Document 4QazQQ7exzXBYXx5v5gwOkD01

NATIONAL TOXICOLOGY PROGRAM Technical Report Series No. 421 TOXICOLOGY AND CARCINOGENESIS STUDIES OF TALC (CAS NO. 14807-96-6) IN F344/N RATS AND B6C3F! MICE (INHALATION STUDIES) U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service National Institutes of Health FOREWORD The National Toxicology Program (NTP) is made up of four charter agencies of the U.S. Department of Health and Human Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health; the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health; the National Center for Toxicological Research (NCTR), Food and Drug Administration; and the National Institute for Occupational Safety and Health (NIOSH), Centers for Disease Control. In July 1981, the Carcinogenesis Bioassay Testing Program, NCI, was transferred to the NIEHS. The NTP coordinates the relevant programs, staff, and resources from these Public Health Service agencies relating to basic and applied research and to biological assay development and validation. The NTP develops, evaluates, and disseminates scientific information about potentially toxic and hazardous chemicals. This knowledge is used for protecting the health of the American people and for the primary prevention of disease. The studies described in this Technical Report were performed under the direction of the NIEHS and were conducted in compliance with NTP laboratory health and safety requirements and must meet or exceed all applicable federal, state, and local health and safety regulations. Animal care and use were in accordance with the Public Health Service Policy on Humane Care and Use of Animals. The prechronic and chronic studies were conducted in compliance with Food and Drug Administration (FDA) Good Laboratory Practice Regulations, and all aspects of the chronic studies were subjected to retrospective quality assurance audits before being presented for public review. These studies are designed and conducted to characterize and evaluate the toxicologic potential, including carcinogenic activity, of selected chemicals in laboratory animals (usually two species, rats and mice). Chemicals selected for NTP toxicology and carcinogenesis studies are chosen primarily on the bases of human exposure, level of production, and chemical structure. Selection per se is not an indicator of a chemical's carcinogenic potential. These NTP Technical Reports are available for sale from the National Technical Information Service, U.S. Department of Commerce, 5285 Port Royal Road, Springfield, VA 22161 (703-487-4650). Single copies of this Technical Report are available without charge while supplies last from the NTP Central Data Management, NIEHS, P.O. Box 12233, MD AO-01, Research Triangle Park, NC 27709 (919-541-1371). NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF TALC (CAS NO. 14807-96-6) IN F344/N RATS AND B6C3F! MICE (INHALATION STUDIES) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 September 1993 NTP TR 421 NIH Publication No. 93-3152 U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service National Institutes of Health 2 Talc, NTP TR 421 CONTRIBUTORS National Toxicology Program Evaluated and interpreted results and reported findings K.M. Abdo, Ph.D. C. J. Alden, Ph.D. G.A. Boorman, D.V.M., Ph.D. D. A. Bridge, B.S. M. Dieter, Ph.D. S. L. Eustis, D.V.M., Ph.D. T. J. Goehl, Ph.D. R.A. Griesemer, D.V.M., Ph.D. J. K. Haseman, Ph.D. R.D. Irwin, Ph D. G.N. Rao, D.V.M., Ph.D. K. L. Witt, M.S., Oak Ridge Associated Universities Lovelace Biomedical and Environmental Research Institute Conducted studies, evaluated pathology findings C.H. Hobbs, D.V.M., Principal Investigator E. B. Barr, M.S. J.M. Benson, Ph.D. N. Gillette, D.V.M., Ph.D. F. F. Hahn, D.V.M., Ph.D. R.K. Jones, M.D. J.L. Mauderly, D.V.M. J.A. Pickrell, D.V.M., Ph.D. Experimental Pathology Laboratories, Inc. Provided pathology quality assurance J.F. Hardisty, D.V.M., Principal Investigator Integrated Laboratory Systems Prepared quality assurance audits S.L. Smith, J.D., Principal Investigator Biotechnical Services, Inc. Prepared Technical Report D.D. Lambright, Ph.D., Principal Investigator P. Chaffin, M.S. G.F. Corley, D.V.M. C.J. Fitz, M.A. A.B. James-Stewart, B.S. NTP Pathology Working Group Evaluated slides, prepared pathology report on rats 14 October 1990) D.G. Goodman, V.M.D., Chair PATH CO, Inc. G.A. Boorman, D.V.M., Ph.D. National Toxicology Program S.L. Eustis, D.V.M., Ph.D. National Toxicology Program J.R. Hailey, D.V.M. National Toxicology Program J.F. Hardisty, D.V.M. Experimental Pathology Laboratories, Inc. J.F. Mahler, D.V.M. National Toxicology Program M.M. McDonald, D.V.M., Ph.D. National Toxicology Program M. Menard, D.V.M. North Carolina Stale University (observer) A. Pinter, M.D., Ph D. National Institute of Hygiene, Hungary C. Van Pelt, D.V.M., Ph.D. E.I. DuPont De Nemours Evaluated slides, prepared pathology report on mice (18 October 1990) M.A. Stedham, D.V.M., Chair PAI G.A. Boorman, D.V.M., Ph.D. National Toxicology Program J.F. Hardisty, D.V.M. Experimental Pathology Laboratories, Inc. C.R. Jeng, B.V.M. North Carolina State University (observer) A.W. Macklin, D.V.M., Ph.D. Burroughs Wellcome M.M. McDonald, D.V.M., Ph.D. National Toxicology Program C.C. Shackelford, D.V.M., M.S., Ph.D. National Toxicology Program 3 CONTENTS ABSTR ACT.................................................................................................................................................................... 5 EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY................................... 9 TECHNICAL REPORTS REVIEW SUBCOMMITTEE.................................................................................... 10 SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS ......................... 11 INTRODUCTION ....................................................................................................................................................... 13 MATERIALS AND METHODS .............................................................................................................................. 19 RESULTS ....................................................................................................................................................., .............. 25 DISCUSSION AND CONCLUSIONS..................................................................................................................... 49 REFERENCES ............................................................................................................................................................ 57 Appendix A Summary of Lesions in Male Rats in the Lifetime Inhalation Study of Talc ............................................................................................................................................. 63 Appendix B Summary of Lesions in Female Rats in the Lifetime Inhalation Study of Talc ............................................................................................................................................. 95 Appendix C Summary of Lesions in Male Mice in the 2-Year Inhalation Study of Talc ............................................................................................................................................. 129 Appendix D Summary of Lesions in Female Mice in the 2-Year Inhalation Study of Talc ............................................................................................................................................. 161 Appendix E Organ Weights and Organ-Weight-to-Body-Weight R a tio s ................................................. 193 Appendix F 4-Week Repeated Inhalation Studies in Rats and M i c e ...................................................... 203 Appendix G Lung Burden, Pulmonary Function, and Lung Biochemistry in R a t s ............................. 215 Appendix H Lung Burden and Lung Biochemistry in M ic e ....................................................................... 243 Appendix I Chemical Characterization, Analysis, and Generation of Chamber Concentrations ...................................................................................................... 261 Appendix J Ingredients, Nutrient Composition, and Contaminant Levels in NIH-07 Rat and Mouse R a tio n ............................................................................................ 277 Appendix K Sentinel Animal Program ............................................................................................................ 283 4 Talc, NTP TR 421 5 ABSTRACT TALC (Non-Asbestiform) CAS No. 14807-96-6 Molecular Formula: Mg,Si4O 10 (OH)2 Molecular Weight: 379.26 Synonyms: talcum; agalite; emtal 596; non-asbestiform talc; non-fibrous talc; steatite; hydrous magnesium silicate Talc ore may contain several other minerals including calcite, dolomite, magnesite, tremolite, anthophyllite, antigorite, quartz, pyrophyllite, micas, or chlorites. Talc products are sold in a multitude of grades which have physical or functional characteristics especially suited for particular applications, so occupational and consumer exposures to talc are complex. Epidemi ology studies have suggested an association between non-fibrous talc and lung cancer risk. Talc was nominated by the National Institute of Occupational Safety and Health (NIOSH) for study by the NTP because of widespread human exposure and because of the lack of adequate information on its chronic toxicity and potential carcinogenicity. Toxicology and carcinogenicity studies of talc (non-asbestiform, cosmetic grade), a finely powdered hydrous magne sium silicate, were conducted by exposing groups of F344/N rats to aerosols for 6 hours per day, 5 days per week for up to 113 weeks (males) or 122 weeks (females). Groups of B6C3F, mice were exposed similarly for up to 104 weeks. Lifetime Study in Rats Groups of 49 or 50 male and 50 female rats were exposed to aerosols of 0, 6, or 18 mg/m3 talc until mortality in any exposure group reached 80% (113 weeks for males and 122 weeks for females). These exposures were selected based on 4-week inhalation studies of the terminal lung talc burden in F344/N rats; concentrations greater than 18 mg/m3 were expected to overwhelm lung clearance mecha nisms and impair lung function. These exposure concentrations provided a dose equivalent of 0, 2.8, or 8.4 mg/kg per day for male rats and 0, 3.2, or 9.6 mg/kg per day for female rats. In a special study, additional groups of 22 male and 22 female rats were similarly exposed and examined for interim pathology evaluations or pulmonary function tests after 6, 11, 18, and 24 months and lung biochemistry and cytolo gy studies after 24 months. The talc aerosols had a median mass aerodynamic diameter of 2.7 im in the 6 mg/m3chamber and a median diameter of 3.2 /xmin the 18 mg/m5 chamber, with geometric standard deviations of 1.9 g.m. However, there was a 7-week period beginning at study week 11 during which the chamber concentration for the 18 mg/m3 rats varied from approximately 30 to 40 mg/m3 because of difficulties with the aerosol concentration monitoring system. Further, there was a 12-week period begin ning at approximately week 70 during which there were difficulties in generating the talc aerosol, and the chamber concentrations for rats and mice were substantially lower than the target concentrations. Survival, Body Weights, and Clinical Findings The survival of male and female rats exposed to talc was similar to that of the controls. Mean body weights of rats exposed to 18 mg/m3 were slightly lower than those of controls after week 65. No clinical findings were attributed to talc exposure. Pathology Findings Absolute and relative lung weights of male rats exposed to 18 mg/m3 were significantly greater than those of controls at the 6-, 11-, and 18-month interim evaluations and at the end of the lifetime study, while those of female rats exposed to 18 mg/m3 were significantly greater at the 11-, 18-, and 24-month interim evaluations and at the end of the lifetime study. Inhalation exposure of rats to talc produced a spectrum of inflammatory, reparative, and prolifera tive processes in the lungs. Granulomatous inflam mation occurred in nearly all exposed rats and the 6 Talc, NTP TR 421 severity increased with exposure duration and concen tration. Hyperplasia of the alveolar epithelium and interstitial fibrosis occurred in or near foci of inflam mation in many exposed rats, while squamous metaplasia of the alveolar epithelium and squamous cysts were also occasionally seen. Accumulations of macrophages (histiocytes), most containing talc particles, were found in the peribronchial lymphoid tissue of the lung and in the bronchial and mediasti nal lymph nodes. In female rats, the incidences of alveolar/bronchiolar adenoma, carcinoma, and adeno ma or carcinoma (combined) in the 18 mg/m3group were significantly greater than those of controls. The incidences of pulmonary neoplasms in exposed male rats were similar to those in controls. Minor alterations attributed to talc exposure were also observed in the upper respiratory tract. Hyper plasia of the respiratory epithelium of the nasal mucosa in males and accumulation of cytoplasmic, eosinophilic droplets in the nasal mucosal epithelium in male and female rats occurred with a concentra tion-related increased incidence in the exposed groups. Adrenal medulla pheochromocytomas [benign, malignant, or complex (combined)] occurred with a significant positive trend in male and female rats, and the incidences in the 18 mg/m3 groups were signifi cantly greater than those of controls. Although adrenal medulla hyperplasia occurred with similar frequency among exposed and control females, the incidences of hyperplasia in exposed males were significantly lower than in controls. Lung Talc Burden Lung talc burdens of male and female rats exposed to 6 mg/m3 were similar and increased progressively from 6 to 24 months. Lung talc burdens of females exposed to 18 mg/m3 also increased progressively from 6 to 24 months, while those of males exposed to 18 mg/m3remained about the same after 18 months. Lung burdens were generally proportional to expo sure concentration at each interim evaluation. Pulmonary Function, Bronchoalveolar Lavage, and Lung Biochemistry In exposed male and female rats there was a concen tration-related impairment of respiratory function which increased in severity with increasing exposure duration. The impairment was characterized by reductions in lung volume (total lung capacity, vital capacity, and forced vital capacity), lung compliance, gas exchange efficiency (carbon monoxide diffusing capacity), and nonuniform intrapulmonary gas distribution. After 24 months, males exposed to 6 mg/m3 talc had a significant increase in B-glucuronidase and poly morphonuclear leukocytes; males exposed 18 mg/m3 had significant increases in B-glucuronidase, lactate dehydrogenase, alkaline phosphatase, and total protein in bronchoalveolar lavage fluid. All exposed females had significantly increased B-glucuronidase, lactate dehydrogenase, alkaline phosphatase, total protein, and polymorphonuclear leukocytes; 18 mg/m3 females also had significantly increased glutathione reductase. Viability and phagocytic activity of macro phages recovered from lavage fluid were not affected by talc exposure. Total lung collagen was significantly increased in rats at both exposure concentrations after 24 months, while collagenous peptides in lavage fluid and the percentages of newly synthesized protein from females, but not males, were also significantly increased at the 6 or 18 mg/m3 levels. In addition, lung proteinase activity, primarily cathepsin D-like activity, was significantly greater in exposed males and females. Rats exposed to talc also had significant increases in collagenous peptides and acid proteinase in lung homogenates. 