Document B82QJz6dM07gBR8Ebb6G9ark4

TR--282-1600 IB**. 2) JULY 1976 PROCEDURES FOR THE I PLAINTIFF'S" J exhibit ASA-103; BIOLOGICAL EVALUATION OF ASBESTOS AND ASBESTOS-LIKE PARTICLES Research Trian^e Park North Carolina 2E2 UNDER CONTRACT NO. 68-02-1567 - ' i- - - Submitted By: -- NORTHROP SERVICES, INC. P. 0. BOX 1484 Huntsville, Alabama 35807 (205) 837-0580 HER 0001315 *1 . Reproduced by the AMERICAN MINING CONGRESS cr r TR-1600 o PROCEDURES FOR THE BIOLOGICAL EVALUATION OF AS8ESTOS AND ASBESTOS-LIKE PARTICLES (REVISED) (* July 1976 by Dr- Lalita D. Palekar PREPARED FOR: ENVIRONMENTAL PROTECTION AGENCY NATIONAL ENVIRONMENTAL RESEARCH CENTER RESEARCH TRIANGLE PARK, N.C. Under Contract 68-02-1567 REVIEWED ANO APPROVED BY: Health Effects Research Stanley G. Colfiff, Program wranager Environmental Research Support (kL. A. L. Grady, Manager Environmental Sciences Group NORTHROP SERVICE, INC. HUNTSVILLE, ALABAMA HER 0001316 NORTHROP SERVICES, INC TR-1600 FOREWORD This report was prepared in response to a request from the Health Effects Research Laboratory of the Environmental Protection Agency (EPA). Included is a detailed plan of the research objectives and procedures to be followed in the accomplishment of Work Order 1.9 under EPA contract 68-02-1567. This protocol was reviewed on July 8, 1976, at the New York Academy of Sciences, New York, by the EPA Ad Hoc Committee of the following participants: Dr. David L. Coffin Environmental Protection Agency Health Effects Research Laboratory MD-52 Research Triangle Park, N. C. 27711 Dr. John Moore National Institutes of Environmental Sciences P. 0. Box 12233 Research Triangle Park, N. C. 27711 Ms. Vandy P. Duffield Environmental Protection Agency Health Effects Research Laboratory MD-83 Research Triangle Park, N. C. 27711 Dr. Colin Harwood IIT Research Institute 10 West 35th Street Chicago, Illinois 60616 Dr. Marvin Kuschner State University of New York Department of Pathology Health Sciences Center Stoney Brook, New York 11790 Dr. Arthur M. Langer Environmental Medicine Mt. Sinai School of Medicine City University of New York 100th Street & 5th Avenue New York, New York 10029 Mr. Fred Miller Environmental Protection Agency Health Effects Research Laboratory MD-55 Research Triangle Park, N. C. 27711 Dr. Lalita D. Palekar Northrop Services, Inc. P. O. Box 12313 Research Triangle Park, N. C. 27711 Dr. Irving J. Selikoff Environmental Medicine Mt. Sinai School of Medicine City University of New York 100th Street & 5th Avenue New York, New York 10029 Dr. Raymond Shapiro National Institutes of Environmental Sciences P. 0. Box 12233 Research Triangle Park, N. C. 27711 Mr. Jack Wagman Environmental Protection Agency Environmental Sciences Research Laboratory MD-59 Research Triangle Park, N. C. 27711 OO^7 ii "I l NORTHROP SERVICES, INC. TR-1600 ABSTRACT This report provides a detailed plan of the research objectives and operating procedures to be followed in future studies on the biological eval uation of asbestos and asbestos-like particles. Included in this report is background information on asbestos and asbestos toxicity, research objectives, capabilities of the research facility, and detailed laboratory operating procedures. iii HER 0001318 NORTHROP SERVICES, INC. TR-1600 occurring in human cases of asbestos mesothelioma and squamous cell carcinoma of the lung, can be induced in numerous species of animals. Therefore, a numbe of species were considered as research animals. The species determined most acceptable for such experimentation is Caesarean-derived (C/D) Wistar rat strains which are free of chronic murine pneumonia and which are kept under barrier controlled conditions. 2.5.2 Experimental Methods and Procedures Six-weeks-old male rats of Caesarean-originated, barrier sustained Fisher strain will be supplied. Upon arrival, the bulk of an order of rats will be housed in a receiving area in an isolator. From each order, five randomly picked rats will be killed and autopsied to determine whether the animals are disease free. In addition, microbial and viral quality control will be estab lished. Once the shipment is satisfactorily cleared, treatment will commence. 2.5.2.1 Preparation of Test Samples. Preweighed test samples will be received in sterile glass tubes. Since the supplier will be collecting the particles aseptically, the samples will not be sterilized upon arrival. However, the sterility of samples will be ascertained by conventional microbiological andviral assays. Immediately before use, sterile physiological saline will be added to the tubes to give concentrations of 2 milligrams per 0.2 milliliter. Care will be taken to avoid settling of the particles which might result in inaccurate dosage. 2.5.2.2 Preparation of Benzo(a)Pyrene Solution. Colloidal suspensions will be made of benzo(a)pyrene (BaP) in physiological saline at concentrations of 3 milligrams per 0.2 milliliter under sterile conditions. The suspension will be stirred continuously to keep BaP particles from settling. 