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Confidential Report 29-55 MELLON INSTITUTE Special Report : 7-8-66 /ffJ 7't* '(> fc 12 pages The Fibrogenic Potential of Asbestos Products via intraperitoneal Injection in Guinea Pigs, Rats and Rabbits and by the Intratracheal Route in the Rat Chemicals Division. Union Carbide Corporation_____ _Industrial Fellowship 274-29 Summary The results of intraperitoneal administration indicate that the asbestos products studied produced fibrotic lesions of the visceral organs in rats and guinea pigs regardless of fiber length. Of the three products, CMS-100 (refined fiber), produces the most severe reaction. JT-100 (standard fiber) and JM-3K (Johns Manville screened fiber) produced similar but less severe reactions. The results on rabbits were inconclusive. Both JT-100 and CMS-100 produced specific lesions in rats by the intra tracheal route but with JT-100 they were more severe. No lesions were found in the rats which received JM-3K fibers. These results indicate that these three asbestos fibers may be fibrogenic by either route. No specific lesions were observed 'in the lungs of the JM-3K intratracheal rats, but only two rats survived to be examined. Samples The following samples were shipped to Mellon Institute at the request of Mr. Paul W. McDaniel who furnished the descriptive information. Designation of Asbestos Product Source Quan tity Date Received M. I. Sample Number *CTS-100 Fiber (same as JT-100 Fiber) Sterling Forest "Standard Fiber" containing impuri ties in the form of clay and magne tite. A short fiber. 8 oz. 8-14-63 26-220 *CMS-100 Fiber "Refined Fiber" with lesser impurities than JT-100. A short fiber. Sterling Forest 8 oz. 8-14-63 26-219 A2v03C * The individual fibers for most part had lengths of 0.9 to 10 microns, but there were some as long as 30 microns and some that were less than 0.5 micron. The diameters of the fiber were generally 0.3 to 1.2 microns, but there were smaller sizes noted. The clay-magnetite particles were mostly 0.5 to 3.0 microns in diameter but there were some present as small as 0.08 micron (by electron microscope). Desienation of Asbestos Product JM-3K Fiber. "Long Fiber" (Brushed through screen to reduce fiber length to 1 mm. or less before injecting.) Source Manville, N.J. Report 29-55 Page 2 Quantitv Date Received M. I. Sample Number 8 oz. 8-26-63 26-227 Intraperitoneal Injections Methods The JM-3K asbestos was brushed through a 1 mm. screen to reduce its fiber length to < 1000 micra (1.0 mm.) in order to facilitate dosing. The CMS-100 and JT-100 asbestos samples were used as received. Each asbestos product was pre pared as a 2.5% (W/V) suspension in 0.85% saline. All needles, syringes and suspensions were sterilized prior to use. The animals were divided into three groups--each consisting of eleven male albino guinea pigs (P), six male albino rats (R), and two male albino New Zealand rabbits (H). The guinea pigs and rab bits received intraperitoneal injections of 2 ml. (50 mg.) and the rats, 1 ml. (25 mg.) of the respective asbestos suspensions. Eleven guinea pigs and six rats received control intraperitoneal injections of saline. There were no rabbit controls. All injections were made in the lower right quadrant of the abdomen. Tables 29-92 through 29-95 contain the results on individual animals. Results CMS-100 produced the most severe reaction in the form of granulomas with giant cells in six of the seven guinea pigs examined micropathologically. Typical granulomas with giant cells were observed in three of four rats and the one rabbit. JT-100 asbestos fiber produced a granulomatous reaction in all of the 8 guinea pigs as well as the one rat subjected to micropathological examination. The rabbits had incidental lesions unrelated to the asbestos fibers. The JM-3K asbestos fibers produced granulomas with fibers visible in five of the five guinea pigs examined. One of two rats had granuloma formation which was visible on gross examination. Methods Intratracheal Injections One per cent (W/V) suspensions of JT-100, CMS-100 JM-3K and a 0.5% suspension of CMS-100 were prepared in 0.85% saline as for the intraperitoneal injections. A 2 < 0 3 3 Report 29-55 Page 3 Female albino rats weighing in excess of 200 grams were given 1 ml. of the respective asbestos suspensions