2-Year Study in M ice Groups of 47 to 49 male and 48 to 50 female mice were exposed to aerosols containing 0,6, or 18 mg/m3 talc for up to 104 weeks. These exposures were selected based on 4-week inhalation studies of the terminal lung talc burden in B6C3F! mice; concentra tions greater than 18 mg/m3 were expected to over whelm lung clearance mechanisms and impair lung function. These exposure concentrations provide a dose equivalent of 0, 2, or 6 mg/kg per day for male mice and 0,1.3, or 3.9 mg/kg per day for female mice. In a special study, additional groups of 39 or 40 male and 39 or 40 female mice similarly exposed were examined for interim pathology evaluations, lung biochemistry, and cytology studies after 6, 12, and 18 months of exposure. The talc aerosols had a median mass aerodynamic diameter of 3.3 tm with a geometric standard deviation of 1.9 fim in the 6 mg/m3chamber, and a median diameter of 3.6 im with a geometric standard deviation of 2.0 fim in the Talc, NTP TR 421 7 18 mg/m3 chamber. Further, there was a 12-week period beginning at approximately week 70 during which there were difficulties in generating the talc aerosol, and the chamber concentrations for rats and mice were substantially lower than the target concen trations. Survival, Body Weights, and Clinical Findings Survival and final mean body weights of male and female mice exposed to talc were similar to those of the controls. There were no clinical findings attribut ed to talc exposure. Pathology Findings Inhalation exposure of mice to talc was associated with chronic active inflammation and the accumu lation of macrophages in the lung. In contrast to rats, hyperplasia of the alveolar epithelium, squamous metaplasia, or interstitial fibrosis were not associated with the inflammatory response in mice, and the incidences of pulmonary neoplasms in exposed and control groups of mice were similar. Accumulations of macrophages (histiocytes) containing talc particles were also present in the bronchial lymph node. In the upper respiratory tract, cytoplasmic alteration, consisting of the accumulation of cytoplasmic eosino philic droplets in the nasal mucosal epithelium, occurred with a concentration-related increased incidence in exposed male and female mice. Lung Talc Burden Lung talc burdens of mice exposed to 6 mg/m3were similar between males and females and increased progressively from 6 to 24 months, except for males at 18 months. The lung talc burdens of mice exposed to 18 mg/m3 were also similar between the sexes at each interim evaluation. Although the talc burdens of males and females increased substantially from 6 to 24 months, the values at 12 and 18 months were similar. Generally, lung burdens of mice exposed to 18 mg/m3were disproportionately greater than those of mice exposed to 6 mg/m3, suggesting that clearance of talc from the lung was impaired, or impaired to a greater extent, in mice exposed to 18 mg/m3 than in mice exposed to 6 mg/m3. Bronchoalveolar Lavage and Lung Biochemistry Increases in total protein, C-glucuronidase, lactate dehydrogenase, glutathione reductase, total nucleated cells, and polymorphonuclear leukocytes in broncho alveolar lavage fluid were observed primarily in mice exposed to 18 mg/m3, although some parameters were also increased in mice exposed to 6 mg/m3. The amount of collagenous peptides in lavage fluid and total lung collagen were increased in male and female mice exposed to 18 mg/m3. Acid proteinase activity, principally cathepsin D-like activity, of lung homogenate supernatant fluid was also significantly increased in mice at the 18 mg/m3 exposure concen tration. Conclusions Under the conditions of these inhalation studies, there was some evidence of carcinogenic activity* of talc in male F344/N rats based on an increased inci dence of benign or malignant pheochromocytomas of the adrenal gland. There was clear evidence of carcinogenic activity of talc in female F344/N rats based on increased incidences of alveolar/bronchiolar adenomas and carcinomas of the lung and benign or malignant pheochromocytomas of the adrenal gland. There was no evidence of carcinogenic activity of talc in male or female B6C3F, mice exposed to 6 or 18 mg/m3. The principal toxic lesions associated with inhalation exposure to the same concentrations of talc in rats included chronic granulomatous inflammation, alveolar epithelial hyperplasia, squamous metaplasia and squamous cysts, and interstitial fibrosis of the lung. These lesions were accompanied by impaired pulmonary function characterized primarily by re duced lung volumes, reduced dynamic and/or quasi static lung compliance, reduced gas exchange efficien cy, and nonuniform intrapulmonary gas distribution. In mice, inhalation exposure to talc produced chronic inflammation of the lung with the accumulation of alveolar macrophages. Explanation of Levels of Evidence of Carcinogenic Activity is on page 9. A summary of Technical Reports Review Subcommittee comments and the public discussion on this Technical Report appears on page 11. 8 Talc, NTP TR 421 Summary of the Lifetime and 2-Year Carcinogenicity Studies of Talc M ale F344/N Rats Fem ale F344/N Rats M ale B6C3F, M ice Exposure levels Body weights Survival rates N onneoplastic effects N eoplastic effects Level o f evidence o f carcinogenic activity 0, 6, or 18 mg/m3 (equivalent to 0, 2.8, or 8.4 m g /k g per day) 18 mg/m3 group slightly lower than controls 9/49, 14/50, 16/50 Lung: granulomatous inflammation (2/49, 50/50, 49/50); interstitial fibrosis (1/49, 16/50, 33/50); alveolar epithelial hyperplasia (5/49, 26/50, 38/50); cyst (0/49, 0/50, 3/50); alveolar squamous metaplasia (0/49, 0/50, 2/50) Adrenal medulla: benign o r malignant pheochromocytoma (26/49, 32/48, 37/47) Some evidence 0, 6, or 18 mg/m3 (equivalent to 0, 3.2, or 9.6 mg/kg per day) 18 mg/m3 group slightly lower than controls 11/50, 13/49, 9/50 Lung: granulomatous inflammation (2/50, 47/48, 50/50); interstitial fibrosis (1/50, 24/48, 44/50); alveolar epithelial hyperplasia (2/50, 27/48, 47/50); cyst (0/50, 1/48, 7/50); alveolar squamous metaplasia (0/50, 0/48, 8/50) Lung: alveolar/ bronchiolar adenoma (1/50, 0/48, 9/50); alveolar/bronchiolar carcinoma (0/50, 0/48, 5/50); alveolar/bronchiolar adenoma or carcinoma (1/50, 0/48, 13/50) Adrenal medulla: benign or malignant pheoch romocytoma (13/48, 14/47, 23/49) Clear evidence 0, 6, o r 18 mg/m3 (equivalent to 0, 2, or 6 mg/kg per day) Exposed groups similar to controls 30/47, 28/48, 32/49 Lung: chronic inflammation (0/45, 16/47, 40/48); macrophage hyperplasia (3/45, 46/47, 48/48) None No evidence Fem ale B6C 3F, M ice 0, 6, or 18 mg/m3 (equivalent to 0, 1.3, or 3.9 mg/kg per day) Exposed groups similar to controls 30/49, 23/48, 25/50 Lung: chronic inflammation (0/46, 25/48, 38/50); macrophage hyperplasia (2/46, 45/48, 43/50) None No evidence Talc, NTP TR 421 9 EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY The National Toxicology Program describes the results of individual experiments on a chemical agent and notes the strength of the evidence for conclusions regarding each study. Negative results, in which the study animals do not have a greater incidence of neoplasia than control animals, do not necessarily mean that a chemical is not a carcinogen, inasmuch as the experiments are conducted under a limited set of conditions. Positive results demonstrate that a chemical is carcinogenic for laboratory animals under the conditions of the study and indicate that exposure to the chemical has the potential for hazard to humans. Other organizations, such as the International Agency for Research on Cancer, assign a strength of evidence for conclusions based on an examination of all available evidence, including animal studies such as those conducted by the NTP, epidemiologic studies, and estimates of exposure. Thus, the actual determination of risk to humans from chemicals found to be carcinogenic in laboratory animals requires a wider analysis that extends beyond the purview of these studies. Five categories of evidence of carcinogenic activity are used in the Technical Report series to summarize the strength of the evidence observed in each experiment: two categories for positive results (clear evidence and some evidence); one category for uncertain findings (equivocal evidence); one category for no observable effects (no evidence); and one category for experiments that cannot be evaluated because of major flaws (inadequate study). These categories of interpretative conclusions were first adopted in June 1983 and then revised in March 198b for use in the Technical Report series to incorporate more specifically the concept of actual weight of evidence of carcinogenic activity. For each separate experiment (male rats, female rats, male mice, female mice), one of the following five categories is selected to describe the findings. These categories refer to the strength of the experimental evidence and not to potency or mechanism. Clear evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a dose-related (i) increase of malignant neoplasms, (ii) increase of a combination of malignant and benign neoplasms, or (iii) marked increase of benign neoplasms if there is an indication from this or other studies of the ability of such tumors to progress to malignancy. Some evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a chemical-related increased incidence of neoplasms (malignant, benign, o r combined) in which the strength of the response is less than that required for clear evidence. Equivocal evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a marginal increase of neoplasms that may be chemical related. No evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing no chemical-related increases in malignant or benign neoplasms. Inadequate study of carcinogenic activity is demonstrated by studies that, because of major qualitative or quantitative limitations, cannot be interpreted as valid for showing either the presence or absence of carcinogenic activity. When a conclusion statement for a particular experiment is selected, consideration must be given to key factors that would extend the actual boundary of an individual category of evidence. Such consideration should allow for incorporation of scientific experience and current understanding of long-term carcinogenesis studies in laboratory animals, especially for those evaluations that may be on the borderline between two adjacent levels. These considerations should include: adequacy of the experimental design and conduct; occurrence of common versus uncommon neoplasia; progression (or lack thereof) from benign to malignant neoplasia as well as from preneoplastic to neoplastic lesions; some benign neoplasms have the capacity to regress but others (of the same morphologic type) progress. At present, it is impossible to identify the difference. Therefore, where progression is known to be a possibility, the most prudent course is to assume that benign neoplasms of those types have the potential to become malignant; combining benign and malignant tumor incidence known or thought to represent stages of progression in the same organ or tissue; latency in tumor induction; multiplicity in site-specific neoplasia; metastases; supporting information from proliferative lesions (hyperplasia) in the same site of neoplasia or in other experiments (same lesion in another sex or species); presence or absence of dose relationships; statistical significance of the observed tumor increase; concurrent control tumor incidence as well as the historical control rate and variability for a specific neoplasm; survival-adjusted analyses and false positive or false negative concerns; structure-activity correlations; and in some cases, genetic toxicology. 10 Talc, NTP TR 421 NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS TECHNICAL REPORTS REVIEW SUBCOMMITTEE The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on talc on June 23, 1992, are listed below. Subcommittee members serve as independent scientists, not as representatives of any institution, company, or governmental agency. In this capacity, subcommittee members have five major responsibilities in reviewing NTP studies: to ascertain that all relevant literature data have been adequately cited and interpreted, to determine if the design and conditions of the NTP studies were appropriate, to ensure that the Technical Report presents the experimental results and conclusions fully and clearly, to judge the significance of the experimental results by scientific criteria, and to assess the evaluation of the evidence of carcinogenic activity and other observed toxic responses. Gary P. Carlson, Ph.D., Chair Department of Pharmacology and Toxicology Purdue University West Lafayette, IN Paul T. Bailey, Ph.D. Toxicology Division Mobil Oil Corporation Princeton, NJ Louis S. Beliczky, M.S., M.P.H.* Department of Industrial Hygiene United Rubber Workers International Union Akron, OH Kowetha A. Davidson, Ph.D. Health and Safety Research Division Oak Ridge National Laboratory Oak Ridge, TN Harold Davis, D.V.M., Ph.D. School of Aerospace Medicine Brooks Air Force Base, TX Jay I. Goodman, Ph.D., Principal Reviewer Department of Pharmacology and Toxicology Michigan State University East Lansing, MI Did not attend David W. Hayden, D.V.M., Ph.D. Department of Veterinary Pathobiology College of Veterinary Medicine University of Minnesota St. Paul, MN Curtis D. Klaassen, Ph.D.* Department of Pharmacology and Toxicology University of Kansas Medical Center Kansas City, KS Daniel S. Longnecker, M.D.* Department of Pathology Dartmouth Medical School Lebanon, NH Barbara McKnight, Ph.D., Principal Reviewer* Department of Biostatistics University of Washington Seattle, WA Ellen K. Silbergeld, Ph.D. University of Maryland Medical School Baltimore, MD Lauren Zeise, Ph.D. California Department of Health Services/RCHAS Berkeley, CA Matthew J. van Zwieten, D.V.M., Ph.D. Principal Reviewer Department of Safety Assessment Merck. Sharp & Dohme Research Laboratories West Point, PA Talc, NTP TR 421 11 SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS On June 23, 1992, the draft Technical Report on the toxicology and carcinogenesis studies of talc received public review by the National Toxicology Program Board of Scientific Counselors' Technical Reports Review Subcommittee. The review meeting was held at the National Institute of Environmental Health Sciences, Research Triangle Park, NC. Dr. K.M. Abdo, NIEHS, introduced the toxicology and carcinogenesis studies of talc by discussing the rationale for study, describing the experimental design, reporting on survival and body weight effects, describing effects on respiratory function, and com menting on compound-related neoplasms in rats and nonneoplastic lesions in rats and mice. The proposed conclusions were some evidence o f carcinogenic activity of talc in male F344/N rats, clear evidence o f carcino genic activity in female F344/N rats, and no evidence o f carcinogenic activity in male or female B6C3F, mice. Dr. van Zwieten, a principal reviewer, agreed with the proposed conclusions. He said that if available, information should be added to the Introduction regarding particle sizes of talc to which humans are exposed during various industrial and cosmetic uses. This would allow a comparison with the aerosol particle size distribution of talc in the animal studies. Dr. Abdo said such information was not available but since the material used was cosmetic grade he as sumed humans were exposed to similar particle sizes. Dr. van Zwieten stated that the section dealing with the histpathologic description of pulmonary neo plasms in rats indicates that uncertainty existed regarding diagnosis of hyperplasia and benign and malignant neoplasia and asked for clarification. Dr. S.L. Eustis, NIEHS, said the pathologists were confident that the lesions diagnosed were neoplasms, but there was difficulty in determining whether or not a small number of lesions of inflammatory or hyper plastic nature were preneoplastic. Dr. Goodman, the second principal reviewer, said his initial position was to disagree with the proposed conclusion. However, he said that he wou|d defer a recommendation pending discussion of whether or not the maximum tolerated dose (MTD) was exceed ed in female rats and consideration of the data con cerning the trend towards an increased incidence of spontaneous pheochromocytomas in rats. Dr. Eustis argued that in this particular study the appearance of lung neoplasms together with impaired pulmonary function is relevant to what might occur in humans with dust overload. Thus, he said, even though the MTD may have been exceeded, the study is valid. Dr. Goodman believed that lung neoplasms produced in female rats following exposure to talc might have been secondary to chronic toxicity. He noted that the recommended time-weighted average human exposure level for talc containing no asbestos fibers is 2 mg/m3 and thought that this dose should have been used in the current study. Dr. Abdo agreed. Because Dr. McKnight, the third principal reviewer, was unable to attend the meeting, Dr. L. Hart, NIEHS, read her review into the record. Dr. McKnight agreed in principle with the conclu sions with the exception that consideration should be given to raising the level of evidence in male rats to clear evidence, since one of the arguments for the level chosen, i.e., no supporting hyperplasia in the adrenal gland, was not warranted. Further, there was strong supporting evidence from the increases in malignant pheochromocytomas and benign and malignant pheochromocytomas (combined) among female rats. Dr. Eustis said because of the high incidence of bilateral pheochromocytomas there was not enough tissue present to find hyperplasia. When considering all the evidence, including a preponder ance of benign neoplasms in male rats, the level of evidence seemed appropriate. Dr. McKnight had commented that evidence from humans suggests that direct effects on the adrenal gland may be possible. Dr. Eustis said that although the possibility cannot be ruled out that talc may reach the adrenal gland, its lack of solubility in aqueous fluids and the way the substance is cleared by the lungs would make a direct effect on the adrenal gland very unlikely. Dr. McKnight thought that a sentence should be added to the conclusions stating that male and female mice might have tolerated higher doses. Dr. Eustis noted that as reported in the conclusions, exposure to talc produced chronic inflammation of the lungs in mice which supported an MTD being reached. Dr. Goodman asked if the conclusion for female rats could be worded "clear evidence of carcinogenic activity only under those circumstances in which there was an indication of chronic toxicity." Dr. Eustis replied that in the discussion the appearance of neo plasms is clearly placed in the context of the chronic toxicity. Dr. Silbergeld said she was increasingly 12 Talc, NTP TR 421 concerned about a rigid criterion whereby evidence of carcinogenicity is discounted if toxicity is present. Dr. Eustis commented that the degree of chronic disease, based on fibrosis and inflammation, was quite similar between male and female rats so it would be difficult to argue that the MTD was exceeded in one sex and not the other. Dr. J.K. Haseman, NIEHS, pointed out that after the levels of evidence there is a paragraph in the conclusion that delineates all toxic lesions associated with chemical exposure in the lung. Dr. J. Haartz, NIOSH, asked that more details be provided for the spatial distribution of the talc in the chambers, and analyses of contaminants such as metals from impurities in the compressed air used. During the public comment period, Dr. Carlson read from a letter from Dr. Frank Mirer, Health and Safety Department, United Auto Workers. Dr. Mirer said the dose selection should be considered in light of current enforceable Permissible Exposure Limits, which are 5 mg/m3 respirable fraction and 15 mg/m3 for total dust. Thus, the low dose selected for this experiment is below the OSHA limit when timeweighted averaging is considered. Dr. Mirer suggest ed that the studies in male rats and male and female mice should be considered inadequate for determina tion of carcinogenicity of talc. Dr. Goodman moved that the conclusion be modified to state that in light of lung toxicity previously noted, the MTD was exceeded in female rats. Dr. Bailey seconded the motion which was defeated by two yes (Drs. Bailey, Goodman) to five no votes with one abstention (Dr. Silbergeld). Dr. Silbergeld abstained because she thought the notion as framed was not informative given that complexities known about the MTD for these types of compounds. Dr. van Zwieten moved that the Technical Report on talc be accepted with the revisions discussed and with the conclusions as written for male rats, some evidence o f carcinogenic activity, for female rats, clear evidence o f carcinogenic activity, and for male and female mice, no evidence of carcinogenic activity. Dr. Hayden seconded the motion. Dr. Goodman offered an amendment to insert a clause in the second sentence of the conclu sions between "rats" and "based" as follows: "under conditions in which there was evidence of chronic lung toxicity." The amendment was tabled for lack of a second. Dr. Silbergeld offered an amendment to insert "the same doses o f between "to" and "talc" in the first sentence of the second paragraph of the conclusions. Dr. Zeise seconded the motion, which was accepted by six yes votes to two no votes (Drs. Davis, Goodman). Dr. Zeise offered an amendment that a sentence be added to the effect that mice may have been able to tolerate higher doses. The amendment was tabled for lack of a second. Dr. van Zwieten's original motion as amended by Dr. Silbergeld was then accepted by seven yes votes to one no vote (Dr. Goodman). 