Document KRR0X76z4jp3kYRr27gXq74Qw

onno PLAINTIFF'S EXHIBIT MOV ~OP v/ In: Pneumoconiosis. Proceedings of the International Conference, Johannesburg. 1969. MESOTHELIOMAS IN RATS FOLLOWING THE INTRA-PLEURAL INOCULATION OF ASBESTOS I J. C. Wagner, G. Berry itnd V. Timbrell Pneumoconiosis Rrseo.cli Unit oj the Medical Research Council. Penarth, Glamorgan Epidemiological studies have suggested that mesotheliomas of the pleura and peritoneum are rcluwi 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 ir.h"-'Cidng the occurrence of such tumours. In ti e. paper the results of three experiments in which asbestos was inoculated intrapleuraliy into rats will be given. Experiments Exi'EiMa.ent A: Cov.i'akison of Titus of Asbestos The treatments were amosiie, c'in soiile, croeido'.ite, oil-cxtrncieri crocldo'iic and a a.uir.e control. There were '6 G.1U''. rats per treatment end the experi ment \t.s duplicated with a similar number of Standaid rats. The dose w.s 20 me. of dust per nt and the experiment was started at the end of 1062. Experiment B: Varying Dosf. Experiment This cxperixenl invoiced only chry'orile and crocldahtc each it 5 doses, 0.5, 1, 2, 4 and S mi;, per . !! v. itli 12 rats per dose per dust. Injection seas f.'jri.oj; .March 1965. nxrr.RiMF.NT C: Comparison of Canadian Chrvsohles Samples of chrysotile from seven Canadian mines were used, the same chrysotile as in experiments A and It and a saline control. The ccr-e wax 20 mg. per rrt, end there v.-e:a :6 rnts per Cir.adie.ti Chrysotile, a: for the 0ri3in.1I sample of chrssottle and 4S controls. Injection was during December 1966. All the experiments were sttnival exjterirr.cr.ts, i.e. the ar.im.ds were only kill,:.-, if they appealed to be distressed, l:\perintvnts .A and B are ccmotetc but 20--. of the rats in Experiment C 'vaac Si.'Mivm^ at tire end of January ic.So s0 ils-.r ike rcu'ls presented of this experiment arc not final. A necropsy examination was carried cut on each anmyd. Of the rats inoavhted with Asbestos cn appreciable proportion developed mesotheliomas in all the experiments out v.e h-.ve nc-.er found 'bis type of tumour with the control treatments. Thus a mesothelioma cm be said to be a direct result of inoculation with asbestos. Method of Analysis The treatment comparisons to ba given arc bared on the incidence of mesothe'tioi ut.x. Tire proportion of animals developing turto- thciioirr.is may be used for comparison vkhin an experiment but it has to be rcaiiced that this proportion is the result of two factors, the first being the natural mentality experienced by a group of animal 5 vtrtkcr mjectcd with asbestos cr not, and the serene! the increased mortality due to the risk of developing a mesothelioma. It is 1:..- latter component we are interested in and we would like to scpaiete it from the for/tier. With large groups of animals such rs <n experiment A this can he achie'.ed b- 1:1*: table methods and the result of the wlet.'A- tion.s depicted bv a survival cur-w sk'/.vVj the. proportion of animals alive a: a r.lven time wheii the aniin.rs were '-t ri:l; cf chn :g a mesothelioma but the risk of nauv.l tie an has b;:n eliminated. I: we knee .i.e mathematical form of this curve v.e i estimate its parameter; and eltectivc'v p \ J ::e a smoother version of the survival :,!= .v. '.I ':1s would be particularly u-ofni tor ottpe. v.'ttfr B and C where sinrli groups cf a.v.rt :l t v.re used and it would be expected that the c.-:-.u- latcd survival curves would show irregidarities. A >nodel relating tite induction ft: tumours with time was given by (1953) and developed by A'nv'tv..' d: , .! (1954) and dike 'icf.6j sr.d 3ns hern . both for analysing ep:Jemio!ccic.d ; . i animal experiments. In our situ. tie... it . . i: the age-speei'tc death rate of e.i'.i't'. ' with a mesc.theiioma a: time 1 J.r tion the model is m -- tk(t--for I > w where c, k and re are const .' . > theoretical approaches lead tu t i it may be considered as the '.it.-.i extreme value distribution eri-i . :. . . 216 f ASARCO ALV 0002155 Mesotheliomas in Rats JI7 number of cells are considered at risk of malignancy ar.d a cancer ceil 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 to the dose and to is the induction or latent period. In our analysis we will also describe the 2ge-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 am given in Table r. We note that the SPF and Standard rats crocidolitc. For each dust the pattern was similar, consisting of an initial period doting which no mesotheliomas wcie found followed by a rapid onset of eases. However, the length of the initial period, i.c. the induction period, was dependent on the dust. For Standard rats similar results were obtained but the diile-ence between chrysotile and crocidolitc was sma:ler. 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 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: Pfuctn-tage of Rats Developing Meso thelioma--Experiment a SPF Amosite............................................ 