by intratracheal injection. The rats were anesthetized with saturated vapor of anhydrous diethyl ether. The trachea was exposed by blunt dissection and the injection was made directly.and the incision closed with a Michael Wound Clamp. Eight rats were injected with 1 ml. of a 1% JT-100 suspension (10 mg. asbestos). Eight rats were injected with 1 ml. of a 1% JM-3K suspension (10 mg. asbestos). Because of the flocculent properties of CMS-100, only three rats were dosed with 1 ml. of a 1Z suspension (10 mg. asbestos). Eight rats were injected with 1 ml. of a 1% CMS-100 suspension (5 mg. asbestos). Six rats injected with physiological saline served as controls. Tables 29-96 through 29-99 contain results on individual animals. Results Specific lesions were found in most of the rats given CMS-100 and JT-100 that consisted of marked granuloma formation with bluish foreign body material present (magnetite?). Marked bronchiolitis obliterans was noted in most rats, also with bluish foreign body material within the lesions. The JT-100 rats had a more severe fibrotic reaction to this foreign material. Multinucleate giant cells were a common feature with the granulomas of the JT-100 rats but were conspicu ously absent in the lungs of the rats given CMS-100. Micropathology was performed on only two rats receiving JM-3K asbestos. Neither of these animals had lesions Rimbr to JT-100 or CMS-100. (? Edwin R. Kinkead, B.S. Research Associate Approved: Acknowledgments: Technical Assistance Pathological Examination Data in Book 868, pp. 63 to 90 and 99-109. Typed: July 11, 1966 - aid Urbano C. Pozzani, M^ Senior Fellow Charles P. Carpenter, Assistant Administr^f Fellow Charles C. Haun, B.S., Junior Fellow John M. King, Ph.D., DVM, Fellow A2h04C Animal Dose of Asbestos Number Intraperitoneal Injections of Asbestos CMS-100 Days to Death or Sacrifice Gross Pathology Micropathology 50 mg. (2 ml. of 2.5% suspension) P. 96813 P. 96803 P. 96815 P. 96806 P. 96802 20 D 75 D 240 D 270 D 286 S P." 96807 286 S P. 96810 286 S P. 96811 286 S P. 96812 286 S P. 96814 286 S P. 96821 286 S Peritonitis; asbestos scattered throughout abdominal cavity. Peritonitis; adhesions of intestines to liver. Autolyzed. No asbestos in abdominal cavity. Multiple small adhesions of the omentum to the mesentery. Adhesions of liver to stomach. Deposits scattered in the intestinal mesentery. Omentumal adhesions to liver with deposits in the omentum. Intestinal adhesions to midline and seminal vessels. Areas of fibrosis in most of left liver lobes. Adhesions--liver to stomach and intestines. Adhesions of abdominal wall to liver, stomach and omentum with deposits. Not examined. Not examined. Not examined. Not examined. Several small, well marked granu lomas with giant cells on the serosal surface of colon. Liver--marked granulomatous re action on the surface. Granulomas--serosal wall. Liver--well marked granulomas. Marked chronic granulomas in the intestine and diaphragm. Intestinal, liver,.skeletal muscle--adhesions to each othe1 Areas of giant cell formation. Colon--large cystic masses fillewith debris. Lined by columns) epithelium.. Liver--fibrosis and granuloma formation. Foreign body granu lomas on abdominal wall. Granulomatous lesions of the stomach, liver and skeletal muscle. H. 96737 H. 96574 60 D 270 S Numerous deposits of asbestos adherred to intestines. Multiple adhesions, ventral colon and urinary bladder. Not examined. Multiple well encapsulated granu lomas. present with innumerable giant cells. (Continued) A2 404", Report 29-55 Page 4 Tab! (Continued) Animal Jose of Asbestos Number ?5 mg. R. 4571 'One ml. of 2.5% ;uspension) R. 4678 R. 4629 R. 4631 R. 4634 R. 4660 Days to Death or Sacrifice Gross Pathology 20 D 170 D 258 S 258 S 258 S 258 S Congested lungs and liver; asbestos scattered throughout abdominal cavity. Asbestos deposits throughout abdominal area. Nodules of white material in mesentery, ventral and abdominal wall. Deposits in the mesentery and ligaments of the testicle. Chronic abscess pneumonia. Scattered deposits in mesentery and wall of small intestines. Adhesion of testes to intestines. Micropathologv Not examined. Not examined. Scattered granulomas in Intestine and skeletal muscle. Multiple granulomas with giant cells and asbestos deposits. Chronic bronchopneumonia. Large granulomas, well encapsulated. 