13 INTRODUCTION TALC (Non-Asbestiform) CAS No. 14807-96-6 Molecular Formula: Mg3Si4O 10(OH)2 Molecular Weight: 379.26 Synonyms: talcum: agalite; emtal 596; non-asbestiform talc; non-fibrous talc; steatite; hydrous magnesium silicate Chemical and P hysical Properties Talc is a fine powder, white to grayish white in color, with a greasy feel and luster. It is insoluble in water, cold acids, and alkalies (Merck Index, 1983) and has a density of 2.7 to 2.8 and a melting point of 900 to 1,000 C (Hawley, 1977). Talc as a pure mineral is composed of 63.5% S i0 2,31.7% MgO,and4.8% H20 (Pooley and Rowlands, 1977). Production, U se, and Human Exposure Talc is produced by open pit or underground mining of talc rocks and processed by crushing, drying, and milling. Contaminating minerals including iron, nickel, manganese, chromium, aluminum, and titani um are separated from talc by flotation or elutriation. Talc is then finely powdered, treated with boiling diluted hydrochloric acid, washed well, and dried (Osol et al., 1980). Geological formation of talc rock results from the alteration of magnesia- and silicarich ultramafic rocks under a range of temperatures and pressures. These hydrothermal alterations may lead to the formation of other mineral phases such as tremolite and serpentine minerals, including chrysotile. These mineral phases may occur as microscopic intergrowths, nodules, or discrete zones within or adjacent to talc (Rohl et at, 1976). United States production of talc for 1985 was esti mated at 1.1 million metric tons, with industrial pattern of use as follows: ceramics, 37%; paints, 19%; paper, 10%; roofing, 10%; plastics, 7%; cosme tics, 5%; rubber, 3%; insecticides, 1%; and other uses, 9% (Bureau of Mines, 1986). Commercial talc is categorized into cosmetic grade, which is free of asbestos, and industrial grade, which contains other minerals including asbestos (Hildick-Smith, 1976). A comprehensive review of the literature before 1987 on the use, exposure, and biological effects of talc was published by IARC (1987). Talc is used as a dusting powder, including baby powder, either alone or with starch or boric acid, for medicinal or toiletry preparations; as an excipient and filler for pills and tablets; and for dusting tablet molds (Merck Index, 1983). It is also used as a filler and pigment for paints, putty, and plaster; as a carrier and diluent for pesticides; as an additive to clay in ceramic manufac ture; in paper coatings; and for the manufacture of rubber and roofing materials (Hawley, 1977). The recommended time-weighted average (TWA) human exposure level for talc containing no asbestos fibers is 2 mg/m3 (ACGIH, 1989). A large segment of the population is potentially exposed to talc. The number of workers exposed to talc has been estimated at 1,371,201, which includes 349,228 females (NIOSH, 1990). In addition, the public is potentially exposed to talc through its many uses in pharmaceuticals and consumer products. Based on its uses, human exposure to talc can occur via inhalation, ingestion, or dermal exposure. Absorption, D istribution, and Excretion Experimental Animals The absorption and disposition of 3H-labeled talc in rats, mice, and guinea pigs administered a single oral dose, as well as its translocation in rabbits adminis tered a single or multiple intravaginal dose was 14 Talc, NTP TR 421 studied by Phillips et al. (1978). The oral doses were 50 mg/kg for rats, 40 mg/kg for mice, and 25 mg/kg for guinea pigs. Rabbits were administered either a single intravaginal dose of 50 mg/kg or the same dose once a day for 6 days. In rats, mice, and guinea pigs, more than 95% of the dose was excreted in the feces 3 to 4 days after dosing. Less than 2% of the radio activity was recovered in the urine. This radioactivity probably reflected contamination of urine samples with feces. No radioactivity was found in the liver or kidneys of these animals. No translocation of talc was found in the ovaries of rabbits. Hanson et al (1985) and Pickrell et al. (1989) studied the lung burden in groups of five male and five female F344/N rats and B6C3F, mice following inhalation exposure to concentrations of talc for 6 hours per day, 5 days per week, for 4 weeks. The mean exposure concentrations used were 2.3, 4.3, or 17 mg/m3 for rats and 2.2, 5.7, or 20.6 mg/m3 for mice. The resulting lung talc burdens were 0.08,0.19, and 0.87 mg/g of lung for rats and 0.1, 0.33, and 1.2 mg/g of lung for mice. These data clearly indicate that the amount of talc retained per unit of lung tissue was proportional to the exposure concentration of talc. Pulmonary deposition, translocation, and clearance of neutron-activated talc was studied in hamsters after a single, 2-hour, nose-only inhalation exposure (Wehner et al., 1977a,b). Deposition of talc in the lung was demonstrated by X-ray fluorescence and X-ray diffraction. An estimated 6% to 8% of the inhaled quantity was deposited in the alveoli. The biological half-life of the talc deposited in the alveoli was estimated at 7 to 10 days. No translocation of talc to liver, kidneys, ovaries, or other parts of the body was found. Humans Talc, a filler in some drugs injected by addicts, was found in the lung (Groth et al, 1972; Lamb and Roberts, 1972; Farber et al, 1981; Crouch and Churg, 1983), spleen, kidney, liver, brain, adrenal gland, thyroid gland (Groth et al, 1972), and retina (Atlee, 1972) of some addicts. In the lung, most of the talc particles were seen within the vessels of the alveolar walls and were often associated with marked foreign body granulomas (Crouch and Churg, 1983). T oxicity Experimental Animals The LDS0 for talc has not been established. Talc caused death in guinea pigs administered 2 or 3 in jections of 25 mg talc in saline (Dogra et al., 1977) and in rats receiving a splenic injection of 1,400 mg/kg body weight (Eger and DaCanalis, 1964). Deaths occurred in rats exposed to a very dense atmosphere of talc (particle size <5 m) 3 hours a day, for 12 days (Policard, 1940). The concentration of talc in the atmosphere was not known and the observed mortality may have been due to suffocation. Wagner et al (1977) reported on the toxic effects of talc in rats exposed orally or by inhalation. No significant decrease in mean life span and no patho logic effects were found in rats fed 100 mg talc for 101 days. Rats exposed to talc atmospheres of 10.8 mg/m3 (particle size, 25 /xm) for 3 months showed minimal lung fibrosis, and no change in severity occurred during the post-exposure period. By contrast, rats exposed to the same atmospheres for a year had minimal to slight fibrosis, and the severity had increased to moderate within a year after cessa tion of exposure. Rats exposed to atmospheres of 30 to 383 mg/m3"industrial" or "pharmaceutical" talc for 9 months developed chronic inflammatory changes, including thickening of the pulmonary artery walls and emphysema (Bethege-Iwanska, 1971). There were no histopathologic changes in the lung, heart, liver, renal tissue, or uterus of hamsters exposed to respirable aerosols containing 8 mg/m3 of cosmetic grade talc for 150 minutes a day, 5 days per week, for 300 days (Wehner, 1980). Rats administered a single intratracheal injection of 50 mg of pure talc in water did not show lung fibrosis or lymph node abnormalities. Those receiving the same dose of "calcined" talc developed lung and lymph node fibrosis (Luchtrath and Schmidt, 1959). These differing results may be related to differences in the crystal structures of "pure" and "calcined" talc. Bronchiolar inflammation occurred in rats 4 days after an intratracheal injection of 25 mg talc (con taining tremolite) in water; collagenous tissue devel oped within a few months after injection (Gross et al, 1970). Introduction 15 Injection of 10 mg of talc containing some asbestos into the pleural cavity of mice produced granulomas (Davis, 1972). A single injection of 20 mg of talc into the right pleural cavity of rats produced granu lomas at the injection site; one lung adenoma was also observed but no other changes related to talc administration were observed in the lung (Wagner et ai, 1977). Rats with abdominal muscle implants of suture materials dusted with talc or talc pellets initially had mild to moderate acute inflammation, followed by chronic inflammation and granuloma formation within 3 days (Sheikh et ai, 1984). Rats with subcutaneous inflammation caused by talc had a decrease in bone formation as evidenced by hypozincemia and a decrease in metaphyseal trabecular surfaces. Both hypozincemia and the decrease in osteoblast trabecular surfaces were directly proportional to the number of granulomas present (Marusic et ai, 1990). Talc produced retinopathy in adult Rhesus monkeys administered intravenous injections of talc once every 2 weeks for 3.5 to 10 months. Talc particles were found lodged in the precapillary arterioles and capillaries, producing a focal occlusion of retinal and choroidal capillaries (Kaga et ai, 1982a,b). Humans Exposure to industrial grade talc dust causes pulmo nary fibrosis; however, reports on cosmetic grade talc dust are conflicting. Hildick-Smith (1976) reported that cosmetic grade talc did not appear to be injuri ous to health, while Vallyathan and Craighead (1981) reported that it was. Four of seven workers exposed to heavy concentrations (0.4 to 36 mg/m3) of cosmetic grade talc for 4 to 27 years had histologic evidence of pulmonary fibrosis at death (Theriault et ai, 1974). Wells et ai (1979) also noted chronic pulmonary degenerative disease in a housewife who reported heavy use of cosmetic talc. Inhalation of pure talc is known to result in a disease known as talcosis, which may include acute or chronic bronchitis and intersti tial inflammation. Radiographically, the lesion appears as a small, irregular nodule, typical of a small-airway obstruction. Intravenous administration of talc-containing oral medications by abusers causes vascular granulomas (Feigin, 1986). Intravenous talcosis was diagnosed in a 36-year-old woman who was a drug abuser (Hill et a i, 1990). Talcosis in this patient was identified by the presence of peripheral nodular lesions on chest X-rays and was confirmed by the presence of birfringent particles in a trans bronchial biopsy. Pulmonary talc granulomatosis was diagnosed in a cocaine sniffer (Oubeid et ai, 1990). Chest X-rays of a heroin addict who later died of respiratory failure showed a progressive massive fibrosis of the lung secondary to intravenous injection of the drug (Crouch and Churg, 1983). Microscopic examination of lung lesions revealed an active granu lomatous reaction with associated vascular oblitera tion. Throughout the lesion, refractile birefringent plates of particulate material were noted. Interstitial perivascular and vascular granulomas were noted in the periphery of the lung. The particulate material was identified as talc by X-ray spectroscopy and diffraction methods. Intravenous injection of talccontaining drugs intended for oral use was the cause of pulmonary granulomatosis and pulmonary hyper tension in 19 patients (Arnett et ai, 1976). In patients with pulmonary hypertension, talc granuloma was found in the pulmonary arteries. In patients without hypertension, talc granuloma was found in the pulmonary interstitium. Patients suffering from talc granulomatosis (confirmed by lung biopsy) as a result of intravenous injection of crushed tablets of pentazocine had dyspnea, increased angiotensin converting enzyme concentrations, and increased lymphocytes by bronchoalveolar lavage (Farber et ai, 1982). Pneumoconiosis (talcosilicosis) was diagnosed in a 54-year-old female confectionery worker who was exposed to talc dust for 5 years (Canessa et ai, 1990). Talc, administered by intrapleural instillation to promote pleural symphysis in the palliation of recur rent malignant pleural effusions, caused adult respira tory distress syndrome (ARDS) in three patients (Rinaldo et ai, 1983). Symptoms of ARDS included fever, dyspnea, and respiratory failure. ARDS occurred in a 16-month-old baby inhaling baby powder. Normal pulmonary function returned in this patient after 6 years, as determined by a follow-up study (Reyes and Brown, 1989). Ca r c in o g e n ic it y Experimental Animals Results of carcinogenicity studies of talc in animals were reviewed by the LARC (1987). The following is an excerpt of this review: No significant difference in neoplasm incidence was observed between two groups of 25 male and 25 female Wistar rats (10 weeks old) that received an equivalent of 50 mg/kg per day of commercial talc (composition not specified) in the diet or the basal diet for life (Gibel et ai, 1976). Similar results were 16 Talc, NTP TR 421 obtained in groups of 16 male and 16 female Wistar rats (21 to 26 weeks old) that received 100 mg of Italian talc (particle size, 25 /m; containing 92% talc, 3% chlorite, 1% carbonate minerals, and 0.5 to 1% quartz) per rat per day in the diet or the basal diet for 5 months and observed for life (Wagner et al, 1977). In both studies small numbers of animals were used. Groups of 24 male and 24 female Wistar rats, 6 to 8 weeks of age, were exposed by inhalation to 10.8 mg/m3Italian talc aerosol 7.5 hours a day, 5 days per week, for 6 or 12 months. Ten days after the end of each exposure period, six rats in each group were killed; an additional four rats were killed one year later. Within 28 months from the beginning of the study, 12 animals in each group had died. No lung neoplasms were observed in rats exposed to talc for 6 months; one lung adenoma occurred in a rat exposed for 12 months. No lung neoplasms were found in the control rats (Wagner et al, 1977). Three groups of 50 male and 50 female hamsters, 4 weeks of age were exposed to talc aerosol (37.1 mg/m3, mean respirable fraction 9.8 mg/m3) for 3, 30, or 150 minutes per day, 5 days a week, for 30 days. Two additional groups of hamsters were exposed to talc aerosol (27.4 mg/m3, mean respirable fraction 8.11 mg/m3) for 30 or 150 minutes per day, for 300 days. Two groups of 25 male and 25 female hamsters were exposed to air and served as controls. No primary neoplasms were found in the respiratory system of any hamster. Twenty-five percent of the hamsters exposed to the aerosols for 30 or 150 minutes for 300 days had alveolar cell hyperplasia compared to 10% in the controls (Wehner et a l, 1977a, 1979). No local neoplasms were found in 50 female R3 mice, 3 to 6 months of age, administered a 0.2 mL subcutaneous injection of talc of unspecified compo sition (80 mg talc in peanut oil) and observed for life (Neukomm and de Trey, 1961). Forty Swiss albino rats, 6 weeks of age (sex unspeci fied) received a single intraperitoneal injection of 20 mg commercial talc (unspecified composition) in saline. Sixteen animals died by the end of 6 months. Of the 24 mice that lived to termination (time not specified) three had peritoneal mesotheliomas com pared to three of 46 of the controls (Ozesmi et al., 1985). This study was considered inadequate because of poor reporting. Forty female Wistar rats, 8 to 12 weeks of age, were given four intraperitoneal injections of 25 mg granu lar talc in 2 mL saline at weekly intervals. Similarly, 80 females were injected with saline and served as controls. The rats were observed until termination or death (average survival time, 602 days). A mesotheli oma occurred in one of 36 rats given talc but none was found in the controls (Pott et al, 1974,1976a,b). No mesothelioma was observed in two groups of 24 male and 24 female Wistar rats administered a single