Document pp96eKj8ozGNqOMeDKGJ0dRGj

Sairkigb Dickinson University RUTHERFORD TEANECK MADISON 25 April 1975 V.K. Rowe, Sc.D. Director, Toxicological Affairs Health & Environmental Research 1803 Building Dow Chemical USA Midland, Mich. 48640 FIORHAM-MADISON CAMPUS 285 Madison Avenue Madison, Now Jersey Am C*4. 701 177-4700 Dear Dr. Rowe: I have been in Minnesota and can only now write to thank you for your letter of the 9th and the paper from your laboratory on "Asbestos in Water in the Chlor-alkali Industry." The methodology, data and comcepts it presents are valuable to us and should be of interest to others working on biologic effects of asbestos. Is there any way of citing it ? For example, if the manuscript is in press, could you tell me the journal ? Trust it is agreeable for me to send copies to Fred Pundsack, Vice-president R & D at Johns-Manville and Ken Haley, Vice-president R & D at Reserve Mining Co. if you have not already done so. Enclosed are some papers from our lab. Among these, my present ation to Bureau of Mines Symposium on Talc offers data in Table 1 to show that carcinogenic responses to asbestos are dose related. Table 2 gives data to show that carcinogenicity of chrysotile is related to fiber size. Also enclosed is a copy of a letter of 3 April that I sent to Dr. Shapiro at FDA re experiments discussed at the meetingyou and I attended in Bethesda last February. As you know, NIEHS is still emending design of those experiments. A marked paragraph in my letter to Dr. Shapiro stresses a point you made at the February meeting. Your letter recalls our good leisurely dinner in New York, which I certainly enjoyed very much. With best regards. Sincerely, WES/elc Enclosures William E. Smith,M.D. Director Health Research Institute ' ST03I2I90 DOW 04801 0 RUTHERFORD TEANECK MADISON 3 April 1975 Dr. Raymond E. Shapiro Epidemiology Unit, HFF-108 Bureau of Foods, Food & Drug Administration 200 C Street, SW, Washington, D.C. 20204 RORHAM-MADISON CAMPUS 285 Madison Avanuo Madison, Naw Jarsay Dear Ray: Our Conversation in New York covered most points I wanted to offer in response to your nice letter of 5th March in which you asked for my comment on design of animal experi ments on oral administration of asbestos. This is to summarize and extend. First, I know of no epidemiologic data to suggest that asbestos may promote the action of some known carcinogen in the gastrointestinal tract. Hence, I see no reason to include in your protocol the many groups on some known carcinogen with and without asbestos. In my opinion, the effort and money spent for such groups could be better invested in the immediately practical question of comparing relative carcinogenicity of long and short fibers administered to experimental animals by mouth. If the experiments are done only with samples, such as UICC Standard Samples of Asbestos, that contain a wide range of fiber sizes with many fibers longer than five microns, positive results would be meaningless for the question of whether millions of dollars should be spent for redesign of municipal drinking water systems in communities throughout the nation where asbestos fibers are said to occur in drinking water. Accordingly, it would seem essential to include groups of animals exposed to samples where the fiber size distribution is comparable to that reported in municipal drinking water, in beer and in soft drinks. Second, as to dose. I can see that high doses, such as one percent asbestos in food, may have a place in small scale experiments designed to test whether some particular method or species will or will not yield gastrointestinal cancers. But for the practical purposes of your experiments, the dose should, in my opinion, be lowered to a level more comparable to expected exposures of human populations, running also some higher dose level as a safety factor. ! ST03I2I9I DOW 0480? Page 2 / Similarly, I can see that administration of asbestos to animals by gavage may have a place in small scale theoretical experiments, but I cannot see that gavage has any relevance to questions that concern regulatory agencies. Third, as to test species. The decision of your group to use rats and hamsters seems reasonable, since lungs of both species are known to be susceptible to carcinogenic actions of asbestos. Hence, negative results in their gastrointestinal tracts or elsewhere should be meaningful, especially since both species are known to be capable of developing tumors of the gastrointestinal tract. The references you asked me to send you on this latter point are: 1. Hoelzel, F. and E. Da Costa. Experimental production of polyposis of the colon in rats. Amer. J. Dig. Dis. 4:23-26, 1937. 2. Fortner, J.G. The influence of castration on spon taneous tumorigenesis in the Syrian (golden) hamster. Cancer Research 21:1491-1498, 1961. 3. Fortner, J.G. et al. Transplantable tumors of the Syrian (golden) hamster. Part I. Tumors of the alimentary tract, endocrine glands and melanomas. Cancer Research 21 (Part 2): 161- , 1961. 4. Fortner, J.G. et al. Transplantable tumors of the hematopoietic tissues, genitourinary organs, mammary glands and sarcomas. Cancer Research 21 (Part 2):199- ,1961. 5. Schabel, F.M., Jr. et al. Experimental evaluations of potential anticancer agents. II. Studies on the growth characteristics, metastases, and djjug response of hamster neoplasms of diverse sites of origin. Cancer Research 21 (Part 2):235- , 1961. 6.Sichuk, G. et al. Spontaneous tumorigenesis in hypophysectomized Syrian (golden) hamsters. Cancer Research 26:2154-2164, 1966. The Hoelzel and Da Costa paper (ref. 1, above) describes numerous intestinal polyps in rats. The other papers describe numerous adenomas and adenocarcinomas of the intestine in hamsters. ; ST0312 192 DOUJ 04803 Last point. Our experience has been that when female hamsters are housed together under laboratory conditions, they fight and often kill each other within the first year. We have rarely seen this with male hamsters. Our breeder (Charles River-Lakeview Hamster Colony, Newfield, N.J.) tells me that they once had an order for year-old female hamsters. They set the females aside, but fighting was so severe that very few survived to the age of one year. Accordingly, I would suggest that any experi ments you plan to do with hamsters be done with males. Fortner (see references above) reported adenomas and adenocarcinomas of the intestine in male as well as female hamsters. Over the past ten years, in autopsies of about 3000 male LVG:LAK hamsters maintained in our laboratory for periods up to 700 days, I have seen seven cases of intestinal adenocarcinomas. These tumors were in animals that died between 347 and 529 days of age. These tumors were scattered through various experiments, hence I cannot relate them to treatments. Trusting that some of this may be of interest and with all best regards. Sincerely, : ST03I2I93 WES/elc cc: David P. Rail, M.D William E. Smith, M.D. Director Health Research Institute Pp. 43 - 48 in PROCEEDINGS OF THE SYMPOSIUM ON TALC. Washington, D.C., May 8, 1973. Information Circular 8639. A. Goodwin, comp. U.S. Bureau of Mines. 