2.5.3 Experimental Design 2.5.3.1 Procedure for Intratracheal Instillation. Before each treatment, the animals will be anesthetized with intraperitoneal injections of 0.4 milliliter of a 1-percent solution of sodium methohexital (Brevital). The abdominal wall will be disinfected with 70-percent ethanol. After the anesthetic is effected, 2-11 |. ' HER 0001332 NORTHROP SERVICES, INC. TR-1600 the rat will be transferred to a hood and placed on a specially designed plat form. Here, the animal's mouth will be opened and the rat will be fixed to the platform by passing a rubber band through the lower incisors and fixing the band to the lower side of the board. The upper incisors will be fixed to the upper side of the board in the same manner. A selected volume of the test substance will then be drawn into a 1-cubic-centimeter tuberculin syringe with a blunt 19-gauge needle bent at a 135 angle. Special illumination in the hood will provide direct beam into the mouth, giving a clear view of the pharynx. The needle will be inserted into the tracheal lumen until a slight resistance is felt. The contents of the syringe will be discharged into the lungs and the animal will be removed from the platform and put into a cage. 2.5.3.2 Intratracheal Experiments. A series of intratracheal experiments is designed to provide information concerning the carcinogenic potential of PMP amphibole alone as well as to evaluate the differences between the synergistic ctrnosHe. effects of PMP amphibole, UICC e&fijewfei&e, and iron oxide particle with BaP. Series I -- Initial Range Finding Study. The dose regimen for Series II will be determined by this series of experiments. Ten animals will receive either 0.5, 1.0, or 2.0 milligrams of the test substance once a week for 12 weeks. The animals will be observed very carefully for any change in their general health and any weight loss for 3 months. After this period, the ani mals will be sacrificed and their pathology will be determined. In case of a severe toxicity, the dose regimen will be reduced by individ ual dose and number of injections. The highest nontoxic dose will be determined. Series II -- Chronic Intratracheal Testing of PMP Amphibole. *_imen will be determined from Series I. I * I. Unknown Sample - PMP Amphibole - 600 Animals II. Asbestos Control - UICC Amosite - 200 Animals III. Negative Control - Saline and Gel - 200 Animals The dose reg- Series III -- Chronic Interaction Studies by Intratracheal Instillations. 59*62 The synergistic effects of chrysotile and BaP have been demonstrated. 2-12 HER 0001333 NORTHROP SERVICES, INC. TR-1600 Shabad et al demonstrated higher incidence of tumors in rats induced by mix ing BaP and India ink powder than from BaP alone. Saffiotti et al7^ also induced lung tumors in hamsters by injecting iron oxide and BaP. Blair 45 has demonstrated similar results on rats. The results of these studies and others of a similar nature led to speculation that it was the presence of particles and not the nature of the particle which contributed to the cocarcinogenic effect. Miller et al 59 , however, pointed out that while chrysotile demonstrated enhanced carcinogenic activity with BaP, amosi,te did not. These results have led to the suggestion that the synergistic effect between asbestos and a carcin ogen may be property of specific types of asbestos. The dose regimen for this series will be 3 milligrams of particle and 3 milligrams of BaP in 0.2-milliliter saline plus gel per week for 5 weeks. This dose is extrapolated from Blair's data to achieve 40-percent tumor incidence in the iron oxide plus BaP group. I. II. III. IV. V. VI. VII. PMP Amphibole plus BaP - 300 Animals UICC Amphibole plus BaP - 300 Animals Iron Oxide plus BaP - 300 Animals PMP Amphibole - 200 Animals UICC Amphibole - 200 Animals Iron Oxide - 200 Animals BaP - 200 Animals Statistical Consideration. A sample size of animals per group is adequate to detect differences between tumor incidence rates that equal or exceed 15 per cent, provided that the lowest incidence rate aJnong the three series is at least 10 percent. These calculations are based on controlling Type I and Type II er rors at 0.05 and 0.20, respectively. A Type I error arises when the researcher declares that the difference is real when, in fact, this difference is zero. On the other hand, a Type II error consists of failing to declare the tumor rates significantly different when, in fact, they are different. A sample size of 600 in Series I is adequate to detect tumor incidence rate as low as 0.16 percent. A general consideration in the statistical analysis of the results in volves determining the number of animals considered to be at risk. Only those animals surviving for a period of time greater than, or equal to, the time of appearance of the first tumor will be included in the final analysis. The 2-13 *1 ' t her 0001334 NORTHROP SERVICES, INC. TR-1600 animals that die early in a study from infectious diseases or other causes will be excluded. 