intrapleural injection of 20 mg Italian talc in saline or saline alone. A pulmonary adenoma oc curred in one rat that died at 25 months. Mean survival time (655 days for the talc group versus 691 for the controls) was not affected (Wagner et al, 1977). Groups of 30 to 50 female Osborne-Mendel rats, 12 to 20 weeks of age, received intrapleural implantation of one of seven grades of refined commercial talc from separate sources in hardened gelatin. Rats were observed for up to 2 years at which time survivors were killed. Pleural sarcoma incidences were: grade 1, 1/26; grade 2, 1/30; grade 3, 1/29; grade 4, 1/29; grade 5, 0/30; grade 6, 0/30; grade 7, 0/29. The incidence of pleural sarcoma was three of 491 in untreated controls, 17 of 615 in controls receiving implants of "nonfibrous" material described by the authors as "noncarcinogenic," and 14 of 29 in rats receiving UICC crocidolite asbestos (Stanton et al, 1981). The IARC Working Group noted that in most of the talc studies, little or no characterization of the mineralogy, fiber content, or particle size of the samples was given. Thus, the group concluded that there was inadequate evidence on the carcinogenicity of talc to experimental animals. Humans An epidemiology study of pottery workers in the United States revealed an association between expo sure to non-fibrous talc and increased mortality and lung cancer incidence (Thomas and Stewart, 1987). Increased incidences of lung cancer occurred exclu sively among pottery workers employed in the manu facture of plumbing fixtures. A later study of Introduction 17 employees in three ceramic plumbing fixture factories showed increased mortality from respiratory disease and from lung cancer. The increased incidence in lung cancer was highest among workers who were simultaneously exposed to silica and talc. The lung cancer mortality risk increased with the number of years of exposure to talc, but showed no pattern by the number of years of exposure to silica. Among men exposed to talc, lung cancer risk decreased with age at first exposure to non-fibrous talc and increased with years since first exposure (Thomas, 1990). Whether or not exposure to silica had a promoting effect on lung cancer is not known. No increased risk for lung cancer or benign respiratory disease was found in millers or miners of non-asbestiform talc (Wergeland et al, 1990). A case-control study found that women who had perineal exposure to deodorizing powders alone or in combination with other talc-containing powders, had a 2.8 times higher risk of developing borderline ovarian neoplasms than women who were not perine ally exposed to powder (Harlow and Weiss, 1989). In an earlier study, the use of talc as a dusting powder on the perineum or on sanitary napkins by women was associated with an increased risk of epithelial ovarian cancer. Women engaged in both practices had a relatively higher risk of developing this type of cancer (Cramer et al., 1982). No information was presented regarding exposure levels or the content of contaminating minerals of the talc used. In another study, the role of exposure to talcum powder, tobac co, alcohol, and coffee, and the histories of tubal sterilization and hysterectomy on ovarian cancer risk was assessed. The study involved 188 women diag nosed with epithelial ovarian cancer and 539 control women. No association was found between the incidence of epithelial ovarian cancer and increasing frequency or duration of talc use. Patients did not differ from control women in the use of talc on sanitary pads, contraceptive diaphragms, or both. (Whittemore et aL, 1988). Reproductive and Teratogenic Effects Experimental Animals Talc produced nonspecific abnormalities in chicken eggs at incidences similar to those caused by thalid omide and sulphadimethoxine (Yang, 1977). No teratologic effects were observed in hamsters, rats, mice, or rabbits after oral administration of talc. The doses used were 1,600 mg/kg for rats and mice on days 6 through 15 of gestation, 1,200 mg/kg for hamsters on days 6 through 10 of gestation, and 900 mg/kg for rabbits on days 6 through 18 of gesta tion (Food and Drug Research Laboratories, 1973). Humans No information on the reproductive or teratogenic effects of talc in humans has been reported. Genetic Toxicity There are no published studies on the genotoxicity of talc. The IARC (1987) review of talc included unpublished results from a 1974 study conducted by Litton Bionetics that showed no mutagenic activity for talc in vitro or in vivo. Talc did not induce mutations in Salmonella typhimurium strains TA1530 or HisG46, or in the yeast, Saccharomyces cerevisiae. No chromosomal aberrations were observed in human fibroblasts treated with talc in vitro. In vivo tests conducted in rats gave negative results for induction of chromosomal aberrations in bone marrow cells and dominant lethal mutations in germinal cells. Study Rationale Talc was nominated by NIOSH in 1978 for testing by NTP because of the paucity of adequate information on its carcinogenicity and because of widespread human exposure. The inhalation route was chosen because it is the most common route for human exposure. Talc, NTP TR 421 19 MATERIALS AND METHODS Procurement and Characterization of Talc Talc(MP 10-52 Grade) was obtained from Walsh and Associates (North Kansas City, MO) in two lots (W101882 and B5415). The talc was manufactured by the Minerals, Pigments, and Metals Division of Pfizer, Inc. and is one of their microtalc series of products. Both lots were from Pfizer's Barretts, Montana, mine which is a strip mine located between Barretts and Three Brother, Montana. This mine is the only source for the MP 10-52 grade talc. The grade designation is for high purity talc that has a top particle size of 10 pm and according to the manufac turer contains no tremolite or any asbestiform minerals. Lot W101882 was used from the beginning of the 2-year studies through January 1986. Lot B5415 was used in the 2-year studies from 27 January 1986 to the end of the studies on 31 October 1986. The talc was extensively characterized by the analytical chemistry laboratory, Midwest Research Institute (Kansas City, MO), and by McCrone Associates (Norcross, GA). The methods and results of these studies are detailed in Appendix I. The study mineral, a finely powdered white solid, was identified as talc by infrared spectroscopy, elemental analysis, Karl Fischer water analysis, thermogravi metric analyses, spark source mass spectrometry, automated scanning electron probe analyses, X-ray diffraction, polarized light microscopy, and transmis sion electron microscopy. Both lots were found to be asbestos free by polarized light microscopy and transmission electron microscopy. Results of auto mated scanning electron microprobe analysis of lot W101882 indicated that the sample was virtually free of silica (1 particle of silica in 1,466 particles examined). Bulk chemical stability studies were not conducted due to the physical and chemical proper ties of talc. During the study the compound was stored in tightly sealed plastic bags at 25 C. (Figure 12). The aerosolized talc particles were then mixed with diluting air before being delivered to the exposure chambers (Hazleton 1000 and 2000, Lab Products, Inc.). A second fluidized-bed generator for the control chamber contained only the stainless steel bed material (Figures 13 and 14). Aerosol concentrations were monitored daily in each chamber by taking three, 2-hour filter samples. Background concentrations of suspended particles were measured daily in the control chamber by taking a 6-hour filter sample. A RAM-S forward light scattering monitor (GCA, Bedford, MA) was used to determine the stability of the aerosol concentrations and the need to adjust the aerosol generation system during the exposure. Determinations were made at the beginning, middle, and end of each filter sampling period. The overall mean concentrations were 6.1 and 18.6 mg/m3 for the rat study and 5.9 and 16.7 mg/m3 for the mouse study. While the overall means were very close to target concentrations, there were problems experienced in maintaining control of chamber concentrations. Weekly mean exposure concentrations for the 2-year studies are presented in Figures 15 through 18. Chamber Atmosphere Characterization Uniformity of the aerosol concentrations in each chamber was determined at approximately 3-month intervals with the RAM-S. The spatial variation as estimated by the relative standard deviation was higher in the mouse study than in the rat study with values from 12% to 44% relative standard deviation for mice and 2% to 31% relative standard deviation for rats. To minimize the variation in talc concentra tions, the animal cages were rotated weekly. The time to reach 90% of the target concentration (Tw) was approximately 10 minutes. Therefore, the length of the exposure was defined at 6 hours plus the T,, of 10 minutes. Generation and M onitoring of Chamber Concentrations Talc aerosols were generated in a single fluidized-bed generator by injecting compressed air into the bed The aerosol size distribution was determined once each month for each chamber using a cascade impactor. The average mass mean aerodynamic diameter and the geometric standard deviation were calculated to be 2.7 1 .9 /itn and 3.2 1 .9 pm for 20 Talc, NTP TR 421 the 6 and 18 mg/m3 rat chambers. The values were 3.3 1.9 jum and 3.6 2.0 jum for the 6 and 18 mg/m3mouse chambers. The individual values are presented in Tables II and 12. Study Design Groups of 50 male and 50 female rats and mice were selected for whole body inhalation to talc at target concentrations of 0, 6, or 18 mg/m3. These exposure concentrations provided a dose equivalent of 0, 2.8, or 8.4 mg/kg per day for male rats, 0, 3.2, or 9.6 mg/kg per day for female rats, 0,2, or 6 mg/kg per day for male mice, and 0, 1.3, or 3.9 mg/kg per day for female mice. Rats were exposed for 6 hours per day, 5 days a week until mortality in any exposure group reached 80% (113 weeks for males and 122 weeks for females). Exposure of rats to talc was extended beyond 2 years based on the report that 80% of pulmonary neoplasms induced in rats by inhalation exposure to diesel exhaust occurred after 2 years (Mauderly et al, 1986). Mice were exposed for 103 or 104 weeks. At the conclusion of the exposures, rats were exposed to filtered air for 10 or 11 days, while mice were exposed to filtered air for 10 to 14 days. All animals were necropsied and received a complete pathology evaluation. Additional special study groups of 22 male and 22 female rats and 40 male and 40 female mice similarly exposed to 0, 6, or 18 mg/m3 were desig nated for interim pathology evaluations; lung talc burden measurements; serial pulmonary function measurements (rats only); and lung biochemistry, cytology, and phagocytosis measurements. Rats were evaluated at 6, 11, 18, and 24 months, while mice were evaluated at 6, 12, and 18 months. Insufficient numbers of rats remained alive at week 103 of exposure for both pulmonary function and/or lung biochemistry/cytology and pathology distribution groups, therefore the remaining rats in these groups were combined. The numbers of rats and mice evaluated for pulmonary function and lung biochem istry, cytology, and phagocytosis and the methods used for each of the parameters are presented in Append G for rats and Appendix H for mice. Source and Specification of Animals Male and female F344/N rats were obtained from Lovelace Inhalation Toxicology Research Institute (Albuquerque, NM). Male and female B6C3F! mice were obtained from Frederick Cancer Research Center (Frederick, MD). Rats and mice were held 3 weeks before the studies began. Rats were 6 to 7 weeks old, and mice were 7 weeks old when the studies began. Animal health was monitored by serologic analyses during the studies under the protocols of the NTT Sentinel Animal Program (Appendix K). Animal Maintenance Rats and mice were housed individually throughout the studies. Drinking water was available ad libitum. Further details of animal maintenance are given in Table 1. Clinical Examinations and Pathology All rats and mice were observed twice daily. Clinical observations and body weights were recorded at the beginning of the studies, weekly for 13 weeks, and monthly thereafter. A necropsy was performed on all rats in the lifetime core study and all mice in the 2-year core study. Organ weights were recorded for the brain, heart, right kidney, liver, and lung at the end of the studies. During necropsy, all organs and tissues were exam ined for grossly visible lesions. A complete histo pathologic examination was performed on all animals. Tissues for microscopic examination were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned to a thickness of 5 /zm, and stained with hematoxylin and eosin. Microscopic evaluations were completed by the study laboratory pathologist and the pathology data were entered into the Toxicology Data Management System. The slides, paraffin blocks, and residual wet tissues were sent to the NTP Archives for inventory, slide/block match, and wet tissue audit for accuracy of labeling and animal identification and for thorough ness of tissue trimming. The slides, individual animal data records, and pathology tables were evaluated by an independent quality assessment laboratory. The individual animal records and tables were compared for accuracy, slides and tissue counts were verified, and histotechnique was evaluated. A quality assess ment pathologist reviewed lung and bronchial and mediastinal lymph nodes in rats and mice and the nose in male mice for accuracy and consistency of lesion diagnosis. The quality assessment report and slides were submit ted to the Pathology Working Group (PWG) chair, who reviewed tissues for which there was a disagree ment in diagnosis between the laboratory and quality assessment pathologists. All pulmonary neoplasms in Materials and Methods 21 female rats and representative histopathology slides of adrenal gland (rats), bronchial lymph node, lung, mediastinal lymph node (rats), and nose, or lesions of general interest were presented by the chair to the PWG for review. The PWG included the quality assessment pathologist as well as other pathologists experienced in rodent toxicologic pathology who examined these tissues without knowledge of dose group or previously rendered diagnoses. When the consensus diagnosis of the PWG differed from that of the laboratory pathologist, the final diagnosis was changed to reflect the opinion of the PWG. Details of these review procedures have been described, in part, by Maronpot and Boorman (1982) and Boorman et al. (1985). For subsequent analysis of pathology data, the diagnosed lesions for each tissue type were evaluated separately or combined according to the guidelines of McConnell et al. (1986). Statistical Methods Survival Analyses The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the Results section of this report. Animals were censored from the survival analyses at the time they were found dead from other than natural causes or were missing, culled, or missexed; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox's (1972) method for testing two groups for equality and Tarone's (1975) life table tests to identify dose-related trends. All reported P values for the survival analysis are two sided. Calculation o f Incidence The incidences of neoplasms or nonneoplastic lesions presented in Tables A l, A4, Bl, B4, Cl, C4, Dl, and D4 are given as the number of animals bearing such lesions at a specific anatomic site and the number of animals with that site examined microscopically. For calculation of statistical significance, the incidences of all nonneoplastic lesions and most neoplasms (Tables A3, B3, C3, and D3) are given as the ratio of the number of affected animals to the number of animals with the site examined microscopically. However, when macroscopic examination was required to detect neoplasms in certain tissues (e.g., skin, intestine, harderian gland, and mammary gland) before micro scopic evaluation, or when neoplasms had multiple potential sites of occurrence (e.g., leukemia or lymphoma), the denominators consist of the number of animals on which a necropsy was performed. Analysis o f Neoplasm Incidences The majority of neoplasms in these studies were considered to be incidental to the cause of death or not rapidly lethal. Thus, the primary statistical method used was logistic regression analysis, which assumed that the diagnosed neoplasms were discov ered as the result of death from an unrelated cause and thus did not affect the risk of death. In this ap proach, neoplasm prevalence was modeled as a logistic function of chemical exposure and time. Both linear and quadratic terms in time were incor porated initially, and the quadratic term was eliminat ed if it did not significantly enhance the fit of the model. The exposed and control groups were com pared on the basis of the likelihood score test for the regression coefficient of dose. This method of adjusting for intercurrent mortality is the prevalence analysis of Dinse and Lagakos (1983), further de scribed and illustrated by Dinse and Haseman (1986). When neoplasms are incidental, this comparison of the time-specific neoplasm prevalences also provides a comparison of the time-specific neoplasm inci dences (McKnight and Crowley, 1984). In addition to logistic regression, alternative methods of statistical analysis were used, and the results of these tests are summarized in the appendixes. These include the life table test (Cox, 1972; Tarone, 1975), appropriate for rapidly lethal neoplasms, and the Fisher exact test and the Cochran-Armitage trend test (Armitage, 1971; Gart et al., 1979), procedures based on the overall proportion of neoplasm-bearing animals. Tests of significance include pairwise comparisons of each exposure group with controls and a test for an overall dose-response trend. Continuity-corrected tests were used in the analysis of neoplasm incidence, and reported P values are one sided. The procedures described above also were used to evaluate selected nonneoplastic lesions. For further discussion of these statistical methods, see Haseman (1984). Analysis on Nonneoplastic Lesion Incidences Because all nonneoplastic lesions in this study were considered to be incidental to the cause of death or not rapidly lethal, the primary statistical analysis used was a logistic regression analysis in which lesion prevalence was modeled as a logistic function of chemical exposure and time. For lesions detected at the interim evaluation, the Fisher exact test was used, a procedure based on the overall proportion of affected animals. 