40 Chrysouk...................................... 64 Crocidolitc...................................... 39 Oil-extracted Crecidolite . . 59 Standard 31 69 6S 64 gave similar results, that amosite 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.c. considering only Fig. 2. Experiment A: Comparison of observed and expected distribution of deaths for crori- dolite in Standard rats. The expected deaths are calculated from the model ttiv- n m the tv::: with a = --10 44, b *= 0 003S6, c = 1-54 x = 3, w = 332. Fig. 1. Experiment A: Survival of S.P.E. rats with mesotheliomas after eliminating ctl'ec. of mortality due to other causes. mesotheliomas as causing death, gives Fig. 1 for SPF rats. For convenience of presentation the oil-extracted crocidolitc has been excluded since its results were so simitar to the natural illustration of the fit of the model, Fig. ?. shows the cumulative number of deaths with and without mesotheliomas for ctr-o'.oiite in Standard rats; there is close uprermont between the observed and expected di.-'ribu- tions. Experiment Id. Averaged over doses, 36';' and 19% of rats developed mesotheliomas for chrysotile and crocidolitc respectively. For each dust the constant tv was assumed independent of dose. This is required on theoretical grounds but with such a smell experiment the dat2 cannot be used to cheek this assumption very rigorously. There is a relationship between the estimate cf the constant c and dose. With only 12 tats per dcse the scatter is l*.6e but it has been shown that the relationship may be taken as linrar, i.e. at any age the risk of developing a mesothelioma is preportiona' to amount of dust injected. The induction period was tea days longer for crocidolitc than chrysolite. I ASARCO ALV 0002156 218 J. C. VPagner, G. Berry and V. Tiinbrcll Fig. 3 shows the observed and expected numbers of mesotheliomas. If we extrapolate the results ol 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 crocidolite. of c 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: Valves of c and Ep.ucite Content Experiment C Mine B D F E A H C ex IP 91 8-7 8-5 6-9 5'4 43 2*0 Brucitc Content 20% Trace Trace Trace to 5% Trace to 3% 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 patt :rr, 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 relatively small area of eastern Canada. Table 3: Intra-Pleural Inoculation or Canadian ClIHVSOTILES Values of c (Carcinogenicity) and Chf.mical Properties 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 ptopottional 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 k 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 prrametcr 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 brucitc, cobalt and chromium contents of the dusts. There is correlation between cobalt and c, but mine C, which has the lowest value Brucitt Mine e x ro' Content Original Sample B D F E '7-7 9-1 8-7 8*5 69 A 54 H 43 C 2*0 _ 20% Trace Trace Trace to 5% Trace to 5% Trace Absent {ppm) _ no 78 78 57 63 43 60 Chrcnriw.i (ppm) _ 7 So 930 730 44 520 480 i?.o 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 ail three experiment; was a super-fine grade from one of these I ASARCO ALV 0002157 Mcsatheiiotv.as it*. Rats 219 mines ()>). The difference observed between the original ebrysotile and the sample fro;n mine D in experiment C is therefore of interest* and could be due to different milling procedures or since the samples were obtained several years aparc 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 ebrysotile in experiment B and C is greater than in experiment A. This could be due to a change in the susceptibility of ibe rats or a change in the dust during storage. An injection experiment was started in 1967 using the U.I.C.C. Reference Samples, repeating the separate Canadian samples and including a pure brucitc treatment, this last treatm :nt 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. Chrvsotile has been shown to be a biologica.il/ active dust but epidemiological studies suggest that crocidoiite is more dangerous. This may be because the spirallike shape of ebrysotile fibres inhibits their inhalation. In 1967 tats 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 intrapleural!)* with samples 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-extractcd forms of crocidoiite. Amosite produced fewer mesotiieiiomas than did ebrysotile and crocidoiite, this being a result of a longer induction petiod. When different doses were applied the risk of developing a mesothelioma at u given age could be taken as proportional to the dose. Samples of chrvsotiic from seven different Canadian mines all produced mesotheliomas. \\"c are grateful for ike help we have received from our colleagues, Mr. D. I;. Mur,day, Mr. W. H. Roberts and .Mr. J. W. Skidmore. ihe trace demen; results were determined by Mr. A. Morgan and his co-workers. References Armitage, P. and Doll, R. (195.*,): Brin J. Career, S. I. Xordling, C. O. ft953): Brit. J. Oncer, 7, Ci. Pike, M. C. (igoi): Biometrics, 22, iga. I ASARCO ALV 0002158