2.5.3.3 Intrapleural Experiment. Tumors of mesothelial origin have been pro duced by many investigators by intrapleural injection of various preparations of particulate matter into experimental animals. ^ Although introduction of particles directly into the pleural space does not reproduce natural route of exposure to inhaled substances, this technique provides a convenient procedure for screening several particulate substances for their relative activity in the production of mesothelioma of the pleura. 2.5.3.4 Procedure for Intrapleural Injection. Rats will be anesthetized by intraperitoneal injection of 0.4 milliliter of a 1-percent solution of sodium methohexital (Brevital) as described earlier. The left thoracic wall will then be shaved properly and disinfected with 70-percent ethanol. A special 18-gauge needle with a blunt hollow trochar is required for this procedure. The needle will be introduced through the thoracic wall of the rat. Once the wall is punc tured, the trochar will be pushed inside and the lung will be pushed aside. The needle will then be pushed farther into the thoracic cavity and the fluid inside the syringe containing the test particles will be discharged into the pleural cavity. After innoculation, the needle wili be retracted from the thoracic cavity and the trochar will be removed. Series IV -- Chronic Intrapleural Testing of PMP Particles. The dose reg imen, will be a single injection of 20 milligrams of the test substance in 0.5milliliters of physiological saline. I. II. III. Unknown Sample - PMP Amphibole - 150 Animals Asbestos Control - UICC Amosite - 150 Animals Negative Control - Saline - 150 Animals Statistical Consideration. A sample size of 150 animals per group is sufficient to duplicate the findings of Wagner and Berry69 with respect to intrapleural injection of various amphibole and chrysotile particles. Again, the Type I and Type II errors are controlled at 0.05 and 0.20, respectively. Untreated Controls. Two Hundred animals from Group III of Series II and 150 animals from Group ill of Series IV will be considered as controls for nett 2-14 le fti HER 0001335 NORTHROP SERVICES, INC. TR-1600 pulmonary tumors. In addition, 250 animals will be maintained in the facility without any treatment. 2.6 LABORATORY OPERATIONS All experiments will be conducted throughout the life span of the animals. The rats will be maintained in a specially designed microbiological barrier which is described in Section III. The three groups (namely, control, PMP par ticles, and asbestos particles) will be housed in separate rooms. Racks, tables, and various other items will be placed in the barrier and will be sterilized with formaldehyde gas before the animals arrive. All items entering the facility af ter the initial sterilization of the barrier will be sterilized by one of the sev eral methods which, are described in Section IV. The animals will be fed autoclaved NIH Formula 31 and sterile water ad lihitim and will be housed in a see-through suspended cage system with an automatic water supply. The cage system will consist of solid-bottom, poly styrene cages suspended in stainless steel racks. This system is equipped with a rear manifold with custom valves specially designed to avoid any water leakage within a cage. Water will be supplied by gravity from a 5-gallon stainless steel tank. This system will also have a special attachment to provide a water bottle if special treatment is required for the animals. The animals will be checked every day. During the treatment period, they will be weighed once a week and subsequently once a month. When each animal dies, it will be autopsied, and its trachea and lungs will be removed and immersed in 10-percent buffered formalin. Other organs (such as liver, kidney, and gastrointestinal tract) will also be examined, and the samples will be preserved in 10-percent formalin. These sections will be sent out to a commercial histology laboratory for processing. Pathological evaluation will then be made on these slides. 2-15 *1 HER 0001336 NORTHROP SERVICES, INC. TR-1600 2.7 REFERENCES 1. Bragg, w. L. and Claringbull, G. F., "Crystal Structure of Minerals," Cornell University Volume IV, p. 226, (1965). 2. Murray, H. M., "Report of the Departmental Committee for Industrial Diseases," HM Stationary Office, London, p. 127, (1907). 3. Vigliani, E. C., Med. Lavoro 5:401, (1968). 4. Meurman, L. O., Acta. Pathol. Microbiol. Scand., (Suppl) 181, p. 327, (1966). 5. Meurman, L. O. and Kiviluoto, R., Int. Conf. Biol. Effects Asbestos, 2nd Dresden, p. 135, (1968). 6. 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