22 Talc, NTP TR 421 Analysis o f Continuous Variables Two approaches were employed to assess the signifi cance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data that had approximately normal distributions were analyzed using the para metric multiple comparison procedures of Williams (1971, 1972) and Dunnett (1955). Lung burden parameters that had skewed distributions were analyzed using the nonparametric multiple compari son methods of Shirley (1977) and Dunn (1964). Jonckheere's test (Jonckheere, 1954) was used to assess the significance of the dose-response trends and to determine whether a trend-sensitive test (Williams'or Shirley's test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-response trend (Dunnett's or Dunn's test). Quality Assurance Methods The lifetime and 2-year studies were conducted in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58). In addition, as study records were submitted to the NTP Archives, they were audited by an independent quality assurance contractor. Separate audits covering completeness and accuracy of the pathology data, pathology specimens, final pathology tables, and preliminary review draft of this NTP Technical Report were conducted. Audit procedures are presented in the reports, which are on file at the NIEHS. The audit findings were reviewed and assessed by the NTP staff so that all discrepancies had been resolved or were otherwise addressed during the preparation of this Technical Report. Materials and Methods 23 Table 1 Experimental Design and Materials and Methods in the Lifetime and 2-Year Inhalation Studies of Talc Study Laboratory Lovelace Inhalation Toxicology Research Institute (Albuquerque, NM) Strain and Species Rats: F344/N Mice: B6C3F] Animal Source Rats: Lovelace Inhalation Toxicology Research Institute (Albuquerque, NM) Mice: Frederick Cancer Research Center (Frederick, MD) Tim e Held Before Studies 3 weeks Average Age W hen Placed on Studies Rats: 6-7 weeks Mice: 7 weeks D ate of First Exposure Rats: 2 July 1984 Mice: 4 June 1984 Duration of Exposure Rats: 6 hours/day, 5 days/week for 113 weeks (males) and 122 weeks (females) Mice: 6 hours/day, 5 days/week for 103-104 weeks Date of Last Exposure Rats: 29 August 1986 (males) and 31 October 1986 (females) Mice: 30 May 1986 Average Age W hen Killed Rats: 120-121 weeks (males) and 129-130 weeks (females) Mice: 112-113 weeks M ethod o f Sacrifice Injection of T-61 solution for all rats in the lifetime study, all rats designated for pathologic evaluation, and all mice. Halothane anesthesia for all rats designated for biochemical interim evaluations. Necropsy Dates Rats: 8-9 September 1986 (males) and 10-11 November 1986 (females) Mice: 9-13 June 1986 (males) and 2-6 June 1986 (females) Size of Study Groups SO males and 50 females M ethod o f Anim al D istribution Assigned to groups by weight and sex using computer-generated random numbers. Anim als per Cage 1 M ethod o f Anim al Identification Toe clip and ear tag Diet NIH-07 Rat and Mouse Ration (Zeigler Bros., Gardner, PA) available a d lib itu m during nonexposure periods M axim um Storage Tim e for Feed 90 days 24 Talc, NTP TR 421 Table 1 Experimental Design and Materials and Methods in the Lifetime and 2-Year Inhalation Studies of Talc (continued) W ater Automatic Watering System (Edstrom), available a d lib itu m Cages Stainless steel mesh cages (Hazleton, Aberdeen, MD) C ham bers Rats: Stainless steel multitiered whole-body exposure chambers (H2000, Hazleton Systems, Aberdeen, MD), washed weekly Mice: Stainless steel multitiered whole-body exposure chambers (H1000, Hazleton Systems, Aberdeen, MD), washed weekly Bedding Untreated paper cage board (Shepherd Specialties Paper, Inc., Kalamazoo, MI), changed twice a day Filters Room Air and Chamber Air High Efficiency Particulate Air (HEPA) Filler (prefilter and exit filler), MIL Spec MIL-F-51068C (Flanders, Washington, DC) Animal Room Environm ent Rats Average temperature: 25 C Relative humidity: 6%-100% Fluorescent light: 12 hours/day Room air changes: minimum of 10 changes/hour Mice Average temperature: 23 C Relative humidity: 0%-100% Fluorescent light: 12 hours/day Room air changes: minimum of 10 changes/hour Exposure Concentrations 0, 6, and 18 mg/m3 by inhalation Type and Frequency o f Observation Observed twice daily, body weights and clinical findings recorded at study initiation, weekly through week 13, and monthly thereafter Necropsy Necropsy performed on all animals. Organ weights recorded for brain, heart, right kidney, liver, and lung. H istopathology Complete histopathologic examinations performed on all animals. In addition to tissue masses and gross lesions, tissues examined included: adrenal gland, bone (including marrow), brain, clitoral gland (female rats), epididymis, esophagus, gallbladder (mice), harderian gland (female rats and mice), heart, kidney, large intestine (cecum, colon, rectum), larynx, liver, lung, lymph nodes (bronchial, mandibular, mediastinal, mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland (male rats), prostate gland, salivary gland, seminal vesicle, skin, small intestine (duodenum, ileum, jejunum), spleen, stomach (forestomach, glandular), testis, thymus, thyroid gland, trachea, urinary bladder, and uterus. 25 RESULTS Rats 4-W eek Study D ose Selection Results of previous studies (Bethege-Iwansha, 1971; Wagner et aL, 1977; Wehner, 1980) indicated that talc produces its toxic effects after prolonged (1 year) exposure. Based on these results it was concluded that lung talc burden and not talc toxicity would be the limiting factor for dose selection for the chronic studies. For this reason the NTP chose to conduct a 4-week lung burden study rather than the conven tional 13-week study. Selection of 6 and 18 mg talc/m3 as the exposure concentrations was based on the results of a 4-week inhalation study in F344/N rats to determine lung talc burden and histopathologic changes associated with talc exposure. These studies indicated that the amount of talc retained in the lung was similar between sexes and proportional to exposure concen tration (Appendix F). Microscopic examination of the lungs revealed an accumulation of alveolar macrophages in the lungs only at the 18 mg/m3 concentration. Based on these findings it was pre dicted that aerosol concentrations greater than 18 mg/m3 would overwhelm lung clearance mecha nisms, impair lung function, and possibly shorten survival. controls throughout the study (Tables 3 and 4, and Figure 2). Mean body weights of male and female rats exposed to 18 mg/m3 were slightly lower than those of controls, particularly after week 65. The final mean body weight of males in the 18 mg/m3 group was 4% lower than that of the controls, while the final mean body weight of females in the 18 mg/m3 group was 14% lower than that of the controls. All serological tests performed prior to the beginning of the study and after 6, 12, and 18 months of exposure were negative. After 24 months and 28 and 30 months (females), serological tests for Kilham rat virus (KRV), Sendai virus, and rat coronavirus/ sialodacryoadenitis virus (RCV/SDA) were positive (Table Kl). The significance of the positive KRV titer is unknown since it was found in only one rat and was not observed at later times. No clinical findings or gross or microscopic lesions that could be attributed to Sendai virus or RCV/SDA infections were observed in the exposed or control groups. Since there was no clinical or pathological evidence of disease and since the infection occurred very late in the study, these subclinical infections are believed to have had no impact on the study results. Lifetime Study Survival Estimates of survival probabilities for male and female rats are shown in Table 2 and in the Kaplan Meier curves in Figure 1. Survival of exposed male and female rats was similar to that of the controls. Body Weights and Clinical Findings The mean body weights of male and female rats exposed to 6 mg talc/m3 were similar to those of Pathology and Statistical Analyses of Results This section describes the statistically significant or biologically noteworthy changes in the incidences of neoplastic or nonneoplastic lesions of the lung, lymph node, nose, and adrenal medulla. Summaries of the incidences of nonneoplastic lesions and neoplasms, the individual animal tumor diagnoses, and the statistical analyses of primary neoplasms that oc curred with an incidence of at least 5% in at least one group are presented in Appendix A for male rats and Appendix B for female rats. 26 Talc, NTP TR 421 Table 2 Survival of Rats in the Lifetime Inhalation Study of Talc 0 mg/m3 6 mg/m3 18 mg/m3 M ale Lifetime Study Groups Animals initially in study 49 Moribund Natural deaths Animals surviving to study termination* Percent probability of survival at end of study*3 Mean survival (days)c 23 17 9 18 696 Survival analysis"* P =0.217N Special Study Groupse Animals initially in study 22 Moribund Natural deaths Scheduled evaluation 9 2 11 50 19 17 14 28 707 P=0.422N 22 5 2 15 50 20 14 16 32 711 P=0.192N 22 6 6 10 Fem ales Lifetime Study Groups Animals initially in study 50 Missing* Moribund Natural deaths Animals surviving to study termination Percent probability of survival at end of study Mean survival (days) 0 28 11 11 22 743 Survival analysis P =0.846 Special Study Groups Animals initially in study 22 Moribund Natural deaths Scheduled evaluation 5 2 15 50 1 17 19 13 28 753 P=0.805N 22 3 1 18 50 0 27 14 9 18 758 P = 0.977 22 8 2 12 3 Includes animals that died during the last week of the study b Kaplan-Meier determinations c Mean of all deaths (uncensored, censored, and terminal sacrifice). "* The result of the life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise comparisons (Cox, 1972) with the controls are in the exposed columns. A negative trend or lower mortality in a group is indicated by N. Not included in survival analyses f Censoied from survival analyses Results 27 PROBABILITY OF SURVIVAL PROBABILITY OF SURVIVAL WEEKS ON STUDY WEEKS ON STUDY Fig u r e 1 Kaplan-Meier Survival Curves for Male and Female Rats Administered Talc by Inhalation for Their Lifetime 28 Talc, NTP TR 421 Table 3 Mean Body Weights and Survival of Male Rats in the Lifetime Inhalation Study of Talc W eeks on Study 0 melm3 Av. WL N um ber o f (g) Survivors Av. WL (g) 6 m s/m 3 W l (% o f controls) Number of Survivors Av. WL (g) 18 m e/m 3 WL (% o f controls) Number of Survivors 1 2 3 4 5 6 7 8 9 10 11 13 17 21 25 29a 33 38 41 45 49a 53 57 61 65 69 73 77 81a 85 89 93 97 101 105a 109 113 118 174 201 225 237 250 265 275 287 297 304 317 339 359 374 380 398 407 413 421 431 434 435 443 450 448 453 452 444 450 447 434 429 410 390 377 358 72 72 72 72 72 72 72 72 72 72 72 72 72 72 71 68 68 68 68 68 63 62 62 62 61 61 60 60 55 49 47 43 40 34 29 18 11 121 174 200 215 239 252 263 270 280 293 300 315 338 355 370 378 393 405 412 420 428 432 432 442 444 440 442 441 434 434 437 429 427 395 391 390 389 103 100 100 95 101 101 99 98 98 99 99 100 100 99 99 99 99 100 100 100 99 100 99 100 99 98 98 98 98 97 98 99 100 96 100 104 109 72 119 72 174 72 202 72 219 72 238 72 251 72 263 72 269 72 281 72 293 72 297 72 312 72 331 72 351 72 367 68 369 68 386 68 393 68 401 68 410 65 418 65 422 65 424 65 430 65 432 65 429 65 432 63 429 57 423 53 424 50 424 48 408 41 407 40 394 35 385 19 376 15 342 101 100 101 97 101 100 99 98 98 99 98 98 98 98 98 97 97 97 97 97 97 97 97 97 % 96 95 95 95 94 95 94 95 96 99 100 96 72 72 72 72 72 72 72 72 72 72 72 72 72 72 72 69 69 68 68 68 65 65 65 65 65 65 63 62 59 57 51 46 40 34 28 24 21 Mean for weeks 1-13 246 14-52 391 53-113 428 244 99 389 99 425 99 243 99 381 97 411 96 a Imerim evaluations occurred during weeks 27, 47, 79, and 105. Results 29 Table 4 Mean Body Weights and Survival of Female Rats in the Lifetime Inhalation Study of Talc W eeks on Study 0 m e/m 3 Av. WL N um ber o f (g) Survivors Av. WL (g) 6 m e/m 3 W t (% o f controls) Number of Survivors Av. WL (g) 18 m e/m 3 WL (% o f controls) Number of Survivors 1 2 3 4 5 6 7 8 9 10 11 13 17 21 25 29b 33 38 41 45 49b 53 57 61 65 69 73 77 81b 85 89 93 97 101 105b 109 113 117 121 123 97 126 136 149 153 160 165 168 174 178 181 186 194 206 213 215 224 233 239 248 256 266 276 285 290 296 300 303 300 306 307 307 303 291 288 290 289 283 277 268 72 72 72 72 72 72 72 72 72 72 72 72 72 72 72 68 68 68 68 68 65 65 62 62 61 61 61 61 57 54 52 49 46 44 37 32 24 18 13 13 101 127 139 144 159 165 169 171 176 182 184 191 201 211 216 219 227 237 242 251 259 270 277 288 288 292 295 297 301 302 305 305 304 296 295 288 273 264 264 260 104 101 102 97 104 103 102 102 101 102 102 103 104 103 101 101 101 102 101 101 101 102 101 101 100 99 98 98 100 99 99 99 100 102 103 99 94 93 95 97 72 98 72 125 72 138 72a 145 72 154 72 160 72 166 72 168 72 173 72 179 72 181 72 187 72 197 72 207 72 214 69 213 69 221 69 229 69 235 69 242 66 252 66 260 66 269 66 276 66 277 66 281 64 284 62 284 55 283 55 283 55 287 53 286 50 281 47 271 43 271 28 273 24 260 18 256 14 231 13 231 101 99 101 97 100 101 101 100 100 101 100 101 101 101 100 99 99 98 98 98 98 98 98 97 96 95 95 94 94 93 94 93 93 93 94 94 90 90 84 86 72 72 72 72 72 72 72 72 72 72 72 72 72 72 72 69 69 69 69 69 66 66 65 65 65 65 65 64 59 57 53 49 43 39 33 26 23 21 13 10 Mean tor weeks 1-13 156 14-52 225 53-123 291 159 102 229 102 288 99 156 100 223 99 271 93 a The number of animals weighed for this week is fewer than the number of animals surviving. b Interim evaluations occurred during weeks 27, 47, 79, and 105. 30 Talc, NTP TR 421 MEAN BODY WEIGHT IN GRAMS 500 450- 300- / 250- 8 S 150 .... ii 50- _ ; 3* i**3 9 8 o AAA mi 5 b A .A E! A mo mo 30 45 60 75 WEEKS ON STUDY MALE RATS CONTROL 0 6HC/U3 A ta M G /uj 105 120 135 500450- 350- 300- 250- 200-.........- 8 B8 is?'? 5 .8 544 I " * 8 8 Ba E A A.f..2Ja? AA '50* j r ...... 1001 t........... 50- --T---------------------1------------------------ 1---------------------- 1---------------------- 1---------------------- 1 15 3 0 45 60 75 90 WEEKS ON STUDY FEMALE RATS CONTROL O 6 MG/U3 A 18 MG/M3__ -- I-- 105 120 135 MEAN BODY WEIGHT IN GRAMS Fig u r e 2 Growth Curves for Male and Female Rats Administered Talc by Inhalation for Their Lifetime Results 31 Lung: Absolute and relative lung weights of male rats exposed to 18 mg/m3 were significantly greater than those of controls at the 6-, 11-, and 18-month interim evaluations and at the end of the study, while those of female rats exposed to 18 mg/m3 were significantly greater than those of controls at the 11-, 18-, and 24-month interim evaluations and at the end of the study (Appendix E). Although lung weights of males exposed to 6 mg/m3 were not significantly different from controls at any of the interim evalua tions, those of females at the 18-month interim evaluation and at the end of the lifetime study were significantly greater. Pulmonary lesions in male and female rats occurring in response to the inhalation of talc aerosols were generally similar at the interim evaluations and the end of the study, but varied in incidence, extent, and severity with exposure concentration and duration (Table 5). At necropsy, the lungs of exposed rats had multiple small, round, pale white lesions visible through the visceral pleura. These lesions were generally larger and more extensive in rats exposed to 18 mg/m3 than in those exposed to 6 mg/m3, and at the end of the study than at the earlier interim evaluations. At the 6-month interim evaluation, the pulmonary lesions consisted of multiple, focal accumulations of alveolar macrophages and infrequent neutrophils within alveolar lumens (inflammation, granuloma tous). When viewed under polarized light, the cytoplasm of the alveolar macrophages contained birfringent particles believed to be talc. In two female rats, the alveolar epithelium in some affected areas had increased numbers of low cuboidal type II pneumocytes (alveolar epithelial hyperplasia), but there was no apparent increase in the amount of collagen within the alveolar septa. The peribronchial lymphoid aggregates of several rats also contained focal accumulations of macrophages that varied from a few to approximately 10 cells in the plane of section (peribronchial hyperplasia, histiocytic). In contrast to the first interim evaluation, hyperplasia of type II pneumocytes was associated with the intra alveolar accumulations of macrophages in all exposed rats examined at 11 months. Moreover, in the most severely affected foci, the alveolar septa were thick ened by the accumulation of reticulin and collagen fibers (interstitial fibrosis). The lesions in rats examined at 18 and 24 months and in core study rats were similar but generally larger and more extensive (Plates 1 and 2). Although alveolar macrophages predominated in the inflammatory lesions, varying numbers of neutrophils were also present and the interstitium contained infiltrates of mononuclear inflammatory cells (lymphocytes and macrophages). Moreover, epithelioid macrophages and multinucleated giant cells were also observed within foci of inflammation at these later time points. In some rats, there were well-delineated areas of fibrosis that completely obliterated the alveoli (Plates 3 and 4). Hyperplasia of the alveolar epithelium was often prominent at the margins of these lesions (Plate 5). The affected cells were cuboidal or columnar with prominent nucleoli and exhibited some pleomorphism. In addition to the changes described above, squamous metaplasia of the alveolar epithelium was observed in two male and eight female rats in the 18 mg/m3 groups of the core study (Table 5). The metaplasia was usually associated with inflammation and was characterized by the replacement of alveolar type I and type II pneumocytes by well-differentiated keratinized squamous cells. Squamous cysts were also observed in three males and seven females in the 18 mg/m3 groups and in one 6 mg/m3 female. The cysts had outer walls of well-differentiated, stratified squamous epithelium without cellular atypia and central lumens often containing sloughed keratin. While it was the consensus of the Pathology Working Group that the squamous cysts represented a form of squamous metaplasia, there was some uncertainty regarding the biological potential of these lesions. Clearly, squamous metaplasia in the upper respiratory tract induced by some chemicals is preneoplastic. Currently, however, there is little known about the potential of these squamous cysts for autonomous growth or for progression to malignancy. Although an alveolar/bronchiolar adenoma was observed in one 6 mg/m3 female at the 18-month interim evaluation, the remainder of the pulmonary neoplasms were seen in rats in the core study (Table 6). The incidences of alveolar/bronchiolar adenoma, carcinoma, and adenoma or carcinoma (combined) in female rats exposed to 18 mg/m3were significantly greater than those of controls. A squa mous cell carcinoma was also observed in an 1G mg/m3 female. Alveolar/bronchiolar neoplasms occurred in two males exposed to talc aerosols, one at each of the exposure concentrations, and none were seen in control males. Because of the low number of affected male rats, these neoplasms could not be attributed to talc exposure. 