4 04 c~ Report 29-55 Page 5 Intraperitoneal Injections of Asbestos JT-100 Animal Dose of Asbestos Number Days to Death or Sacrifice Gross Pathology Micropatholoev 50 mg. (2 ml. of 2.5% suspension) P. 96844 P. 96851 P. 96849 P. 96834 11 D 150 D 185 D 286 S P. 96835 P. 96837 P. 96839 P. 96840 P. 96841 P. 96847 286 S 286 S 286 S 286 S 286 S 286 S P. 96848 286 S Peritonitis, asbestos scattered throughout abdominal cavity. Entire gut adherred together. Liver adherred to ventral wall and diaphragm. Multiple visceral adhesions of the asbestos to the ventral abdominal wall. Gray deposits in the ventral abdominal wall. Severe multiple adhesions with pus incorporated. Multiple intestinal adhesions. Gray deposits in the abdominal wall. Omental adhesions to the liver with tiny deposits in this mesentery. Spleen aidherred to ventral wall, stomach to diaphragm. Multiple tiny deposits on left liver lobes and left abdominal wall. Omental adhesions to the ventral tips of the liver. Deposits on the intestinal mesentery and abdominal wall with omental adhesions. Not examined. Not examined. Not examined. Scattered foreign body granulomas with slightly elongated fibers of asbestos in them. Typical granulomas. Adherred masses with many granulomas on the serosal surface. Typical connective tissue granulomas. Typical connective tissue granulomas. Numerous granulomas with foreign bodies present in them. Typical granulomas with foreign bodies., giant cells, fibrosis present. Typical connective tissue granulas. H. 96729 H. 96734 270 S 270 S Heart covered by chronic connective tissue. Multiple serosal deposits scattered primarily on the colon and ventral wall. Essentially normal. Essentially normal. i\ 2 rV o1 r> (Continued) Report 29-55 Page 6 Dose of Asbestos Animal Number 25 mg. (One ml. of 2.5Z suspension) R. 4576 R. 4536 R. 4534 R. 4617 R. 4596 Table 29-y3 (Continued) Days to Death or Sacrifice Gross PatholoRv 36 D 160 D 200 D 242 D 258 S Cannibalized. Autolyzed. Peritonitis, pleuritls, some fibrosis in pleural cavity. Pneumonia. Asbestos particles scattered throughout entire abdominal cavity. SljLght deposits in the mesentery and omentum. MicropatholoRY Not examined. Not examined. Not examined. Not examined. Intestine--typical granulomas with foreign bodies. Report 29-55 Page 7 Intraperitoneal Injections of Asbestos JM-3K Animal Dose of Asbestos Number 50 mg. (2 ml. of 2.5% suspension) P. 96829 P. 96833 P. 96830 P. 96826 P. 96828 P. 96832 P. 96822 P. 96823 fO -r* o P. 96824 V' P. 96827 P. 96831 Days to Death or Sacrifice Gross Pathology MicropatholoRy 18 D 43 D 64 D 76 D 78 D 86 D 236 S 286 S 286 S 286 S 286 S Peritonitis, asbestos adherred to ven tral wall and small intestine. Intestines adherred to ventral wall. Scattered adhesions to ventral abdominal wall. Peritonitis, liver and stomach adhferred to ventral abdominal wall. Peritonitis, asbestos scattered through out abdominal cavity. Intestines adherred to ventral abdominal wall. Abscessed at point of adhesion. Multiple adhesions of the liver to the stomach, omentum, and intestines. Deposits on right seminal vessel. Greenish deposits in the ventral abdominal wall and omentum with adhesions to liver. Midline omental adhesions to greenish deposits. Liver adherred to ventral abdominal wall. Not examined. Not examined. Not examined. Not examined. Not examined. Not examined. Foreign bodies. Granuloma attached to intestinal wall. Some granu lomas in liver. Seminal vessel--granulomatous re action with foreign material in soft tissue and mesentery. Foreign body granulomas with foreign material present. Deposits of foreign material and large granulomas on muscle. Tip of liver infectious; abscess formation, granuloma formation. R eport 29-55, Page 8 H. 96764 H. 96697 25 mg. R. 4628 (One ml. of 2.5% suspension) R. 4622 R. 4623 R. `4615 R. 4578 R. 4583 60 D 270 S 35 D 230 D 234 D 241 D 258 S 258 S No asbestos in peritoneal cavity. Asbestos at site of injection. Not examined. Essentially normal. Pneumonia. Intestine adherred to the ventral wall. Pasturella Pneumonia. Intestines adherred to abdominal wall with each other. Autolyzed. Pasturella Pneumonia. Adhesions of the omentum and mesentery. Middle ear infection. Not examined. Not examined. Not examined. Not examined. Essentially normal. Essentially normal. Table 29-95 Intraperitoneal Injections of Saline Control Dose of Saline 2 ml. Animal Number P. 96857 P. 96856 P. 96853 P. 96858 P. 96852 P. 96859 P. 96860 P. 96861 P. 96862 P. 96863 P. 96864 Days to Death or Sacrifice Gross Pathology 88 D 156 D 170 D 207 D 286 S 286 S Pneumonia. Pneumonia. Pneumonia. Pneumonia. Essentially normal. Essentially normal. 