilc and the sample from mine D in experiment C is therefore of interest and couid be due to different milling procedures or since the samples were obcained several years apart due to coming from a different part of the mine. It is also of interest to note that the mesothelioma race due co 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, repeating the separate Canadian samples and including a pure brucite treatment, this last treatm att being of interest in view of the results of experiment C. The procedure of intra-plcural inoculation is unrealistic when compared with human experience and results* obtained from inhalation experiments would be more infor mative. Chrvsoti'.e has been shown to be a biologicall/ active dust but epidemiological studies suggest that crocidoiitc is more dangerous. This may be because the spiraiIike shape of chry:o;iie fibres inhibits their inhalation. In 1967 rats were exposed in chambers to dust clouds of the reference samples and 3 months ago another inhalation experimant was started. Summary Rats have been inoculated intrapieurally 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 crocidoiitc. Amosi:: produced fewer mesotheiiomas than did chrysotilc and croddoiite, this being a result of a longer induction period. When different doses were applied the risk of developing a mesotheiioma ac a given age could be taken as proportional to the dose. Sampics of chrysotilc from seven different Canadian mines all produced- mesotheliomas. Wc are grateful for the help we have received from our colleagues, Mr. 0. E. Midday, .Vtr. V7. H. Roberts and .Mr. J. W. Skidmore. The trace clement results were determined by Mr. A. Morgan and hts co-workers. Referexces Armitage, P. and Doll, R. (roj,f): 3de J. Cancer, 3. t. Kordling, C. O. (1953): Brit. J- Cancer, 7, Ci. Pike, M. C (rs36): Biometrics, 22, 14:. ASARCO ELP 0003556