32 Talc, NTP TR 421 Table 5 Incidences of Selected Lung Lesions in Rats in the Lifetime Inhalation Study of Talc 0 mg/m3 M ale 6 mg/m3 18 mg/tn3 0 mg/m3 Fem ale 6 mg/m3 18 mg/m3 6-M onth Interim Evaluation Lung3 Inflammation, Granulomatous6 Peribronchial Hyperplasia, Histiocytic Hyperplasia, Alveolar Epithelium 3 0 0 0 11-M onth Interim Evaluation Lung Inflammation, Granulomatous Peribronchial Hyperplasia, Histiocytic Hyperplasia, Alveolar Epithelium Interstitium, Fibrosis, Focal 2 0 0 0 0 18-M onth Interim Evaluation Lung Inflammation, Granulomatous Peribronchial Hyperplasia, Histiocytic Hyperplasia, Alveolar Epithelium lnterstilium, Fibrosis, Focal 3 1 (1.0) 0 1 (1.0) 0 Alveolar/bronchiolar Adenoma 0 24-M onth Interim Evaluation Lung Inflammation, Granulomatous Peribronchial Hyperplasia, Histiocytic Hyperplasia, Alveolar Epithelium Interstitium, Fibrosis, Focal 3 0 0 0 0 Core Study Lung Inflammation, Granulomatous Peribronchial Hyperplasia, Histiocytic Alveolar Epithelium, Hyperplasia Alveolus, Metaplasia, Squamous Interstitium, Fibrosis, Focal Cyst (Squamous) 49 2 (1.0) 0 5 (2.0) 0 1 (1.0) 0 3 3* (1.3)c 1 (1.0) 0 3 3* (2.3) 2 (2.0) 0 3 0 0 0 3 3* (1.7) 0 3* (2.0) 2 (1.0) 3 3* (3.0) 0 3* (1.7) 3* (1.0) 3 0 0 0 0 3 3 (1.3) 2 (1.0) 3 (1.0) 3* (1.0) 0 2 2 (2.0) 2 (1.0) 2 (1.0) 2 (1.5) 0 3 0 0 1 (1.0) 0 0 6 6* (1.5) 1 (1.0) 6* (1.0) 5* (1.0) 2 2 (2.0) 1 (2.0) 2 (1.5) 2 (1.5) 5 1 (1.0) 0 1 (1.0) 0 50 50**(1.6) 12* *(1.3) 26**(1.3) 0 16**(1.2) 0 50 49* *(2.3) 8** (1.9) 38**(1.7) 2 (1.0) 33**(1.8) 3 50 2 (1.5) 0 2 (1.0) 0 1 (1.0) 0 3 3* (1.3) 1 (1.0) 1 (1.0) 3 3* (3.0) 2 (1.0) 1 (1.0) 3 3* (1.7) 1 (1.0) 3* (1.0) 2 (1.0) 3 3* (2.7) 2 (1.5) 3* (2.3) 3* (1.0) 3 3* (1.7) 1 (1.0) 3 (1.0) 3* (1.3) 1 3 3* (2.0) 2 (1.0) 3 (1.3) 3* (1.7) 0 9 9**(1.4) 2 (1.0) 9**(1.4) 8**(1.4) 3 3 (1.7) 0 3 (2.3) 3* (3.0) 48 47**(1.5) 8** (1.3) 27* *(1.2) 0 24**(1.5) 1 50 50* *(2.8) 9* *(1.3) 47**(2.1) 8** (1.1) 45* *(2.1) 7*. Significantly different (P<0.05) from the control by Fisher's exact test (interim evaluation) or logistic regression (lifetime study) PiO.Ol 3 Number of animals with lung examined microscopically. 6 Number of animals with lesion. c Average severity grades of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked Results 33 Table 6 Incidences of Lung Neoplasms in Rats in the Lifetime Inhalation Study of Talc 0 m g/m 3 M ale 6 mg/m3 18 m g/m 3 0 m g/m 3 Fem ale 6 mg/m3 18 mg/m3 Core Study Alveolar/bronchiolar Adenoma Overall rales* Terminal rates'* First incidence (days) Logistic regression testc 0/49 (0%) 0/9 (0%) P=0.494 Alveolar/bronchiolar Carcinoma Overall rates Terminal rates First incidence (days) Logistic regression test 0/49 (0%) 0/9 (0%) P=0.370 Alveolar/bronchiolar Adenoma or Carcinoma Overall rates 0/49 (0%) Terminal rates 0/9 (0%) First incidence (days) - Logistic regression test P=0.494 Squamous Cell Carcinoma Overall rates 0/49 (0%) 1/50 (2%) 0/14 (0%) 781 P =0.527 0/50 (0%) 0/14 (0%) - 1/50 (2%) 0/14 (0%) 781 P=0.527 0/50 (0%) 1/50 (2%) 1/16 (6%) 799 (T) P=0.615 1/50 (2%) 1/16 (6%) 799 (T) P=0.615 1/50 (2%) 1/16 (6%) 799 (T) P=0.615 0/50 (0%) 1/50 (2%) 0/11 (0%) 805 P < 0.001 0/50 (0%) 0/11 (0%) P =0.003 1/50 (2%) 0/11 (0%) 805 P <0.001 0/50 (0%) 0/48 (0%) 0/13 (0%) P=0.503N 0/48 (0%) 0/13 (0%) - 0/48 (0%) 0/13 (0%) P=0.503N 0/48 (0%) 9/50 (18%) 1/9 (11%) 716 P =0.010 5/50 (10%) 3/9 (33%) 828 P =0.028 13/50 (26%) 4/9 (44%) 716 P <0.001 1/50 (2%) (T) Terminal sacrifice * Number of neoplasm-bearing animals/number of animals examined microscopically. b Observed incidence at terminal kill c Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The logistic regression test regards these lesions as nonfalal. A lower incidence in an exposure group is indicated by N. d Not applicable; no neoplasms in animal group The adenomas were irregular, circumscribed masses consisting of cuboidal to columnar epithelium ar ranged in alveolar, tubular, or papillary formations and separated by varying amounts of collagenous connective tissue. The neoplastic epithelium general ly formed a single layer and was relatively uniform with minimal cellular atypia. The carcinomas were distinguished from the adenomas on the basis of having greater cellular pleomorphism and atypia, but they exhibited little evidence of invasion and none metastasized (Plates 6 and 7). In several benign and malignant neoplasms, the central portion of the mass was composed primarily of dense collagen and the epithelial component was located at the periphery. The extent of fibrosis in these neoplasms is not typical of spontaneous alveolar/bronchiolar neo plasms in control F344/N rats. The fibrous connec tive tissue was not interpreted as being a primary scirrhous response to the neoplastic epithelium, but rather a component of the prolonged inflammatory reaction to talc. Lymph node: Histiocytic hyperplasia, consisting of accumulations of macrophages in the subscapular and medullary sinuses, occurred in the bronchial lymph nodes (male: 0 mg/m3, 0/41; 6 mg/m3, 44/48; 18 mg/m3, 46/49; female: 0/46, 40/47, 45/47) and in the mediastinal lymph nodes of rats exposed to talc (male: 0/48, 40/49, 43/47; female: 0/47, 33/44, 40/47) (Tables A4 and B4). The macrophages had foamy cytoplasm filled with birfringent particles of talc. Nose: Hyperplasia of the respiratory epithelium of the nasal mucosa occurred in three male rats exposed to 6 mg/m3 and 14 male rats exposed to 18 mg/m3, but not in the control group (Table A4). The lesion consisted of an increase in the number of goblet cells 34 Talc, NTP TR 421 primarily in the mucosa of the nasal septum. Hyper plasia of the respiratory epithelium also occurred in several female rats, but the incidences in the exposed groups were not significantly increased (Table B4). During the pathology review process, it was noted that male and female rats in control and exposed groups had large eosinophilic droplets in the cyto plasm of the olfactory and, to a lesser extent, the respiratory epithelium. The lesion (cytoplasmic alteration) was focal or multifocal and usually located near the junction of the two epithelial types. Al though present in the controls, the incidences were increased in exposed rats (males: 3/49, 18/48, 40/47; females: 5/48, 23/45, 46/48). Adrenal medulla: Focal adrenal medulla hyperplasia or pheochromocytoma were observed in rats at the various interim evaluations, but the number of affected rats was too small to draw definitive conclu sions. However, in the core study, benign, malignant, or complex (combined) pheochromocytomas occurred with a significant positive trend in male and female rats, and the incidences in the 18 mg/m3groups were significantly greater than those of controls by pairwise comparisons (Table 7). Moreover, bilateral pheo chromocytomas were more frequent in exposed male rats than in controls (Tables A3 and B3). Although adrenal medulla hyperplasia occurred with similar frequency among exposed and control female rats, the incidences of hyperplasia in exposed males were significantly lower than controls. The lower inci dences in exposed males are possibly due, in part, to the reduced amount of normal medullary tissue (e.g., medullary tissue without a pheochromocytoma) in which to observe hyperplasia. aggregates, large solid clusters, and/or trabecular cords several layers thick separated by a delicate fibrovascular stroma. The larger neoplasms usually exhibited greater cellular pleomorphism and atypia than smaller neoplasms. Because the only morpho logical criterion that unambiguously distinguishes malignant from benign pheochromocytomas is frank invasion or metastasis, a diagnosis of malignant pheochromocytoma was made only when there was invasion of the capsule. Complex pheochromocyto mas consisted of an admixture of neoplastic pheochromocytes and neuroblasts, ganglion cells, and/or Schwann cells. Lung Talc Burden The lung talc burdens of exposed rats, normalized to control lung weight or exposure level, are presented in Tables G2 and G3. The lung talc burden normal ized to control lung weight (mg talc/g control lung) adjusts for differences in lung weight between sexes or at different ages. The lung burden normalized to control lung weight and exposure level adjusts for exposure level to determine the effect of exposure concentration on talc clearance from the lung. Hie data, normalized to control lung weight, show that talc burdens of rats exposed to 6 mg/m3 were similar between males and females and increased progressively from 6 to 24 months (Table G2). Lung talc burdens in females exposed to 18 mg/m3 also increased progressively from 6 to 24 months. In contrast, lung talc burdens of males at the 18 mg/m3 exposure concentration increased from 6 to 18 months, but remained about the same at 18 and 24 months. Focal hyperplasia and pheochromocytoma constitute a morphological continuum. Focal hyperplasia consisted of irregular, small foci of small to normal sized medullary cells arranged in packets or solid clusters slightly larger than normal; compression of the surrounding tissue was minimal or absent. Pheochromocytomas were generally larger than focal hyperplasia and caused variable compression of the surrounding parenchyma; many obscured much or all of any remaining normal medullary tissue. The neoplastic cells were arranged in variably sized The exposure-normalized data show that lung talc burdens were generally proportional to exposure concentration at each interim evaluation. The exposure-normalized lung burdens of rats exposed to 6 or 18 mg/m3 were generally similar at each of the interim evaluations except for slight increases for males at 6 and 11 months and females at 6 months (Table G3). This suggests that either clearance of talc was not substantially impaired by increasing the exposure concentration, or that clearance of talc was impaired similarly at both exposure levels. Results 35 Table 7 Incidences or Neoplasms and Nonneoplastic Lesions of the Adrenal Medulla in Rats in the Lifetime Inhalation Study of Talc 0 mg/m3 M ale 6 mg/m3 18 m g/m 3 0 m g/m 3 Fem ale 6 mg/m3 18 mg/m3 11-M onth Interim Evaluation Adrenal Medulla* 233333 Hyperplasia1* ' 000000 Pheochromocytoma, Benign 1 0 0 0 0 0 18-M onth Interim Evaluation Adrenal Medulla 332233 Hyperplasia 0 100 11 Pheochromocytoma, Benign 0 0 1 0 0 0 24-M onth Interim Evaluation Adrenal Medulla 362593 Hyperplasia 220300 Pheochromocytoma, Benign 0 2 0 0 4 0 Pheochromocytoma, Benign, Bilateral 1120 13 Core Study Adrenal Medulla Hyperplasia 49 48 47 48 47 49 20 8** 9* 22 20 16 Pheochromocytoma, Benign Overall rates0 Terminal rates'* First incidence (days) Logistic regression testc 25/49 (51%) 6/9 (67%) 429 P =0.007 30/48 (63%) 11/14 (79%) 558 P=0.213 Pheochromocytoma, Malignant Overall rates Terminal rates First incidence (days) Logistic regression test 3/49 (6%) 1/9 (11%) 670 P =0.096 3/48 (6%) 1/14 (7%) 544 P=0.662 Pheochromocytoma, Complex Overall rates Terminal rates First incidence (days) Logistic regression test 0/49 (0%) 0/9 (0%) - P=0.486 2/48 (4%) 1/14 (7%) 558 P = 0.230 Pheochromocytoma, Benign, Malignant, or Complex Overall rates 26/49 (53%) 32/48 (67%) Terminal rates 7/9 (78%) 12/14 (86%) First incidence (days) 429 544 Logistic regression test P =0.007 P=0.147 36/47 (77%) 16/16 (100%) 614 P = 0.009 13/48 (27%) 5/11 (45%) 678 P=0.185 7/47 (15%) 3A6 (19%) 645 P=0.178 0/48 (0%) 0/11 (0%) P < 0.001 1/47 (2%) 0/16 (0%) 743 P = 0.503 0/48 (0%) 0/11 (0%) - 37/47 (79%) 16/16 (100%) 614 P-0.006 13/48 (27%) 5A1 (45%) 678 P-0.014 14/47 (30%) 5/13 (38%) 705 P =0.541 1/47 (2%) 0/13 (0%) 849 P = 0.509 0/47 (0%) 0A3 (0%) - 14/47 (30%) 5A3 (38%) 705 P-0.541 18/49 (37%) 6/9 (67%) 697 P =0.225 10/49 (20%) 3/9 (33%) 784 P = 0.001 0/49 (0%) 0/9 (0%) - 23/49 (47%) 8/9(89% ) 697 P -0.024 Significantly different (PS0.05) from the control by logistic regression PsO.Ol * Number of animals with adrenal medulla examined.microscopically. b Number of animals with lesion. c Number of animals with neoplasm per number of animals with adrenal medulla examined microscopically. d Observed incidence at terminal kill e Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The logistic regression test regards these lesions as nonfatal. f Not applicable; no neoplasms in animal group 36 Talc, NTP TR 421 Pulmonary Function Results of the respiratory function measurements are presented in Tables G9 through G41. A progressive dose and time-related impairment of respiratory function was observed in both male and female rats exposed to talc. The impairment was restrictive in nature, consisting of reduced lung volume, increased lung stiffness, reduced gas exchange efficiency, and nonuniform intrapulmonary gas distribution. 6-Month Interim Evaluation: At 6 months there were few significant differences between values for rats exposed to 18 mg/m3and controls, and no significant differences between values for rats exposed to 6 mg/m3 and controls. There were, however, slight trends among both males and females toward smaller lung volumes and reduced forced expiratory flow. Total lung capacity, vital capacity, and forced vital capacity were all slightly smaller in the 18 mg/m3 groups, but only the forced vital capacity of females differed significantly from controls. All forced expiratory flow rates were lower in the 18 mg/m3 groups, but only those of males were significantly lower than those of the controls. The reduced flow rates were partly related to the smaller lungs, but even volume-normalized flow tended to be reduced in the exposed rats. The reduced flow rates most likely reflected changes in small airways. Total pulmonary resistance, which primarily reflects flow resistance in large airways, was unaffected. 11-Month Interim Evaluation: Functional alterations were clearly apparent in exposed males and females after 11 months. Total lung capacity, vital capacity, and forced vital capacity were significantly lower in males and females exposed to 18 mg/m3 and males exposed to 6 mg/m3. The reduced volume was accompanied by significant reductions in quasistatic lung compliance in males, and both dynamic and quasistatic lung compliance in females. The volume and compliance changes indicate a stiffening of the lung (or increase in elastic recoil). Forced expiratory flows during mid to late expiration were slightly lower in exposed males than in controls, but the differences were not significant. A reduction of alveolar-capillary gas exchange effi ciency was reflected by a significant reduction of carbon monoxide diffusing capacity in the 18 mg/m3 male and female rat groups. Although diffusing capacity is somewhat volume dependent, the reduced lung volume did not completely account for the change. Volume-normalized diffusing capacity was also significantly reduced in male and female rats exposed to 18 mg/m3. 