19?4 EXPERIMENTAL STUDIES ON BIOLOGICAL EFFECTS OF TREMOUTE TALC ON HAMSTERS by William E. Smith, M.D.1 My name is William E. Smith. I am director of Health Research Institute at Fairleigh Dickinson University, Madison, N.J. I am a doctor of medicine. Over the past 23 years, I have studied more than 200 chemical materials for carcinogenicity in animals and have reported results of these tests in scien tific journals. In the past 8 years my associates and I exposed animali to various mineral dusts, maintained them over their natural life spans, and studied them for pathologic changes by gross and microscopic examination of their tissues and organs. The principal purpose of these latter studies has been to develop infor mation on possible fibrogenic and/or carcinogenic properties of various prepa rations of mineral dusts. The principal support for this work was provided by Research Grant EC 00226 from the Bureau of Occupational Safety and Health, U.S. Public Health Service. Thus far, we have published five papers (listed in the bibliography section of this paper) describing some of our findings in animals exposed to various preparations of asbestos. Today, 1 would like to offer a brief summary of our experiments with asbestos and describe as yet unpublished data from studies we have made with a sample of talc containing a large amount of tremolite. From our work, we identified the Golden Syrian hamster as a species that was capable of developing pulmonary fibrosis resembling asbestosis seen in man. Those experiments were done by repeated intratracheal injections of chrysotile or amosite asbestos. The method used for those experiments, weekly intratracheal injections, is technically tedious and time consuming. We found that we could compare the relative fibrogenicity and carcinogencity of mineral dusts more conveniently by depositing the dusts by a single injection into the pleural space of hamsters. After single intrapleural Injection of preparations of the chrysotile, amosite, anthophyllite, or crocidolite types of asbestos, we found that ham sters developed extensive pleural adhesions. These adhesions were composed mainly of round cells and multinucleated giant cells in the first few months after injection. Within 5 months, these adhesions became densely fibrotlc. Adhesions induced by the chrysotile, amosite, anthophyllite, or crocidolite types of asbestos tended to extend over large areas of the surfaces of the lungs. 1 Director, Health Research Institute, Fairleigh Dickinson University, Madison, N.J. DOW'04905 16121E01S 44 In concrasc, we saw very little reaction when we gave hamsters intra pleural injections of a sample of tremolite talc, which I shall Identify by cur sample number, FD-14. After intrapleural injection of this tremolite talc, the particles were gathered into small depots on the lung surface. These depots contained round cells-and multinucleated giant cells, but there was verv little formation of fibrous tissue. The experiments thus show that fiErogenic activity of tremolite talc is very much less than the fibrogenic action of the four principal types of asbestos. Also, in hamsters that had been given intrapleural injections of the four principal types of asbestos, we found intrathoracic tumors that we designate a:, mesotheliomas (table 1). TABLE 1. - Number of mesotheliomas in groups of 50 hamsters Dose. me 25 10 1 Chrysotile 9 4 0 Amosite - 3 0 Anthoohvllite - 3 - Crocidolite - 10 2 From the data in table 1, it can be seen that the yield of mesotheliomas, in groups of 50 hamsters per sample, was related to dose. In response to a large dose (25 mg) of chrysotile, nine hamsters developed mesotheliomas. In response to a smaller dose (10 mg) of chrysotile, four hamsters developed meso theliomas. In response to a still smaller dose (1 mg), there were no mesothe liomas. This experiment provides evidence to show that the carcinogenic action of chrysotile is related to dose, hence should be controllable by appropriate industrial hygiene measures to control dose. At the 10 mg dose level, amosite and anthophyllite gave about the same yield of mesotheliomas as did chrysotile, but crocidolite induced more than double the number of mesotheliomas gotten with the other types of asbestos. Also, crocidolite induced mesotheliomas in two hamsters treated with the lowest dose level (1 mg) whereas no mesotheliomas occurred in animals treated with that dose of chrysotile or amosite. Table 2 shows results of tests for carcinogencity of six preparations of chrysotile in groups of 50 hamsters each. Each of these preparations was tested at our highest dose level (25 mg per injection). The table gives data i'n the mean fiber length and mean fiber diameter of these samples. It can be s<.on that 8 to 10 mesotheliomas were induced by three of these samples: harsh chrysotile, soft chrysotile, and the same sample of soft chrysotile heattreated to achieve some of the properties of harsh chrysotile. These three samples that proved carcinogenic had mean fiber length of 5.3 to 6.9 microm otors and moan fiber diameter of 0.2 to 0.8 micrometer. These three active preparations of chrysotile were subjected to further milling to reduce the majority of particles to submicroscopic dimensions. The l S6UU01S : DOU 04806 45 cable shows that Chis reduction in particle size eliminated the carcinogenic effect of these samples. It also greatly reduced their fibrogenic action. TABLE 2. - Tests for carclnocenlcltv of six preparations of chrvsotile in erouns of 50 hamsters each Chrysotile preparation Soft..................... Soft (heated). Harsh................... Mean fiber leneth Mean fiber diameter 0M, EM, 0M, EM, micrometers micrometers micrometers micrometers 32.5 6.9 2.2 0.18 -- - - 38.9 5.3 2.9 .2 No. of mesotheliomas 8 10 9 Soft..................... .9 .03 0 Soft (heated). - - - - 0 Harsh................... - .4 - .07 0 OM Optical microscope. EM Electron microscope. Now, we have tested a sample of tremolite talc for carcinogenicity in hamsters. Electron diffraction studies of this sample showed it to contain 50 percent fibrous tremolite and 35 percent talc (table 3). A majority of the talc particles were platy, but some were rolled or fibrous. TABLE 3. - FD-14 (tremolite talc): Composition Percent Selected area electron diffraction: Fibrous tremolite............................................ 50 Antigorite............................................................. 10 Talc-........................................................................ 35 Chlorite......... ....................................................... 5 :0f this about 75 percent is platy and 25 percent is rolled or fibrous. In this sample, the fibrous particles had an average length of 5.7 microm eters and an average diameter of 1.6 micrometers (table 4). They are there fore in a size range that might be carcinogenic in our experiments with chrysotile. However, this sample of tremolite talc induced no mesotheliomas after intrapleural injection into hamsters. TABLE 4. - FD-14 (tremolite talc): Size measurement1 (Phase microscopy lOx ocular, lOOx objective) Leneth. micrometers Ranee Averaee Fibrous particles.............. 