Document e5yqM5m6Bx87zQyyGqjR54Brq

BIOLOGIC TESTS OFTREMOLITE IN HAMSTERS William E. Smith, Doras D. Hubert Health Research Institute, Farleigh Dickinson University, Madison, New Jersey 07940. Harold J. Sobei, Eugene Marquet Veterans Administration Hospital, East Orange, New Jersey 07019, and College of Medicine and Dentistry, Newark, New Jersey. Some years ago, we began to test specially prepared samples of minerals for carcinogenicity and fibrogenicity by intrapleural injection into hamsters. In these tests, we found that we could get tumors resembling mesotheliomas by injecting preparations of chrysotile asbestos fibers, provided that we used large enough doses, and provided that we used preparations with large numbers of fibers longer than 20 /im (Smith, 1974; Smith and Hubert, 1974). From experiments with glass fibers, we learned that not only the length, but also the diameter of fibers was important, tumors resulting in hamsters after the injection of fibers 0.75 /xm in diameter, but not with fibers 5 /xm in diameter (Smith and Hubert, 1974). Our long-fiber preparations of chrysotile induced extensive, fibrous pleural a d h e sio n s, and the o cca sio n al tu m o rs ca m e later; w h e re a s our short fib e r p re p a ra tions did not. A question then arose as to whether the tumors were a non-specific result of mesothelial cells becoming trapped in fibrous pleural adhesions where their oxygen supply could be impaired, and malignant change might occur, according to Warburg's theory of carcinogenesis (Warburg, 1956). TABLE 1. Tests of Samples of Tremolite by Intrapleural injection In Hamsters (Suspension of ail samples except 72N were autoclaved before injection.) Sample Number Tumors/survivors' Dose: 25 mg Tumors/survivors1 D ose: 10 m g 350 days 500 days 600 days 350 days 500 days 600 days 14 -- JV.A- 0/35 275 0/31 31 2/28 72 3/20 72N 4/22 0/27 0/15 4 /9 5/6 9/10 0/20 0 /3 6/5 5/1 11/2 0 /3 4 1/41 0/13 0/25 0 /1 4 1/19 1/6 0/19 0/61 1/11 3/2 6/9 `Numerator = cumulative number of hamsters with tumors related to treatment. Denominator = number s u rv iv o rs -2 additional animals survive :6 additional animals survive Reprinted from OUSTS and DISEASE CPuobplyisrihgehdt by PagtyboSloocxiePtyubfolieshOecrsc,u1p9a7ti9onal and Envhomnental Health PLAINTIFF'S E X H IB IT WCD-91 336 To explore that eslen e a n e c i o talc had occasionally been used m su g ry SMITH T AL. w b0 Ugbt a commercially - -- TM. W hittaker. Clark S Dame s. ,s d e s c rib e d by the (alc fro m N e w Y o rk S ta te . It b e ca m e o u r s a m p le ch em ica| characteristics have been previously de- contain 50'. Lm oiite. 35% talc. 10% antigonte and 5% chlorite. .. .; : : ;; Since then we have carried out intrapleural tests in hamsters with 3 different samles of tremolite specially prepared by various milling and separation tech niques. Biologic responses to these samples have differed, as shown in Table 1. The top line in Table 1 is data from our previously published tests of tremolitic Ole (FD-14). It was tested only at our highest dose level (25 mg), and, as shown, no tumors resulted. The other samples (275, 31 and 72) listed in Table 1 are the preparations made from tremolitic ores. Sample 275 was isolated from a sample of tremolite taken from a tremolitic talc ore body similar to those from which FD-14 was produced. Sample 31 was prepared from a sample of tremolite taken from a deposit of tremolitic talc in the western United States. Sample 72 was prepared from a s p e c im e n of asbestiform tremolite. As shown, no tumors related to treatment were found in animals injected with sample 275 at either the 25 mg or 10 mg dose level. A few animals treated with that sample are still living; however, from comparison with the other samples, it appears to be non-carcinogenic. (Animals surviving at time of presentation of this paper were subsequently necropsied. no tumors were found in them). In contrast, tumors related to treatment were found m some animals injected with samples 31 and 72 at the 25 mg dose level, and less often, at the 10 mg dose ; level. The first tumor was found 184 days after injection, and most of the tumors by a year or longer after starting the tests. To compare the carcinogenicity of these samples, one must therefore bear in mind the number of animals that survived long into the p e rio d of the e x p e rim e n ts . T he n u m b e r o f s u rv iv o rs at v a rio u s tim es, and the cumulative number of animals with tumors related to treatment are shown in Table 1. We conclude that sample 31 is less carcinogenic than sample 72. As shown, at the 10 mg dose, only a single tumor arose in response to sample 31, despite the relative number of animals that survived into late periods of the experiment. Pleural fibrosis was extensive in animals treated with sample 72, less so with 31, and very slight with 275 and FD-14. The fibrogenicity of these samples thus paralleled their carcinogen icity. _ For intrapleural injection, our routine procedure has been to suspend mineral samples in saline, and sterilize the suspensions by autoclaving, before injection. In the present work, we injected one group of hamsters with a suspension of sample 