18-Month Interim Evaluation: Total lung capacity, vital capacity, and forced vital capacity of all exposed groups of male and female rats were significantly lower than those of controls at 18 months, except for vital capacity of males exposed to 6 mg/m3. In females exposed to 18 mg/m3, these decreases were accompanied by significant increases in resting (functional residual capacity) and minimum (residual) volumes. The decrease in volume at maximum inflations (total lung capacity, vital capacity, and forced vital capacity) reflected the inability of the stiffened lungs to stretch normally. Volumenormalized forced expiratory flows of exposed male and female rats were generally greater than those of controls, due to the reduced lung volume and little or no reduction in flow. All parameters of lung compliance in male and female rats exposed to 18 mg/m3were also significant ly lower than controls at 18 months, while two of the three compliance parameters were significantly lower at the 6 mg/m3exposure level. The carbon monoxide diffusing capacities in males and females exposed to 18 mg/m3 were significantly lower than controls at 18 months, which is consistent with the findings at 11 months. The slope of phase III of the single-breath N2wash out of male and female rats exposed to 18 mg/m3was significantly greater than controls, apparently due to uneven mixing of oxygen with residual nitrogen in the lung during maximal inflation. This finding reflects a nonuniform distribution of inhaled air. 24-Month Interim Evaluation: Because of reduced survival in all groups of male and female rats, fewer animals remained alive at 24 months for evaluation of pulmonary function. Because of the smaller group sizes (three rats each from the control and 18 mg/m3 groups were evaluated), few of the differences were statistically significant. Nevertheless, there were reductions in lung volume parameters (total lung capacity, vital capacity, and forced vital capacity), lung compliance, and carbon monoxide diffusing capacity in exposed male and female rats consistent with the findings at the earlier time periods. Results 37 The progression of the functional impairments over the course of the study are illustrated in Figure 3, which plots the data for three functional parameters obtained from the three male and three female rats in the 18 mg/m3 exposure groups surviving until 24 months. Bronchoalveolar Lavage and Lung Biochemistry Following the completion of the pulmonary function tests at the 24-month interim evaluation, broncho alveolar lavage was performed on the remaining rats in these groups and the lavage fluid was evaluated for enzymes, protein, and cell content as shown in Tables G4 and G5. Values for glucose-6-phosphate dehydrogenase and glutathione peroxidase are not reported because they were below the limits of detection. The values for C-glucuronidase, alkaline phosphatase, lactate dehydrogenase, and total protein in both male and female rats exposed to 18 mg/m3 talc were significantly greater than those of controls. In addition, females in this group had a significantly higher value of glutathione reductase. Both male and female rats exposed to 6 mg/m3talc had significantly greater B-glucuronidase values, but only female rats exposed to 6 mg/m3 had higher values of alkaline phosphatase, lactate dehydrogenase, and protein. The percentages of polymorphonuclear leukocytes in the lavage fluid were also significantly greater in male and female rats exposed to talc at both concentration levels. The increases in enzymes, total protein, and leukocytes are consistent with the morphological findings of a chronic active inflammatory process and cellular degenerative changes. The viability and phagocytic activity of alveolar macrophages recovered from the lungs of rats ex posed to 6 or 18 mg talc/m3 or from the chamber controls ranged from approximately 60% to 80%. Neither the viability nor phagocytic activity were significantly affected by exposure to talc (Table G6). Table G7 summarizes the effects of talc exposure on collagen metabolism and protein synthesis. Collage nous peptides in lavage fluid and collagen production (% newly synthesized protein) from female rats, but not males, exposed to 6 or 18 mg/m3were significant ly greater than controls. Total lung collagen from males and females at both exposure levels was also significantly greater. Values for non-collagenous protein synthesis were significantly greater in males exposed to 6 or 18 mg/m3 and in females exposed to 18 mg/m3than in controls. Lung proteinase activity, as determined from lavage fluid and homogenate supernatant fluid, is shown in Table G8. Acid proteinase activity, primarily cathepsin D, was significantly greater in both males and females exposed to 6 or 18 mg/m3 than in controls. Neutral proteinase activity in homogenate superna tant fluid was also greater in rats exposed to talc. The activity was mostly serine proteinase, like that of polymorphonuclear leukocyte elastase and cathepsin G. 38 JOT Talc, NTP TR 421 P e r c e n t of C o n t r o l M e a n V a l u e (+- SE) -1t0- --H ao 40 00 00 70 w e e k s of E x p o s u r e --- O u a s i s t a t i c C o m p l i a n c e " T o t al L u n g C a p a c i t y A.. C O D i f f u s i n g C a p a c i t y Figure 3 Effect of 18 mg Talc/m3 Exposure on Respiratory Function of Male and Female Rats Surviving to 104 Weeks Results 39 M ice 4-W eek Study D ose S election Selection of 6 and 18 mg talc/m3 as the exposure concentrations was based on the results of a 4-week inhalation study in B6C3F, mice to determine lung talc burden and histopathologic changes associated with talc exposure. These studies indicated that the amount of talc retained in the lung was similar between sexes and proportional to exposure concen tration (Appendix K). Microscopic examination of the lungs revealed an accumulation of alveolar macrophages in the lungs only at 18 mg/m3. Based on these findings it was predicted that aerosol con centrations greater than 18 mg/m3would overwhelm lung clearance mechanisms, impair lung function, and possibly shorten survival. 2-Year Study Survival Estimates of survival probabilities for male and female mice are shown in Table 8 and in the Kaplan-Meier curves in Figure 4. Survival of male and female mice exposed to talc was similar to that of the controls throughout most of the study. One female mouse exposed to 18 mg/m3 died on day 20 and six others died of undetermined causes on day 28 of the study. Body Weights and Clinical Findings Mean body weights of male and female mice exposed to talc were similar to controls throughout the study (Tables 9 and 10, and Figure 5). There were no clinical findings in exposed mice that could be attrib uted to exposure to talc. Prior to the start of the study and after 6 months of exposure, all serological tests were negative. At 12 months, 8/24 mice were positive for mouse hepati tis virus (MHV), but retesting of the serum by the enzyme linked immunosorbent assay (ELISA) showed all to be negative (Table Kl). At the end of the study, 7/30 were positive for Mycoplasma arthritidis and 21/30 were positive for epizootic diarrhea of infant mice (EDIM). No clinical signs or gross or microscopic evidence of disease associated with M. arthritidis was observed. EDIM does not cause clinical disease or pathology in adult mice. 40 Talc, NTP TR 421 Table 8 Survival of Mice in the 2-Year Inhalation Study of Talc 0 mg/ra3 6 mg/m3 18 mg/m3 M ale Core Study Groups Animals initially in study 50 Missexed Missing Moribund Natural deaths Animals surviving to study termination Percent probability of survival at end of studyb Mean survival (days)c 1 2 1 16 30 65 648 Survival analysis'1 P=0.886N Special Study Groupse Animals initially in study Missing Moribund Natural deaths Scheduled evaluation 39 0 0 4 35 50 1 1 2 18 28 58 648 P=0.771 40 1 1 5 33 50 0 1 3 14 32 66 645 P = 1.000N 40 1 1 7 31 F em ales Core Study Groups Animals initially in study 50 Culled Missing Moribund Natural deaths Animals surviving to study termination Percent probability of survival at end of study Mean survival (days) 0 1 2 17 30 62 663 Survival analysis P =0.321 Special Study Groups Animals initially in study 39 Moribund Natural deaths Scheduled evaluation 2 7 30 50 1 1 4 21 23 48 648 P = 0.322 40 5 5 30 50 0 0 4 21 25 50 590 P =0.286 40 1 10 29 Censored from survival analyses b Kaplan-Meier determinations c Mean of all deaths (uncensored, censored, and terminal sacrifice). d The result of the life table trend lest (Tarone, 1975) is in the control column, and the results of the life table pairwise comparisons (Cox, 1972) with the controls are in the exposed columns. A negative trend or lower mortality in an exposure group is indicated by N. e Not included in survival analyses Results 41 PROBABILITY OF SURVIVAL 0 15 30 *3 0 75 9 0 105 120 WEEKS ON STUDY Figure 4 Kaplan-Meier Survival Curves for Male and Female Mice Administered Talc by Inhalation for 2 Years 42 Talc, NTP TR 421 Table 9 Mean Body Weights and Survival of Male Mice in the 2-Year Inhalation Study of Talc Week on Study _____ 0 me/m1_____ ______________ 6 ma/m3_______________ ____________ 18 mg/m3 Av. W t N u m b e r o f Av. WL WL (% o f N u m b e r o f Av. WL WL (% of (8) Survivors (8) controls) Survivors (g) controls) Number of Survivors i 23.3 50 23.8 102 2 24.0 48 23.9 100 3 25.0 47 25.4 102 4 25.4 47 26.4 104 5 26.1 47 26.2 100 6 27.3 47 27.4 100 7 27.8 47 27.4 99 8 25.8 47 27.9 108 9 28.1 47 28.3 101 10 28.8 47 28.5 99 11 29.1 47 29.5 101 12 29.0 47 29.2 101 13 30.1 47 30.5 101 17 31.5 47 30.8 98 21 32.2 47 30.9 96 25 33.4 47 31.8 95 29 33.0 47 32.3 98 33 33.9 47 33.3 98 37 34.7 47 34.2 99 42 35.7 47 35.4 99 45 36.9 47 36.0 98 49 36.4 47 35.5 98 53 36.4 47 36.6 101 57 36.9 47 35.8 97 61 36.8 46 37.6 102 65 37.2 44 37.1 100 69 36.5 44 37.1 102 73 37.2 42 36.5 98 77 36.9 41 35.1 95 81 37.6 40 36.8 98 85 37.0 35 37.1 100 89 36.7 35 35.9 98 93 34.9 34 36.3 104 97 34.2 33 35.2 103 101 33.9 31 34.1 101 Mean f o r weeks 1-13 26.9 14-52 34.2 53-101 36.3 27.3 101 33.4 98 36.2 100 50 23.7 49 24.3 49 24.8 49 25.0 49 26.6 49 26.9 49 27.5 49 29.7 48 28.5 48 28.7 48 28.3 48 28.7 48 29.8 48 31.0 48 31.4 48 32.5 48 32.7 48 33.2 46 33.8 46 34.7 46 35.7 45 35.5 44 36.3 44 35.7 43 36.6 43 36.4 43 36.0 43 35.1 43 35.0 40 35.2 37 35.2 37 34.8 34 33.4 34 33.3 31 33.3 102 101 99 98 102 99 99 115 101 100 97 99 99 98 98 97 99 98 97 97 97 98 100 97 100 98 99 94 95 94 95 95 % 97 98 27.1 101 33.4 98 35.1 97 50 50 50 50 49 49 49 49 49 49 49 49 49 47 47 46 46 46 46 46 46 46 46 46 45 44 42 42 42 39 39 38 38 36 36 Results 43 Table 10 Mean Body Weights and Survival of Female Mice in the 2-Year Inhalation Study of Talc W eek OD Study ______ 0 mg/m*_______ _______________ 6 m e/m 3__________________ Av. W t. N u m b er of Av. W t. W t. (% of N um ber of (8 ) Survivors (8) controls) Survivors ______________ 18 m g/m 3 Av. W t. W t. (% of (8) controls) Number of Survivors i 19.3 50 19.3 100 2 19.9 50 20.1 101 3 21.0 50 213 101 4 22.4 50 22.5 100 5 22.5 49 22.7 101 6 24.4 49 23.7 97 7 24.6 49 24.5 100 8 22.1 49 24.2 110 9 24.6 49 24.9 101 10 25.2 49 25.4 101 11 25.6 49 26.2 102 12 25.5 49 25.1 98 13 26.3 49 26.4 100 17 27.5 49 26.7 97 21 28.4 49 27.2 96 25 29.5 49 28.1 95 29 29.8 49 28.6 96 33 30.1 49 29.7 99 37 30.7 49 29.9 97 42 31.7 49 30.8 97 45 32.4 49 31.7 98 49 32.2 49 31.2 97 53 32.7 49 31.4 96 57 32.7 49 31.0 95 61 33.1 49 32.9 99 65 33.0 48 32.4 98 69 32.7 47 32.4 99 73 32.8 43 32.1 98 77 32.6 43 31.3 96 81 33.5 41 32.7 98 85 32.5 40 33.0 102 89 32.7 39 32.1 98 93 31.7 37 31.7 100 97 31.5 35 31.7 101 101 31.8 31 31.4 99 Mean for weeks 1-13 23.3 14-52 30.3 53-101 32.6 23.6 101 29.3 97 32.0 98 50 19.6 102 50 20.5 103 50 21.1 101 49 21.5 96 49 23.2 103 49 23.8 98 49 24.3 99 49 26.8 121 49 25.2 102 49 25.3 100 49 25.0 98 49 25.2 99 49 25.9 99 47 27.3 99 47 27.7 98 47 28.9 98 47 28.9 97 47 29.5 98 47 29.9 97 47 30.3 96 47 31.1 96 47 31.0 96 47 31.9 98 47 31.2 95 46 32.3 98 46 32.7 99 46 32.1 98 44 31.0 95 43 31.3 96 39 32.1 96 39 32.7 101 36 32.1 98 33 31.2 98 30 30.6 97 27 31.0 98 23.6 101 29.4 97 31.7 97 50 50 50 49 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 43 42 41 40 37 35 35 33 30 27 44 Talc, NTP TR 421 MEAN BODY WEIGHT IN GRAMS MEAN BODY WEIGHT IN GRAMS 33- 30- IB 'a " ! .......... I ' * B " ........ co .. a ..... O <3 n mo .............. 8* 23- / 20- i . 15 FEMALE MICE C0K70L O IMCA(J A ta MO/M3 30 43 60 75 WEEKS ON STUDY ~1-- 90 105 F ig u r e 5 Growth Curves for Male and Female Mice Administered Talc by Inhalation For 2 Years Results 45 Pathology and Statistical Analyses o f Results This section describes the statistically significant or biologically noteworthy changes in the incidences of neoplastic or nonneoplastic lesions of the lung, lymph node, and nose. Summaries of the incidences of nonneoplastic lesions and neoplasms, the individual animal tumor diagnoses, and the statistical analyses of primary neoplasms that occurred with an incidence of at least 5% in at least one group are presented in Appendix C for male mice and Appendix D for female mice. granular, eosinophilic cytoplasm filled with birefrin gent particles of talc. Nose: The incidences of focal cytoplasmic alteration were increased in groups of mice exposed to talc (male: 5/45, 23/46, 40/47; female: 29/46, 37/46, 40/50; Tables C4 and D4). Focal cytoplasmic alteration was characterized by the formation of large eosinophilic droplets in the cytoplasm of olfactory and respiratory epithelial cells and was similar to that observed in rats. Lung: Absolute and relative lung weights of male and female mice exposed to 18 mg talc/m3 were significantly greater than those of the controls at the 12-month interim evaluation and at the end of the study. Absolute lung weights of 18 mg/m3males and absolute and relative lung weights of 18 mg/m3 females were significantly greater at the 18-month interim evaluation. Lung weights of mice exposed to 6 mg/m3 were similar to controls at each of the interim evaluations. Lung Talc Burden . The lung talc burdens, normalized to control lung weight or exposure level, are presented in Tables H2 and H3. Lung talc burden normalized to control lung weights (mg talc/g control lung) adjusts for differences in lung weight between sexes or at differ ent ages. The lung burden normalized to control lung weight and exposure level adjusts for exposure level to determine the effect of exposure concentra tion on talc clearance from the lung. The pulmonary lesions in mice exposed to talc were similar at the interim evaluations and at the end of the study, but the lesions varied in extent and severity with exposure concentration and duration (Table 11). The principal lung lesion occurring in exposed mice was an accumulation of alveolar macrophages in the alveoli surrounding terminal bronchioles (hyper plasia, macrophage) (Plate 8). The macrophages had abundant, slightly foamy to granular, eosinophilic cytoplasm containing birefringent talc particles. Small numbers of neutrophils were sometimes observed in the affected areas, and the interstitium contained infiltrates of mononuclear inflammatory cells (inflammation, chronic active) (Plates 9 and 10). In contrast to the pulmonary lesions in rats, hyper plasia of type II pneumocytes or fibrosis were not prominent components of the lesions in mice. The incidences of pulmonary neoplasms were similar among exposed groups and controls. Lymph node: The bronchial lymph nodes of mice exposed to talc contained accumulations of macro phages in the medullary sinuses (hyperplasia, histio cytic - male: 0 mg/m3, 1/32; 6 mg/m3, 32/39; 18 mg/m3, 42/44; female: 0/38, 25/37, 39/43; Tables C4 and D4). The macrophages had abundant, slightly foamy to The data, normalized to control lung weight, show that talc burdens of mice exposed to 6 mg/m3 were similar between males and females and increased progressively from 6 to 24 months, except for males at 18 months (Table H2). However, because of the small sample size of males at 18 months (two ani mals), the lung talc burden of this sample may not be representative of the group as a whole. The lung talc burdens of mice exposed to 18 mg/m3 were also similar between sexes at each interim evaluation. Although the talc burdens of males and females increased substantially from 6 to 24 months, the values at 12 and 18 months were similar. The exposure-normalized data show that lung talc burdens of mice exposed to 18 mg/m3were dispropor tionately greater than those of mice exposed to 6 mg/m3 (Table H2). The slight increases in expo sure-normalized lung talc burden were statistically significant in males and females at 12 and 24 months, but not at 6 or 18 months. The lack of statistical significance at 18 months might be explained, in part, by the small sample size. These data suggest that clearance of talc from the lung was impaired, or impaired to a greater extent, in mice exposed to 18 mg/m3 than in mice exposed to 6 mg/m3. 