2.5-16.5 5.7 Platv oarticles................... - - 1 Ratio fibrous to platy particles: 3 to 1. Diameter. micrometers Ranee Averaee 1- 5 1.6 1-10 2.0 ST03I2I96 DOU 04807 46 Tabic 5 gives detailed data on these tests which were conducted at our highest dose level (25 mg). In view of the long latent period before develop* cent of tumors, if we wish to compare relative carcinogenicity of different samples, validity of the comparisons depends on the number of animals surviv ing into the later periods of the tests. In table 5, the number of survivors in each group at each time period is shown as the denominator. The cumulative number of animals bearing mesotheliomas is shown as the numerator. TABLE 5. - Yields of mesotheliomas in hamsters after intrapleural inlection at 25-mg dose level1 a Sample 151 340 372 450 500 550 600 650 700 davs davs davs davs davs davs davs davs davs Soft chrysotile................... 0/44 0/33 1/30 1/20 4/11 4/9 7/3 8/1 (?) Harsh chrysotile................ 1/41 4/26 4/21 7/16 7/13 7/9 8/5 9/2 C*) Heat-treated soft chrysotile............................ 0/37 1/35 1/32 1/27 4/17 5/15 8/4 10/1 (3) Tremolite talc..................... 0/52 0/35 0/34 0/29 0/27 0/23 0/20 0/15 0/7 1 Numerator: Cumulative number of hamsters with mesotheliomas. rDenominator: Number of hamsters surviving on stated day. ^Survivors at 650 days in chrysotile groups were killed on that day. Pleural adhesions, but no tumors, were found in them. We started with about 50 hamsters in each group. The first tumor was found at 151 days in an animal that had been treated with harsh chrysotile. Now let us look at the situation as it was 500 days after start of tests. At that time, 4 mesotheliomas had occurred in the group treated with soft chrysotile and only 11 animals survived. In the group treated with harsh chrysotile, 7 mesotheliomas had occurred and 13 hamsters were still living. In the group given heat-treated soft chrysotile, there were 4 mesothe liomas and 17 survivors. By comparison, no mesotheliomas had occurred in the group treated with trcmolite talc, and there were 27 survivors. The group on tremolite talc thus had many more animals at risk in the late stages of the tests, yet no mesotheliomas developed in them. In the three groups on preparations of chrysotile, all of the animals had died or were killed to close out those groups by 650 days. At that time, the final yield of mesotheliomas in those groups was 8, 9, and 10, respectively. a By comparison, the group on tremolite talc had developed no mesotheliomas by 650 days, and there were still 15 animals living and at risk in that group at that time. i t I 1 I l I t I I i i 1 i < 4 i \i ST03I2I91 DOU 04808 47 Eight of these survivors died of various causes between 650 and 700 days. No mesotheliomas were found in them. The remaining seven survivors on tremolite talc were killed*for examina tion at 700 days. No mesotheliomas were found in them. Throughout the tests, animals treated with the preparations of chrysotile in table 5 were found to have thick fibrous pleural adhesions extending over large areas of the lung surfaces, whereas animals treated with tremolite talc never showed more than small depots of the mineral particles with relatively little tissue reaction. In summary, our experiments show profound differences between the bio logic effects of tremolite talc, in respect to both fibrogenicity and carcino genicity, as compared with the biologic effects of the four principal types of asbestos: chrysotile, amosite, anthophyllite, and crocidollte. i I I I ; ST03I2I98 DOW 04809 48 Bibliography 1. Miller, L., W. E. Smith, and S. W. Berliner. Tests for Effect of Asbestos on Benzo(a)pyrene Carcinogenesis in the Respiratory Tract. Annals of the New York Academy of Sci., v. 132, article 1, 1965, pp. 489-500. 2. Smith, W. E. Asbestos in Relation to Tumors of the Respiratory Tract. Ch. in Lung Tumors in Animals, ed. by L. Severi. University Perugia, Italy, 1966, pp. 565-570. 3. Smith, U. E., D. D. Hubert, and M. Badollet. Biologic Differences in Response to Long and Short Asbestos Fibers. Am. Industrial Hygiene Assoc. J., v. 33, February 1972, p. A162. 4. Smith, W. E., L. Miller, and J. Churg. An Experimental Model for Study of Cocarcinogenesis in the Respiratory Tract. AEC Symp. ser. 21, 1970, pp. 299-316. 5. Smith, W. E., L. Miller, R. E. Elsasser, and D. D. Hubert. Tests for Carcinogenicity of Asbestos. Annals of the New York Academy of Sci., v. 132, article 1, 1965, pp. 456-488. DOW 04810 ; ST0312I99 AMER. INDUS! HYGIENE ASSN. J. 34 : 277-228 (MAY 1973) w Industrial Hygiene Summary Reports TUi Journal welcomes brief comments derived from daily activities which may bo useful to other members of the profession. Address your communications tot Richard F. Scherberger, Health and Safety Laboratory, Building 320, Kodak Pmfc Works, Eastman Kodak Company, Rochester, New York 14050. Asbestos, Talc and Nitrites in Relation to Gastric Cancer Recently, Merliss1 suggested that the high incidence of gastric cancer in Japan may be associated with treatment of rice with talc in Japan. He noted that some commercial "talcs" contain asbestos, which he proposed as an etiologic agent in gastric cancer. Mer liss cited a report of a small excess of gastric cancer in a group of insulation work ers exposed to dusts containing asbestos and a larger excess of pulmonary carcinomas and pleural mesotheliomas in miners who appear to have had mixed exposures to as bestos and talc. He noted that some geo logic deposits of talc are associated with tremolite, anthophyllite and chrysotile varieties of asbestos, accounting for the presence of fibrous particles of-asbestos to gether with platy particles of talc in some commercial preparations of "talc." Our group has carried out tests for car cinogenicity of various preparations of as bestos through experiments with animals. The present paper summarizes some results and reports a new experiment with a prep aration of talc containing asbestos. A pos sible alternative mechanism involving ni trites in gastric carcinogenesis is discussed. In 45 hamsters maintained throughout their lives on diets containing 1% of the chrysotile or amosite varieties of asbestos, wc found no gastric carcinomas and no tu- Thii work was supported by USPHS Research Grant EC 00224 from National Institute of Occupational Safety and Health, and by a grant from Fannit E. Rippcl FbundfttiML mors in the gastrointestinal tract except for a neoplasm in the mesentery of the colon.2 We are unable to relate that lesion to the treatment, since ashing has revealed no as bestos fibers in it. After intrapleural injection of 25 mg chrysotile or amosite into hamsters, we re ported tumors diagnosed as pleural mesothe liomas.2 Eight to 10 such tumors were found in each of 3 groups of 50 hamsters treated at this dose level.3 We have now completed an experiment in which the hamster-intrapleural method was used to test for