286 S 286 S 286 S 286 S 286 S Emaciated. Liver massively reduced in size. Essentially normal. Essentially normal. Chronic nephritis in both kidneys. Essentially normal. One ml. R. 4484 R. 4483 R. 4531 R. 4540 R. 4542 R. 4557 12 D 258 S 258 S 258 S 258 S 258 S Pneumonia. Chronic abscessed pneumonia. Essentially normal. Essentially normal. Essentially normal. Essentially normal. MicronatholoEV Not examined.. Not examined. Not examined. Not examined. Essentially normal. Scattered areas of interstitial pneumonia. Liver necrosed and abscessed. Essentially normal. Essentially normal. Chronic interstitial nephritis. Essentially normal. Not examined. Massive abscessed pneumonia. Essentially normal. Essentially normal. Essentially normal. Essentially normal. A 2 A o; c Report 29-55 Page 9 Table 29-96 Intratracheal Injections of Asbestos CMS-100 to Rats Rat Dose of Asbestos Number 10 mg. (One ml. of 1% suspension) 84636 84645 Days to Death or Sacrifice 30 S Pneumonia. 90 S Pneumonia. Gross Pathology 84702 180 S Chronic pneumonia. Micropathologv Pneumonia, numerous granulomas scattered throughout lung. Pneumonia, lesions of bronchiolitis obliterans in several bronchi` and bronchioles..' Scattered granulomas; lesions of broncfiiolitis obliterans with foreign material and thromboticlike areas. 5 mg. (One ml. of 0.5% suspension) 72531 81427 81433 81440 72528 77679 77680 81600 12 D 30 S Cannibalized. Lung--appeared normal. 90 S No lesions. 90 S 180 S 180 S 180 S 180"S Chronic pneumonia. Not recorded. Lipid foci in lungs. Gray foci in lungs. Pneumonia. Not examined. Multiple granulomas; bluish ma terial present in all granu lomas . Scattered small granulomas. Some bronchiolitis obliterans also apparent. Pneumonia; scattered granulomas; bronchiolitis obliterans. Scattered small granulomas. Bronchiolitis obliterans. Chronic granulomas sans giant cells. Numerous scattered granulomas. Multiple granulomas scattered throughout lung. Bronchiolitis obliterans. A24CU7 Report 29-55 Page 10 Table 2. Intratracheal Injections of Asbestos JT-100 to Rats Rat Dose of Asbestos Number 10 mg. (One ml. of 1% suspension) 84629 84672 84669 84633 84659 Days to Death or Sacrifice Gross Pathology 60 D 123 D 151 D 30 S Large white areas on lung; atelectasis of the right lung. Pneumonia. Pasturella Pneumonia. Chronic pneumonia. 30 S Chronic pneumonia. 84674 84676 84631 90 S 90 S 180 S Pneumonia. Chronic abscessed pneumonia. Not recorded. Micropatholoev Bronchiectasis. Areas of acute bronchopneumonia. Not examined. Not examined. Multiple granulomas and bronchi olitis obliterans. Pneumonia; granulomas, large lesions of bronchiolitis obliterans. Numerous granulomas, well marked bronchiolitis obliterans. Scattered granulomas. Granulomas, some bronchiolitis obliterans. A24048 Table 29-98 Intratracheal Injections of Asbestos JM-3K to Rats Dose of Asbestos 10 mg. (One ml. of 1% suspension) Rat Number 84695 84635 81214 81171 81161 84660 Days to Death or Sacrifice Gross Pathology 10 D 13 D 15 D 50 D 30 S 180 S Entire lung was dark red. Chronic pneumonia. Pneumonia and emphysema. Chronic pneumonia; bronchial lymph nodes, enlarged and gray. Chronic pneumonia. Chronic pneumonia and enlarged lymph nodes. Micropatholoev Not examined. Not examined. Not examined. Not examined. Chronic bronchopneumonia. Chronic bronchopneumonia, a few scattered granulomas. Report 29-55 Dose of Saline One ml. Table 29-99 Intratracheal Injections of Saline to Rats Rat Number 78253 78273 78302 78717 84639 84646 8*651 84677 Days to Death or Sacrifice Gross PatholoEv 30 S 30 S 90 S 90 S 180 S 180 S 180 S 180 S Essentially normal. Essentially normal. Essentially normal. Azygous ibscessed. Essentially normal. Essentially normal. Essentially normal. Essentially normal. mr-tm Micropatholoev Essentially normal. Essentially normal. Essentially normal. Few areas of chronic pneumonia. Essentially normal. Essentially normal. Essentially normal. Essentially normal. A24049 Report 29-55 Page 12