72, autoclaved in our usual manner, and we injected another group of hamsters with a suspension of sample 72 that had not been autoclaved. Table 1 shows that more tumors occurred in the group given material that had not been autoclaved, but this may not be significant because the number of survivors in these 2 groups are so different. When the samples are compared microscopically, morphologic differences can be seen. Figure 1 shows scanning electron micrographs of each sample at x 1250. Comparative measurements of fiber size distributions in those samples are not yet BIOLOGICI FIGURE 1. Sc left) and 72 (lc ", jwSsa 3 3 8 SMITH EXAL. available, but differences can be visualized from Figure 1, in which each micrograph bears a 5 nm scale. The sample that induced most tumors (Number 72) is seen to contain numer-: ous long, thin fibers with parallel sides. Average diameter of these fibers has been calculated as 0.4^m. By reference to the 5 tim scale on the micrograph, it can be seen that many of these fibers are longer than 20 rtm. The less carcinogenic sample (Number 31) also shows many long, thin parti cles. Average diameter is 0.5 Aim. Soma of these particles appear to have parallel sides, but others, although elongated, appear to be rather roughly shaped, resembl ing acicular fragments rather than crystalline fibers. Sample 275, which induced no tumors, shows the paucity of long, thin particles so evident in samples 31 and 72. The average diameter of particles in 275 is 0.4 Atm. In sample 31, as in 275, some of the elongated particles appear to be fibrous-shaped with parallel sides, but others are rather roughly shaped acidular fragments. . The other sample that proved non-carcinogenic, FD-14, shows long fibers, some thin and some thick, and many platy or amorphous particles. Recall that it contains about 35 % talc, measurements of only the fibrous-shaped particles by optical microscopy at x1000 were earlier reported to show an average diameter of 1.6 Atm (Smith, 1974). Measurements of fibrous-shaped particles in the presently available scanning electron micrographs at x1250 show an average diameter of 1 /i.m. ' ', , ' .. ...:.. '.. . The negative results with FD-14 may be explained by its lesser content of tremolite,which was 50%, although a relatively low content of tremolite would not explain the negative results with 275. X-ray diffraction studies of 275, 31 and 72 show their tremoiite content to be, respectively, about 95%, 90% and 95%. To what can we attribute the positive results with 72 and 31? Since they contain at least 5% of material other than tremolite, we cannot be sure that their activity is due wholly, or even in part, to tremolite. If we assume that their activity is due to tremolite, then the experiments indicate that appropriately high doses of long, thin particles of tremolite induced tum ors, w hereas high doses of shorter particles did not. This would, of course, be consistent with previous findings by ourselves and others with other materials, such as chrysotile and glass fibers. From the point of view of industrial hygiene, it is noteworthy that the experi ments show clear-cut, dose-related responses to both preparations that induced tumors. In addition, for estimation of biologic activity of materials containing tremo lite, the experiments show that consideration must be given, not merely to the amount of tremolite, but also to other factors, such as the morphologic characterist ics of the mineral. Factors of host susceptibility must also be considered. Most tumors in these experiments were diagnosed as mesotheliomas, of which some were examined by electron microscopy and found to contain Type C virus particles (Sobel ef a/., 1978). Observation of virus particles in the cells of these tumors suggests further work to learn whether a virus is involved in the causation of mesotheliomas. ACKNOWLEDGMENTS This work was sup p o rte d by a grant from institute o f O ccupational and En vironmental Health (Montreal) and a grant from R.T. Vanderbilt Company; Prepara tions of tremolitic ores used in these experiments were provided by Dr. James P. Leineweber, Johns-Manville Corporation, Denver, Colorado, and Dr. Allan M. Harvey. R.T. Vanderbilt Company, East Norwalk, Connecticut. ,. BIOLOGIC TE REFERENC Smith, W.E. L Proceeding comp. U S. Smith. W E., i 92-101 m E. Springer-Vs Sobel. M, Marc similarities A 1 0 0 .1978 Warburg. O. O BIOLOGIC TESTS OF TREMOUTE IN HAMSTERS 339 REFERENCES Smith, W E.: Experimental studies on biological effects of tremolite talc on hamsters. Pp. 43-48 in Proceedings of the Symposium on Talc. Washington, O.C. Information Circular 8639. A Goodwin, comp. U.S. Bureau ot Mines. 1974. Smith. W.E., and Hubert, D Q.: The intrapleural route as a means for estimating carcinogenicity. Pp. 92-101 in Expenmental Lung Cancer. Carcinogenesis and Bioassays. Ed. by E. Kartoe and J.F Park. Springer-Verlag, Berlin/New York, 1974. Sobel. H, Marquet, E., Smith, W.E.. and Hubert, D.D.: Asbestos-induced mesotheliomas in hamsters: similarities to human mesotheliomas and presence of type C virus particles. Federation Proc., 37: A100, 1978. Warburg, 0 .. On the origin of cancer cells. Science 123: 309-314 1956