carcinogenicity of a sample of talc containing asbestos. For this work, we obtained samples of commercial talcs from Whittaker, Clark & Daniels, Inc., New York Gty. A sample of cosmetic talcum powder grade was seen by microscopic ex amination to be composed of platy particles typical of the mineral, talc (hydrous mag nesium silicate). Another sample of a grade used for some industrial purposes ("#13 Talc") was found to be a mixture of platy and fibrous particles. X-ray diffraction analysis revealed the presence of major amounts of tremolite asbestos and talc and a minor amount of a serpentine mineral (antigorite). Each of 50 hamsters were given right intrapleural injection of 25 mg of this sam ple suspended in 0.5 ml saline. The animals were followed for their life spans. No tu mors attributable to the treatment were found. Granulomatous lesions were corn- 227 ST03IZ200 DOW 04811 22t mon on pleural. surfaces, but these lesions tended to be small and focal in contrast to larger and more fibrotic lesions induced in hamsters by chrysotile or amosite. The negative results of this test for car cinogenicity of a talc containing asbestos, and our earlier test on feeding asbestos to hamsters, do not offer support to the view that asbestos-containing talc contributes to the incidence of gastric cancer in Japan. Although death rates reported for gastric cancer are notably high in Japan in com parison to other countries, the highest re ported rate comes from Chile.4 Studies of gastric cancer in Chile have been reported from our laboratory with an hypothesis that high rates for esophageal and gastric cancer may be due to conversion of nitrates to nitrites by soil bacteria with subsequent synthesis of nitrosamine carcinogens in plants used as food or through interaction in the stomach with secondary amines in food.5 Chile has the world's largest known ge ologic deposits of nitrates, which are used as fertilizers. Nitrosamine has been reported in wheat plants and wheat grains.6 Nitrosamines can form through interaction of nitrites with secondary or tertiary amines in t Ik- stomach, and have been shown to induce esophageal and gastric cancers in rats.7-1 High concentrations of secondary amines have been reported from Japan in dried fish.'' The situation invites attention to a po tential role of the nitrogen cycle in environ mental carcinogenesis. A source for direct ingestion of nitrites by human populations May, 1973 arises from use of nitrites as food additives. Other potential factors in gastric carcino genesis have been recently reviewed by Haenszel et of.10 who found no support for the talc-asbestos hypothesis on epidemiologic grounds. 1. Merliss, R. R.: Talc-Treated Rice and Japanese Stomach Cancer. Science 173:1141 (Sept 17, 1971). 2. Smith, W. E., L. Miller, R. Elsasser and D. Hubert: Tests for Carcinogenicity of Asbestos. Ann. New York Acad. Science 132:456 (Dec. 31, 1965). 3. Smith, W. E., D. Hubert, and M. Badollet: Biologic Differences in Response to Long and Short Asbestos Fibers. Amer. Ind. Nyg. Assoc. 1. 33: Abstract #162 (Feb. 1972). 4. Macdonald, E., and P. Wolf: in Epidemiologi cal, Experimental and Clinical Studies on Gastric Cancer. Japanese Cancer Assn. GANN Monograph 3 (1968). 5. Zaldtvar, R.: Geographic Pathology of Oral, Esophageal, Gastric and Intestinal Cancer in Chile. Ztsch. Krebsforsch. 75:1 (1970). 6. Hedler, L,, and P. Marquardt: Ueber das Vorkommen von Nitrosaminen in Nahrungsund Futtermitteln. Naunyn-Schmiedebergs Arch. Pharm. exp. Path. 259:176 (1968). 7. Sander, J-, F. Schweinsberg, and H. Menz: Untersuchungen ueber die Entstehung cancerogener Nitrosamine. im Magen, Hoppe-Seyler's Ztsch. physiol. Chem. 349.1691 (1968). 8. Druckrey, H., S. Ivankovic. and D. Schmaehl: Organotrope carcinogene Wirkungen bei 65 verschiedenen N-nitroso-Verbindungen an BDRatten. Ztsch. Krebsforsch. 69.103 (1967). 9. Kawamura, T., K. Sakai, and F. Miyazawa: Studies on nitrosamines in foods (IV). Dis tribution of secondary amines in foods. J. Food Hyg. Soc. (Japan) 12:192 (1971). 10. Haenszel, W., M. Kurihara, M. Segi, and R. Lee: Stomach Cancer among Japanese in Hawaii. J. Nat"l Cancer Inst. 49:969 (1972). William E. Smith, MJ). Health Research Institute Fairleigh Dickinson University Madison, NJ. 07940 ST03I220I DOW 04812 Reprint from Experimental Lung Cancer. Carcinogenesis and Bioassays Edited by E. Karbe and J. F. Park Springer-Verlag Berlin Heidelberg New York 1974 Printed in Germany Not for Sale DOW 04813 ST03I2202 The Intrapleural Route as a Means for Estimating Carcinogenicity* William E Smith and Doras D. Hubert Health Research Institute, Fairleigh Dickinson University, Madison, Ml 07940, USA ABSTRACT Xntrathoracic tumors have been reported from several laboratories after intrapleural injection of various preparations of mineral sub stances into experimental animals. Application of information from such studies to problems of pulmonary carcinogenesis involves con sideration of whether tumors so induced arise from mesothelial cells of the pleura or from other cells of the lungs or thoracic cage. Another question stems from the fact that direct introduction of test substances into the pleural space does not reproduce natural routes of exposure to inhaled carcinogens, however, the intrapleural route may serve a useful role as a convenient screening procedure in com parison to laborious, repeated intratracheal injections or the large space requirements of inhalation exposures. In experiments with hamsters exposed to a variety of preparations of asbestiform and other minerals, we have given each animal only a single intrapleural injection. Multiple injections present undesirable vari ables, since early granulomatous responses to a single injection en tail variations in deposition of subsequent injections. After single intrapleural injection of hamsters, we have seen upward of 80 animals with intrathoracie tumors spreading along pleural sur faces in a manner characteristic of mesotheliomas. For purposes of this paper, these tumors will be designated as mesotheliomas, although we were able to trace their derivation from the mesothelium only in rare cases, most of the tumors being too large to identify their pre cise origin. Data from these experiments show that tumor response to chrysotile, amosite, and crocidolite is related to dose. Tumors regarded as meso theliomas arose in response to preparations of chrysotile containing numerous fibers visible by optical microscopy but not in response to preparations in which the great majority of fibers were visible only by electron microscopy. This research was supported by Research Grant EC 00226 from the u. FOblic Health Service and by grants from Johns-Manville Fund and Fannie E. Rippel Foundation. DOU qAS''- ^ G0Z21COlS. ST03I220U Introduction -J Beginning in 1952, HUEPER and associates reported a series of ,.x. perlmants in which they attempted to utilize the pleural cavit-. . study of biologic responses to various minerals that had been as- ' r ed with pulmonary carcinogenesis in epidemiologic studies of eomu ' occupational groups. Test materials were suspended in lanolin or gelatin and injected into animals through the supraclavicular foss.< or implanted through thoracotomy. Epidermoid carcinomas, round ecu carcinomas, adenocarcinomas, and sarcomas were reported in lungs ot rats and mice after what was said to be intrapleural deposition of powdered chromite roast and a number of chromates, whereas no lung tumors were found in rats exposed by inhalation to chromite ore with particle size averaging less than 4 urn (HOEPER and PAYNE, 1959, I960; HUEPER and CONWAY, 1964). Intrapleural deposition of powdered metallic nickel and uranium was said to induce sarcomatous responses from the lung and pleural tissues of rats (HUEPER and CONWAY, 1964). These sarcomas, however, appear to have been in the thoracic wall or sub cutaneous tissues at sites of injection (HUEPER, 1952, 19S5; HUEPER et al., 1952). For intrapleural experiments, multiple injections were often given over a period of months. Pleural adhesions resulting from early injections may have caused later injections to be deposited in to the lungs, which might account for some tumors described as being found in the lungs. HUEPER (1954) found no tumors related to treatment in 25 OsborneMendel rats, each of which received 6 monthly injections of about 15 mg of a preparation of asbestos described as g commercial-grade medium fiber. This material was suspended in lanolin. HUEPER and PAYNE (1962) mention a pleural mesothelioma in 1 of 21B 'Bethesda black rats' (from the National Institutes of Health) and state that it is a tumor that appears without any intervention in rats of that strain. In the same paper, they report an experiment in which chromic acetate in a gelatin capsule was implanted into the right pleural cavity of 42 rats and into thigh muscles of 35 rats of that strain. Aside from 1 tumor at the intramuscular site,' they did not consider tumors occurring elsewhere in these animals to have any connection with the treatment. These other tumors Included 3 meso theliomas of the pleura. WACNER (1962, 1966) has developed a method for intrapleural Injections of materials suspended in saline solution using a needle attached to a two-way tap introduced under ether anesthesia into the right axilla of rats at the level of the second teat. One end of the tap is attached to a capillary manometer, which gives a negative reading when the needle reaches the pleural cavity. A syringe containing the inoculum is then attached to the other arm of the tap and a volume of 0.4 ml is injected. With this technique, WAGNER and BERRY (1969) Injected 20 mg of prepara tions of amosite, crocidolite, chrysotile, and silica into specific pathogen-free (SPF) Wistar rats. The suspensions in saline were steril ized in an autoclave. There were 96 rats per sample, equally divided as to sex. With the 3 types of asbestos, they reported tumors that they diagnosed as mesotheliomas in 38, 61, and 55 rats, respectively. These tiusors ranged in size from large masses enveloping the right lung to small nodules on the parietal pleura. They were equally distributed among males and females. Histologically, these tumors were said to be composed most frequently of spindle cells with occasional clefts lined by epithelial cells and were therefore considered as mixed- DOW 04815 #4 pattern mesotheliomas. Purely spindle-cell tumors or tumors described as t-ubulopapillary were less common. In 9 of the rats injected with asbestos, subcutaneous sarcomas were found at injection sites. They were attributed to deposition of the inoculum in the chest wall rather than in the pleural cavity. In the 96 rats injected with silica, with a particle size of less than 5 um, about half developed intrathoracic tumors diagnosed as histocytic reticulum cell-sarcomas. These tumors are discussed and con trasted with tumors diagnosed as mesotheliomas (WAGNER, 1966). No mesotheliomas were found in 96 control rats injected only with saline solution. These experiments with SPF rats were repeated with standard rats and generally comparable results were obtained. From subsequent experiments with the same technique, WAGNER et al. (1973) reported that development of mesotheliomas in SPF Wistar rats was approximately proportional to dose of asbestos. In groups of 30 to 35 rats, they found no mesotheliomas in response to a sample of glass fibers, but single mesotheliomas in response to glass powder, nonfibrous aluminum oxide, and barium sulphate, 3 mesotheliomas in response to aluminium silicate fibers, and 18 in response to brucite. They state that their sample of brucite also contained chrysotile. After Intrapleural injection of 1 ml of 1:15 suspensions of asbestos fibers into 102 Sprague-Dawley rats, DONNA (1970) saw tumors in 12 animals after 13 to 20 months. These tumors were described as mesothellal in 3 rats treated with chrysotile, whereas in 4 rats treated with crocidolite and 5 rats treated with amosite, there were said to be undifferentiated pulmonary carcinomas. The possibility that some of the inoculum may have been deposited into the lungs must be con sidered. A technique designed to achieve widespread deposition and retention of particulates in the pleural cavity has been developed by STANTON and WRENCH (1972) . They suspended preparations of asbestos in liquid gelatin, which was then allowed to harden on a pledget of coarse glass fibers measuring about 30 x 20 x 2 mm. The pledgets were intro duced into the left pleural cavity of female Osborne-Mendel (om) rats through a thoracotomy under ether anesthesia. In rats thus treated, they found numerous tumors that they described as pleural mesotheliomas of spindle cell or pleomorphic types, sometimes with tubulopapillary features. In groups of 30 rats treated with pledgets containing 40 mg of UICC (Union Internationale Contre le Cancer) standard reference samples (TIMBRELL, 1969) of chrysotile A, amosite, and crocidolite, tumors designated as mesotheliomas were seen in 15, 15, and 14 ani mals respectively. Milling of this sample of crocidolite to reduce particle size gave a product that induced mesotheliomas in only 8 animals. With pledgets containing 10 mg or 1 mg UICC crocidolite, the number of mesothelioma-bearing rats was reported as 11 and 2, respectively. When 10 mg of the same crocidolite was suspended in saline and in jected without pledgets of glass fibers, mesotheliomas were found in 9 rats. The closely similar yield of these tumors with and without pledgets indicates no advantage of this special method to influence distribution of particulates within the pleural cavity. No tumors resulted from control implants of the coarse glass fiber pledgets. Single mesotheliomas were found in groups with pledgets impregnated with 40 mg of glass fibers 1 um to 25 um in diameter. In 2 groups treated with 40 mg of glass fibers, 0.06 um to 3 um in diameter, 3 and 5 tumors diagnosed as mesotheliomas were found. ST03I2205 c r. c & " DOW 04816  :<aori or tumors described : the rets injected with t injection sites. They a in the chest well rether perticle size of less then ors diagnosed as histoare discussed end con es (WAGNER, 1966). No injected only with saline re repeated with standard btained. chnlque, WAGNER et al. iomas in SPF Wistar rats oestos. In groups of 30 ssponse to a sample of iponse to glass powder, ze, 3 mesotheliomas in 3 in response to brucite. contained chrysotlle. suspensions of asbestos 1970) saw tumors in 12 >ere described as mesotereas in 4 rats treated ilte, there were said to ! possibility that some the lungs must be con- position and retention n developed by STANTON s of asbestos in liquid a pledget of coarse The pledgets were introOsborne-Mendel (om) rats In rats thus treated, d as pleural mesotheliomas es with tubulopapillary ledgets containing 40 mg r) standard reference site, and crocidolite, in 15, 15, and 14 anicrocidolite to reduce 3the1iomas in only 8 rocidolite, the number 11 and 2, respectively, led in saline and inzheliomas were found in nors with and without tl method to influence il cavity. te coarse glass fiber iroups with pledgets * 25 urn in diameter. In >.06 um to 3 ws> in diatas were found. 96 Materials and Methods Our group has carried out experiments in which various preparations of minerals have been injected into the pleural cavity of Syrian gol den hamsters. Throughout, we have used male hamsters of the LVGsLAK strain obtained from LaReview Hamster Colony, Newfield, New Jersey. Test materials have been suspended in 0.9% NaCl solution, sterilized in an autoclave, and injected in a volume of 0.5 ml with a syringe fitted with a 20-gauge needle, except for thick slurries for which an 18-gauge needle was used. Each animal received only 1 injection, which was made into the right chest in the mid-axillary line about 1/4 inch above the lower end of the sternum. Depending on the nature and dose of teat materials, granulomatous responses can largely ob literate the pleural cavity within a few weeks. Accordingly, we have not used and do not recommend repeated injections. In our early work (SMITH et al., 1965b), we 'stabbed* the right chest 2 days before injection in an attempt to induce pneumothorax and thus favor Intrapleural deposition and spread of inoculum. With experience, we found that comparable deposition and spread can be achieved by direct injection. Hence, preliminary 'stabbing* has been omitted in all of our intrapleural work since 1965. Following attempts at intrapleural injections, part of the inoculum was sometimes found subcutaneously in the chest wall. Sarcomas occa sionally arose at such sites. We have not included such subcutaneous tumors in comparing yields of lntrathoracic tumors in response to different test materials. To avoid this problem, the occasional ani mal that shows a subcutaneous swelling at the time of injection is discarded and replaced. With this precaution, examination of trial animals immediately after injection has shorn the inoculum in the pleural space. The technique of intrapleural injection is thus not more difficult than the commonly used technique of intraperltoneal injection. No anesthesia is required. The operator holds the animal in 1 hand and makes the Injection with the other, holding a finger on the xiphoid to assure proper positioning of the needle. \ Results After intrapleural injection of a carcinogenic dose, we have occa sionally seen small tumors whose origin from mesothelium could be traced. The majority of lntrathoracic tumors found in our experiments, however, have been large masses whose origin could not be precisely ascertained. Such masses compress the lung, sometimes largely fill . the right chest, spread along pleural surfaces, but Invade the lungs only superficially. Some penetrate the diaphragm. Occasionally, metastaaes are found. Gross and microscopic appearance of such tumors in hamsters have been described and illustrated (SMITH et al., 1965a; SMITH et al., 1965b). In man, mesotheliomas have been described as tumors that spread along mesothelial surfaces and they have been subdivided into epitheliallike, sarcomatous, or mixed types (CHURG and SELIKOFF, 1968). The majority of lntrathoracic tumors in our experiments with hamsters appear aarcomatous while others resemble the so-called epithelial or mixed types. Dse of the term 'mesothelioma* for many experimentally induced tumors reported in animals has been questioned by GROSS (1973) ST03I2206 DO^ 0 96 In an axchange of views including STANTON* a contribution. For the present, we use the tern "mesothelioma" for purposes of comparing experimental yields of malignant tumors that appear to be primary in the chest, spread along mesothelial surfaces, and histologically bear some resemblance to one or another type of tumor presently diag nosed as mesothelioma in man. Thus defined, we found that the yield of mesotheliomas after intra pleural Injection of hamsters was related to the dose of chrysotile or amosite. In groups of 50 hamsters, the number of tumors considered to be mesotheliomas were 9, 4, and 0 in response to 25 mg, 10 mg, and 1 mg chrysotile; 4 and 0 in response to 10 mg and 1 mg amosite (SMITH et al., 1968). No mesotheliomas were found in response to 25 mg of a preparation of talc that contained 50% fibrous tremolite (SMITH, 1973). We saw no mesotheliomas in 100 untreated hamsters maintained as control groups in these experiments. 3 preparations of chrysotile with mean fiber length of 5.3 wm to 6.9 urn, as measured by electron microscopy, were milled to reduce fiber size to mean lengths of 0.37 wm to 0.86 ym. These 6 preparations were tested at a dose level of 25 mg in groups of 50 hamsters each. The 3 original preparations induced extensive pleural adhesions and yielded 8, 9, and 10 mesotheliomas, whereas the 3 samples that had been subjected to further milling induced relatively thin pleural adhesions and no mesotheliomas (SMITH et al., 1972). Fiber diameter in the original preparations averaged 0.18 ym to 0.2 ym and in the milled preparations 0.03 ym to 0.07 ym. This evidence that carcinogenicity of asbestos is related to physical properties is supported by an as-yet unpublished experiment of ours in which 50 hamsters were given an intrapleural injection of 25 mg serpentine rock dust (antigorite). This material has the chemical composition of chrysotile but is not crystallized in fibrous form.. Only 1 mesothelioma resulted.* com; typ' the spa in The ham cor BEF anc (IS 9 at the anc our the *PI Si: of ti.. mo: Uic th< da: po bo 19* . C/5 The following information is available from additional aa-yet unpub lished experiments with groups of 50 hamsters: Since the external surface of chrysotile fibers is thought to consist of magnesium hydroxide (SPEIL and LEINEWEBER, 1969), an experiment was designed to ask whether such a surface might play a role in asbestos carcinogenesis. For this purpose, a fibrous magnesium hydroxide (nemalite) was tested. At the 25-mg dose level, no mesotheliomas resulted. CO ro no Another experiment was designed to test whether carcinogenic activity night be shown by fibers comparable to asbestos fibers in size but different in chemical composition. For this purpose, we used silicon dioxide fibers with an average diameter of 0.75 ym. At a dose of 10 mg, r.csotheliomas developed in 4 hamsters. No mesotheliomas occurred in 50 hamsters injected with borosilicate glass fibers with an average diameter of 5 ym, or in 50 hamsters injected with such glass fibers coated with phenolformaldehyde (a binder used in some preparations c! fiberglass). di t: tl 1? D: A standard reference (UICC) sample of crocidolite (TXMBRELL, 1969) was tested in 2 groups of SO hamsters each. At a dose of 10 mg, it in duced mesotheliomas in 10 hamsters. At a dose of 1 mg, it induced meso theliomas in 2 hamsters. 3 of 50 hamsters developed mesotheliomas after 10 r; of a standard reference (UICC) sample of anthophyllite. These findings show that intrapleural injection of hamsters with various mineral substances suspended in saline provides a convenient means for Ti v. Hi O O B P DOW 04818 attribution. For the rposes of comparing ppear to be primary a, and hiatologically t tumor preaently dlag- heliomas after intrahe dose of chrysotile er of tumors considered se to 25 mg, 10 mg, and and 1 mg amosite (SMITH response to 25 mg of us tremolite (SMITH, d hamsters maintained ength of 5.3 um to re milled to reduce wm. These 6 preparations of 50 hamsters each, pleural adhesions and a 3 samples that had tiveiy thin pleural 1972). Fiber diameter co 0.2 wm and in the is related to physical td experiment of ours I injection of 25 mg II has the chemical tad in fibrous form. iitional as-yet unpub- > is thought to consist .969), an experiment was .ay a role in asbestos inesium hydroxide (nemalesotheliomas resulted. - carcinogenic activity : fibers in size but -pose, we used silicon wm. At a dose of 10 mg, .heliomas occurred in .bars with an average th such glass fibers n some preparations te (TIMBRELL, 1969) was >se of 10 mg, it inf 1 mg, it induced mesooped mesotheliomas after anthophyllite. of hamsters with various a convenient means for 97 compariaon of relative carcinogenicity. In reaponae to the principal typea of asbestos, hamsters have shown a lesser'incidence of meso theliomas than rats, but, as might be expected from the shorter life span of hamsters, mesotheliomas tend to appear earlier in them than in rats. The earliest tumor that we considered a mesothelioma was found in a hamster examined 151 days after Injection of 25 mg chrysotile. In contrast, the earliest mesothelioma reported in rats by WAGNER and BERRY (1969) was at 353 days after injection of 20 mg chrysotile, and the earliest mesothelioma seen in rats by STANTON and WRENCH (1972) appears to have been at about 350 days after Implantation of a glass pledget containing 40 mg amosite. In tests of UICC crocidolite at 10 mg, STANTON and WRENCH'S data from rats show the earliest meso thelioma at about 560 days when the material was Injected in saline and about 525 days after implantation in a glass pledget, whereas our test of 10 mg UICC crocidolite in hamsters gave the first meso thelioma at 418 days. By the intrapleural route, the hamster thus appears to afford a more rapid test model than the rat. Since tests for carcinogenicity are customarily run over the life span of test species, the rate at which results can be obtained is of prac tical importance. To learn whether mice might give useful information more rapidly than hamsters, we made intrapleural injections of 5 mg UICC crocidolite suspended in saline into 40 male BALB/c mice. Xntrathoracic tumors were found in 3 of these mice at 301, 301, and 340 days. These 3 tumors resemble plasma cell tumors that have been re ported in BALB/c mice after intraperitoneal injection of Plexiglass borings. Plexiglass discs, Millipore filters, or mineral oil (POTTER, 1968). Plasma cell tumors have been reported in White Leghorn fowls after introduction of tremolite into axillary air sacs (PEACOCK and PEACOCK, 1966). Both pleural and peritoneal mesotheliomas have been reported in female CBA mice after subcutaneous injection of massive (60-mg) amounts Of asbestos in divided doses (ROE at al., 1967). Among 5 groups of 20 mice treated with crocidolite, amosite, or chrysotile there were 6 mice with sarcomas at injection sites and 10 mice with mesotheliomas. These were distributed among the treatment groups. The lesions diag nosed as mesotheliomas appear to have been small papillomatous pro liferations of mesothelial cells. Heavy deposits of fibers were seen in submesothelial tissues of mice with mesotheliomas. It was thought that fibers had been actively transported from the subcutaneous site to the submesothelial site, but subsequent studies by the same group did not provide evidence for this. Passive movement of fibers along tissue planes or as a consequence of inflammation and necrosis is now thought the store probable explanation (ROE, personal communication, 1974). Discussion The literature on tumors induced by intraperitoneal injection of various plastic, metallic and other materials has been reviewed by HUEPER (1964a) and HUEPER and CONWAY (1964). Discussions of mechanisms of carcinogenesis by this route and comparison of results of tests by other routes has been presented by BISCHOFF and BRYSON (1964) and BRYSON and BISCHOFF (1967, 1969). Most of the tumors reported as ex perimentally induced at intraperitoneal sites have been sarcomas in ST03I2208 98 rats. After intraperitoneal Implantation of polyurethan foam into Bathesda black rats, HUEPER (1964b) reported tumors including an un specified number of mesotheliomas, but he also states that peritoneal mesotheliomas occur spontaneously in rats of this strain. Induction of sarcomas in rats after intraperitoneal injection of as bestos was reported by SCHMXHL (1958). KLOSTERK0TTER and ROBOCK (1970) reported experiments in which they made intraperitoneal injections of 50 mg asbestos into groups of 30 rats. With preparations containing fibers up to 50 vm in length, they found fibrosis in response to chrysotile, amosite, and crocidolite. Mesotheliomas were said to have been found in 2 rats of the group on amosite and in 4 rats of the group on crocidolite. No tumors were found in the chrysotile group. In other groups treated with these* 3 samples of asbestos after further milling, there was little fibrogenicity and no tumors. Most particles in these milled products were said to be less than 1 urn in size. The animals were observed for periods of 6 to 12 months. POTT et al. (1972) reported results from groups of 30 female Wlstar rats given 4 intraperitoneal injections of 25 mg UICC chrysotile A or the same material after further milling. 'Samples were suspended in saline and injected at weekly intervals. The animals were followed for periods up to 2 years. Extensive adhesions were found in the peri toneal cavity of animals given UICC chrysotile but only slight fibrosis in those that had received the milled product. The incidence of tumors was about 40% in both.groups, but the first tumor was found at 7 months in the group on UICC chrysotile in contrast to 13 months for the milled product. Most of the tumors were diagnosed as sarcomas but 7 were list ed as mesotheliomas. After intraperitoneal injections of groups of 40 female rats with 6.25 mg, 25 mg, and 100 mg chrysotile, POTT and FRIEDRICHS (1972) found 17, 19, and 16 animals, respectively, with abdominal tumors within 530 days. This apparent plateau of response suggests to us that dose-response studies.using intraperitoneal injection may require lesser doses than the intrapleural route. Since the surface area of the peritoneum is much more extensive than the surface area of the pleural cavity, larger numbers of mesothelial cells are at risk. In testing materials for carcinogenicity in animals, evaluations depend upon observation of tumors of types or at sites not found in controls, or at a frequency or speed of appearance greater than seen "spontaneous ly" in untreated control animals. For studies on mesotheliomas, there is only limited information on their spontaneous incidence or pathology in animals. A transplantable mesothelioma has been reported in a mouse (SHAPIRO and WARREN, 1949). A mesothelioma was listed among tumors seen in untreated control hamsters (FORTNER, 1961). As mentioned above, both pleural and peritoneal mesotheliomas have been said to occur spontaneous ly in Bethesda black rats (HUEPER and PAYNE, 1962; HUEPER, 1964b). MORRIS et al. (1961) state that papillary mesotheliomaa of the testes or epididymis occurred in 3 Buffalo rats fed N-2-fluorenylacetamide and in 2 untreated control Buffalo rats. Pathologic material from these and other Buffalo rats was studied by H.L. STEWART (personal communi cation, 1974) who advises that he had not seen spontaneous pleural meso theliomas but that he had seen mesotheliomas arise spontaneously in that strain from an extension of the peritoneal mesothelium (tunica vaginalis of the testis). He states that these tumors are papillary, sometimes tubular, and they occasionally spread to the peritoneal cavity. In a discussion of spontaneous lesions of rats, RIBELIN and MCCOY (1965) state that they omentum and sere for their photor They state that they may be four ACI, Buffalo, ar The cited report treated control: nisms in testin> mesothelial tis: chickens after . 1970). Resembla: to cases of mulof asbestos sug chemicals assoc of a virus (SMr References BISCHOFF, F., B Prog. exp. Tu BRYSON, G., BIS Tumor Res. 9, BRYSON, G T<; exp. Tv CHABOT, : C/3 of perl --4 / avian 1 m CHURG, J. lioma. CO Baltimc roDONNA, A. rodolite 1-32 (1 FORTNER, genesis to (1961). GROSS, P. glass. J. nat HUEPER, W.C.s cancers in re HUEPER, W.C.s reactions in pensions of : Cancer Inst. HUEPER, W.C.: Cancer produ^ Cancer Inst. HUEPER, W.C.s liferants. N- HUEPER, W.C.s plastics. J* HUEPER, W.C., Springfield, HUEPER, W.C., chromium com health. Amer 0482 ooW 100 HUEPER, W.C. PAYNE, W.W.: Experimental studies on chromium compounds. Proceedings of the 13th International Congress on Occupational Health, N.Y., Booh Craftsmen Ass., pp. 473-486, 1960. HUEPER, W.C., PAYNE, W.W.: Experimental studies in metal carcino genesis: chromium, nickel, iron, arsenic. Arch. Environ. Health , 445-462 (1962). HUEPER, W.C., ZUEFLE, J.H., LINK, A.M., JOHNSON, M.G.< Experimental studies in metal cancerigenesis. II. Experimental uranium cancers in rats. J. nat. Cancer Inst. 13, 291-306 (1952). KLOSTERKOTTER, W., ROBOCK, X.: Experimentelle Untersuchungen sum Wirkungsmechanismus von Asbest unter besonderer BerUcksichtigung der FaserlXnge. Schriftenreihe Arbeitsmedizin, Sozlalmedizin, Arbeitshygiene 36, 111-130 (1970). * MORRIS, H.P., WAGNER, B.P., RAY, F.E., SNELL, K.C., STEWART, H.L.: Comparative study of cancer and other lesions of rats fed N,N'-2,7fluorenylenebisacetamide or N-2-fluorenylacetamide. National Cancer Inst. Monograph 1, 1-54 (1961). PEACOCK, P.R., PEACOCK, A.: Asbestos induced tumours in fowls. In: Lung Tumours in Animals (L. Severi, ed.), pp. 571-588. Perugia 1966. POTT, F., FRIEDRICHS, K.H.: Tumoren der Ratte nach i.p.-Injektion faserfdrmiger StXube. Naturwissenschaften 9, 318 (1972). POTT, E.F., HUTH, F., FRIEDRICHS, K.H.: Tumoren der Ratte nach i.p.Injektion von gemahlenem Chrysotil und Benzo(a)pyrene. Zbl. Bakteriologie, Parasitenkunde, Infektionskrankhelten und Hygiene, I Abt. Orig., Relhe B 15, No. 5-6, pp. 463-469 (1972). POTTER, M.: A resume of the current status of the development of plasma-cell tumors in mice. Cancer Res. 28, 1891-1896 (1968). RIBELIN, W.E., MCCOY, J.R.: The Pathology of Laboratory Animals, p. 268. Springfield, 111.: Charles C. Thomas 1965. ROE, F.J.C., CARTER, R.L., WALTERS, M.A., HARINGTON, J.S.: The pathological effects of subcutaneous injections of asbestos fibres in mice: Migration of fibres to submesothellal tissues and induc tion of mesotheliemata. Int. J. Cancer i, 628-638 (1967). ROE, F.J.C.: personal communication, 1974. SCHMXHL, d.: Cancerogene Wirkung von Asbest bei Implantation an Ratten. Z. Krebsforsch. 62, 561-567 (1958). SHAPIRO, D.M., WARREN, S.: Cancer Innervation.jCancer Res. 9, 707- 711 (1949). SMITH, W.E.: Asbestos, talc and nitrites in relation to gastric cancer. Amer. ind. Hyg. Ass. J. 34, 227-228 (1973). SMITH, W.E.: Multiple pulmonary carcinomas and mesotheliomas in hamsters. In: Multiple Primary Malignant Tumours (L. Severi, ed.), Perugia, in press. SMITH, W.E., HUBERT, D.D., BADOLLET, M.S.: Biologic differences in response to long and short asbestos fibers. Amer. ind. Hyg. Ass. J. 33, A162 (1972). SMITH, W.E., HUBERT, D.D., MILLER, L., BADOLLET, M.S., CHURG, J.: Tests for threshold levels of carcinogenicity of asbestos. In: Internationale Xonferenz ttber die biologischen Wirkungen dea Asbestes (Anspach, ed.), pp. 240-242. Dresden 1968. SMITH, W.E., MILLER, L., CHURG, J., SELIKOFF, I.J.: Mesotheliomas in hamsters following intrapleural injection of asbestos. J. Mt. Sinai Hosp. 32, 1-8 (1965a). SMITH, W.E., MILLER, L., ELSASSER, R.E., HUBERT, D.D.: Tests for carcinogenicity of asbestos. Ann. N.Y. Acad. Sei. 132. 456-488 (1965b). SPEIL, S., LEINEWEBER, J.P.: Asbestos minerals in modern technology. Environ. Res. 2, 166-208 (1969). STANTON, M.F., WRENCH, C.: Mechanisms of mesothelioma induction with asbestos and fibrous glass. J. nat. Cancer Inst. 48, 797-821 (1972). STEWART, H.L.: personal communication, 1974. I timbrel: Cancel Intern burg 1 WAGNER, pleura 180-18 WAGNER, of var (L. Se WAGNER, tion w WAGNER, InocuL 173-18 I 1 01221COlSt DOU 04821 f l on chromium compounds, on Occupational Health, in metal carcinoEnviron. Health 5, M.G.s Experimental ).:al uranium cancers 2 itersuchungen rum c BerUcksichtigung Sozialmedizin, Arbeits- 2., STEWART, H.L.: of rats fed N,N'-2,7mide. National Cancer mours in fowls. In: S71-588. Perugia 1966. ach i.p.-Injection 318 (1972). der Ratte nach i.p..) pyrene. Zbl. Bakten und Hygiene, I (1972). 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Sever!, ed.), pp. 389-606. Perugia 1966. WAGNER, J.C., BERRY, G.s Mesotheliomas in rats following inocula tion with asbestos. British J. Cancer 2j, 567-581 (1969). WAGNER, J.C., BERRY, G., TIMBRELL, V. Mesotheliomata in rats after inoculation with asbestos and other materials. Brit. J. Cancer 2, 173-185 (1973. 1 1 2 2 1 (0 1 8 * ti t DOW 04822