46 Talc, NTP TR 421 Table 11 Incidences of Selected Lung Lesions in Mice in the 2-Year Inhalation Study of Talc 0 mg/m3 M ale 6 mg/m3 18 mg/m3 0 mg/m3 Fem ale 6 mg/m3 18 mg/m3 6-M onth Interim Evaluation Lung* Hyperplasia, Macrophage Inflammation, Chronic Active 4 0 0 4 3 (1.0)c 0 4 4* (1.0) 1 (1.0) 4 0 0 4 0 0 12-M outh Interim Evaluation Lung Hyperplasia, Macrophage Inflammation, Chronic Active 4 0 0 4 4* (1.0) 0 4 4* (1.8) 2 (2.0) 3 0 0 4 4* (1.0) 0 18-M onth Interim Evaluation Lung Hyperplasia, Macrophage Inflammation, Chronic Active Alveolar/bronchiolar Adenoma Alveolar/bronchiolar Carcinoma 4 0 0 0 .1 4 4* (1.3) 0 1 0 4 4* <2.5) 2 (1.5) 0 0 4 0 0 1 0 4 4* (L3) 0 0 0 2-Year Study Lung Hyperplasia, Macrophage Inflammation, Chronic Active Alveolar Epithelium, Hyperplasia Alveolar/bronchiolar Adenoma Overall rates" Logistic regression te st' 4 3 (23) 0 1 (1.6) 47 46**(1.4) 16**(1.1) 0 48 48* *(2.8) 40* *(2.2) 0 46 2 (2.5) 0 0 48 45**(1.6) 25**(1.4) 0 6/45 (13%) P-0.251 4/47 (9%) P=0.411N 9/48 (19%) 3/46 (7%) P-0371 P-0.467N 2/49 (4%) P-0.499N Alveolar/bronchiolar Carcinoma Overall rates Logistic regression test 7/45 (16%) P=0.069N 2/47 (4%) P-0.073N 2/48 (4%) P-0.070N 2/46 (4%) P --0325N 4/49 (8%) P --0.356 Alveolar/bronchiolar Adenoma or Carcinoma Overall rates 12/45 (27%) Logistic regression test P-0.522N 5/47 (11%) P-0.043N 11/48 (23%) 5/46 (11%) P-0.423N P --0.269N 6/49 (12%) P -0 3 1 9 4 4* (1.0) 0 4 4* (2.0) 1 (3.0) 4 4* (Z5) 0 0 0 50 43**(2.8) 38 *(23) 1 (1.0) 2/50 (4%) P-0.515N 1/50 (2%) P-0.500N 3/50 (6%) P-0.367N Significantly different (P<0.05) from the control by Fisher's exact test (interim evaluation) or logistic regression (2-year study) PsO.Ol * Number of animals with lung examined microscopically. b Number of animals with lesion. c Average severity grades of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked d Number of animals with neoplasm per number of animals examined microscopically. e Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the control and that exposed group. The logistic regression test regards these lesions as nonfatai. A negative trend or a lower incidence in an exposure group is indicated by N. Results 47 Bronchoalveolar Lavage and Lung Biochemistry Bronchoalveolar lavage was performed and lung homogenate supernatants collected for analyses at 6, 12, 18, and 24 months. A summary of the changes occurring in bronchoalveolar fluid enzymes, protein and cells are shown in Tables H4 through H22. Values for glucose-6-phosphate dehydrogenase, glutathione peroxidase, and alkaline phosphatase were not reported because they were below the limit of detection. B-Glucuronidase activity of lavage fluid from male and female mice exposed to 18 mg/m3 was greater than that of controls at 12, 18, and 24 months, but not at 6 months. In mice exposed to 6 mg/m3, fi-glucuronidase activity was greater than that of controls only at the 24-month interim evaluation. Lactate dehydrogenase and glutathione reductase activities in male and female mice exposed to 18 mg/m3 were significantly greater than those of controls at 18 and 24 months. Glutathione activity of males exposed to 18 mg/m3was also greater than that of controls at 12 months. Values for total protein in lavage fluid from males and females in the 18 mg/m3 groups were significantly greater than those of controls at 18 months; at 24 months only that of males was significantly greater. Significant differences in total and differential cell counts between exposed and control mice were observed only at 18 and 24 months at the high concentration level (Tables H8 to H ll). The num bers of total nucleated cells, polymorphonuclear leukocytes, and macrophages were significantly greater in males and females exposed to 18 mg/m3 than in controls. Exposure of mice to 6 or 18 mg talc/m3 produced a concentration-related decrease in phagocytic activity of macrophages derived from lavage fluid (Tables H12 to H14). The number of macrophages containing phagocytized sheep erythrocytes from male and female mice exposed to 18 mg/m3was significantly lower than that from control mice at 12, 18, and 24 months. Although phagocytic activity of macrophages from mice exposed to 6 mg/m3 was intermediate between controls and the high concentration groups, only the difference between the exposed and control males at 12 months was statistically significant. The effects of talc exposure on lavage fluid collage nous peptides and total lung collagen are shown in Tables H15 through H18. The amount of collage nous peptides in lavage fluid from male mice exposed to 18 mg/m3 was significantly greater than that of controls at 12, 18, and 24 months, while collagenous peptides of females exposed to 18 mg/m3were signifi cantly increased only at 24 months. Consistent with these findings, total lung collagen was significantly greater in 18 mg/m3 at 18 and 24 months and in females at 24 months. Collagenous peptides and total lung collagen from mice exposed to 6 mg/m3 were similar to controls at each of the interim evaluations. The acid and neutral proteinase activity of lung homogenate supernatant fluid and the acid proteinase activity of lavage fluid are shown in Tables H19 through H22. Although there were no consistent exposure-related changes in lavage fluid acid protein ase activity at any of the interim evaluations, acid proteinase activity in supernatant fluid from male and female mice exposed to 18 mg/m3 was significantly greater than controls at 12, 18, and 24 months. The increase in acid proteinase activity was primarily due to cathepsin D-like activity. There were no consis tent exposure-related changes in neutral proteinase activity at any of the interim evaluations. 48 Talc, NTP TR 421 o Plate 1 Mild focal inflammation with thickening of the alveolar septa and distortion of the alveoli in the lung of a male F344/N rat exposed to 18 mg talc/m3 at the 18-month interim evaluation of the lifetime inhalation study. H&E, 25X Plate 2 Lung of a male F344/N rat exposed to 18 mg talc/m3 at the 18-month interim evaluation of the lifetime inhalation study. Note the accumulation of alveolar macrophages with pale granular cytoplasm in the alveolar duct and slight thickening of the septal walls. H&E, 80X P late 3 Individual and confluent foci of interstitial fibrosis extend throughout the pulmonary parenchyma of a male F344/N rat exposed to 18 mg talc/m3 at the 24-month interim evaluation of the lifetime inhalation study. H&E, 6.6X Plate 4 Higher magnification of Plate 3 showing accumulation of fibrous tissue and interspersed inflammatory cells which obliterate the alveoli. H&E, 33X Plate 5 Squamous metaplasia and hyperplasia of the alveolar epithelium adjacent to an area of chronic inflammation and interstitial fibrosis in the lung of a male F344/N rat exposed to 18 mg talc/m3 in the lifetime inhalation study. H&E, 40X Plate 6 Alveolar/bronchiolar carcinoma in a male F344/N rat exposed to 18 mg talc/m3 in the lifetime inhalation study. Note the large mass obliterating the pulmonary parenchyma. H&E, 2.5X Plate 7 Higher magnification of the alveolar/bronchiolar carcinoma shown in Plate 6. Note the neoplastic epithelium arranged in irregular papillary formations. H&E, 50X Plate 8 Minimal focal accumulation of alveolar macrophages in the lung of a male B6C3F] mouse exposed to 18 mg lalc/m3 at the 12-month interim evaluation of the 2-year inhalation study. H&E, SOX Plate 9 Mild chronic active inflammation with slight thickening of the alveolar septa in the lung of a female B6C3F, mouse exposed to 18 mg talc/m-1 in the 2-year inhalation study. H&E, 50X P late 10 Alveolar macrophages in alveoli and mononuclear cells in the interstitium of the lung of a male B6C3Fj mouse exposed to 18 mg talc/m3 in the 2-year inhalation study. H&E, 100X 49 DISCUSSION AND CONCLUSIONS Talc ore may contain several other minerals, including calcite, dolomite, magnesite, tremolite, anthophyllite, antigorite, quartz, pyrophyllite, micas, or chlorites. Since talc products are sold in a multitude of grades which have physical or functional characteristics especially suited for particular applications, occupational and consumer exposures to talc are complex. Exposure to industrial grade talc is known to cause pulmonary fibrosis, but the limited data on exposure to cosmetic grade talc are conflicting. Recently, epidemiology studies have suggested an association between nonfibrous talc and lung cancer risk (Thomas and Stewart, 1987). Talc was nominated by NIOSH for study by the NTP because of widespread human exposure and because of the lack of adequate information on its chronic toxicity and potential carcinogenicity. The NTP toxicity and carcinogenicity studies of nonasbestiform, cosmetic grade talc, a finely powdered hydrous magnesium silicate, were conducted by exposing groups of male and female F344/N rats and B6C3F, mice to target aerosol concentrations of 0,6, or 18 mg talc/m3 for 6 hours per day, 5 days per week. Rats were exposed to talc until mortality in any group reached 80% (113 weeks for males and 122 weeks for females). Mice were exposed for 103 or 104 weeks. Exposure concentrations for the long term studies were based on talc deposition and clearance patterns obtained from 4-week inhalation studies (Hanson et al., 1985). In these studies, the amount of talc retained per gram of lung tissue was 79, 190, or 840 xg for male rats and 76, 185, or 770 /xg for female rats exposed to 2, 6, or 18 mg/m3, respectively. The amount of talc retained per gram of lung tissue in mice exposed at the same concentra tion levels were 130, 330, or 1,140 xg for males and 110, 330, or 1,160 xg for females. Only rats and mice at the highest exposure level had talc-containing macrophages within the alveolar spaces. Because there was a direct relationship between chamber concentration and lung talc burden and because of the talc-containing alveolar macrophages at the 18 mg/m3concentration, it was predicted that higher levels would overwhelm lung clearance mechanisms in both species and cause deterioration of lung functions. Thus, 18 mg/m3 was chosen as the top exposure concentration for the NTP long-term studies. The overall mean chamber concentrations achieved in the NTP long-term studies were 6.1 and 18.6 mg/m3 for the rat study and 5.9 and 16.7 mg/m3 for the mouse study. The average mass mean aerodynamic diameter of the talc particles was calculated to be 2.7 yu.mand 3.2 yum for the 6 and 18 mg/m3rat cham bers and 3.3 yum and 3.6 yum for the 6 and 18 mg/m3 mouse chambers, respectively. Seventy-five percent of the talc particles counted in four samples were in the 1 to 3 xm range. Monodisperse aluminosilicate particles larger than 10 yum are nearly all removed by inertia] impaction in the nasal chamber or at bifurcation of the airways in rats, while particle deposition in the alveolar ducts and alveoli rises from almost zero for 10 yum particles to about 10% for 1 yum particles (Raabe et al, 1977). Thus, the large proportion of talc particles in these NTP studies were in the respirable range. Because of difficulties with the aerosol concentration monitoring system for the 18 mg/m3 rat chamber, there was a 7-week period beginning at study week 11 during which the chamber concentration for the high dose rats varied from approximately 30 to 40 mg/m3. Further, there was a 12-week period beginning at approximately week 70 during which there were difficulties in generating the talc aerosol and the chamber concentrations for rats and mice were substantially lower than the target concentrations (Figures 15 to 18). Although the exposure concen trations varied substantially from target concentra tions during these periods, this does not preclude drawing conclusions regarding the chronic toxicity and carcinogenicity of talc. Since talc is a relatively inert particle, the amount of talc deposited and retained at the target site (lung talc burden) is a more relevant measure of talc exposure than chamber concentration. The problems with maintaining the target concentrations in the NTP studies had no apparent substantive effect on lung talc burdens. 50 Talc, NTP TR 421 The lung talc burden represents the difference between the amount of talc deposited in the lung and the amount removed by the clearance mechanisms. Inhaled particles deposited on the mucosal surface of the trachea, bronchi, or bronchioles are transported up the airways and from the lung through the ciliary activity of the respiratory epithelium, while particles reaching the alveolar region are phagocytized by alveolar macrophages and, to a lesser extent, other phagocytic inflammatory cells. Some alveolar macro phages migrate to the ciliated epithelium of the airways while others cross the alveolar epithelium to enter the interstitium and finally the lymphatics. Phagocytic cells reaching the lymphatics are trans ported in the lymph to the bronchial and mediastinal lymph nodes. Depending on the physiochemical properties of the inhaled particles, they may be partially or completely degraded within phagoly sosomes of the macrophages and soluble components released from the cell. Talc is insoluble in water, cold acids, and alkalies and is likely to be insoluble in biological fluids. Talc particles were observed within macrophages in the lung and bronchial and mediasti nal lymph nodes of rats and mice in these inhalation studies. The lung talc burden of rats was greater than that of mice at each of the exposure concentrations and interim evaluations. The difference in lung talc burden is most likely related to species anatomical and physiological differences known to influence particle deposition and retention including air flow pattern and velocity, respiratory rate, tidal volume, and clearance rate (McMahon et aL, 1977; Raabe et aL, 1977). The lung talc burdens of exposed rats and mice were generally similar between males and females at each exposure concentration and increased progressively with exposure duration. This indicated that the amount of talc deposited in the lung exceed ed the clearance from the lung. The lung talc burden of rats was also generally proportional to exposure concentration at each interim evaluation, indicating that clearance of talc was not substantially impaired by increasing the exposure concentration, or that clearance of talc was impaired similarly at both exposure levels. In contrast, the lung talc burden of mice exposed to 18 mg/m3 was disproportionately greater than that of mice exposed to 6 mg/ih3, indicat ing that clearance of talc from the lung was impaired, or impaired to a greater extent, in mice exposed to the higher concentration. Analysis of bronchoalveolar lavage fluid has been used in human medicine for diagnosing the type or stage of various forms of interstitial lung disease and more recently as a rapid in vivo method of evaluating lung injury in toxicologic studies (Henderson et aL, 1985). Bronchoalveolar lavage was performed on rats and mice exposed to talc to evaluate its usefulness in chronic toxicology studies. Qualitatively similar changes in lavage fluid enzymes and cytology were observed in both species. Increases in neutrophils and total protein in lavage fluid are sensitive indica tors of inflammation, and the increases in these parameters in rats and mice exposed to talc are consistent with the inflammation observed histologi cally in the lungs. Increases in cytoplasmic (lactate dehydrogenase and glutathione reductase) and lyso somal (B-glucuronidase) enzymes, which are indica tive of cellular injury, were also observed in both species. Whether lactate dehydrogenase and glutathi one reductase were derived from parenchymal cells or inflammatory cells is unknown. The increase in glutathione reductase activity suggests that cellular injury may have involved an oxidative process involv ing free radicals produced during phagocytosis. The phagocytic ability of alveolar macrophages recovered from lavage fluid was not impaired in rats exposed to talc for 24 months, as indicated by the lack of a significant difference in the number of viable macrophages and the percentage of cells phagocytizing sheep erythrocytes in exposed and control rats. In contrast, both the viability and the phagocytic ability of alveolar macrophages from exposed mice were significantly lower than those of macrophages from controls. The percentage of macrophages containing phagocytized erythrocytes decreased as aerosol concentration and exposure duration increased. Since alveolar macrophages play a major role in the clearance of particles from the lung, the decreased viability and phagocytic ability of these cells may explain the disproportionately greater lung talc burden in mice exposed to 18 mg/m3than in mice exposed to 6 mg/m3 and the difference in talc lung burdens between exposed rats and mice. Due to limitations in chamber size and the number of animals that could be exposed, the numbers of animals utilized in the lung biochemistry studies were generally small. Therefore, some of the apparent inconsistencies in the results of these studies can be attributed to the small sample sizes and the biologic Discussion and Conclusions 51 variation in pulmonary response among individuals. Despite these limitations, increases in lavage fluid collagenous peptides and total lung collagen were observed in both rats and mice exposed to 18 mg talc/m3. In rats, these changes were also accompanied by increases in noncollagenous protein synthesis (total ,4C-proline incorporated into lung tissue minus that incorporated into collagen), and, in females only, an increase in collagen production (fraction of total 14C-proline incorporated into collagen). Some parameters were also significantly increased in rats exposed to 6 mg talc/m3. While these results are consistent with the fibrosis observed histologically in rats, fibrosis was not seen histologically in mice. Talc exposure was associated with a dose- and timerelated impairment of respiratory functions in male and female rats. Although only slight trends were observed at 6 months in rats exposed to 18 mg/m3, functional alterations in rats at the high concen tration were clearly evident after 11 months. In rats exposed to 6 mg/m3, decrements in respiratory func tion were observed in males at 11 months and in males and females at 18 months. The functional impairment was characterized by reduced lung vol umes and reduced dynamic and/or quasistatic lung compliance, indicating an increase in elastic recoil (increased lung stiffness). Further, reduced gas exchange efficiency and nonuniform intrapulmonary gas distribution were also observed. These changes are consistent with the multifocal fibrosis and inflam mation that was located in the centriacinar region of the lung. Deposition of talc in the lungs of rats and mice produced an inflammatory response characterized primarily by the accumulation of alveolar macro phages and, to a lesser extent, neutrophils and monocytes within alveolar lumens. Smaller numbers of lymphocytes and plasma cells were also observed in the interstitial tissue surrounding airways, blood vessels, and alveolar septa. The lesions developed at the junction of the alveolar ducts and terminal bronchioles where particles of the size range used are known to be deposited (Brody and Roe, 1983). Although the inflammatory response was basically similar in rats and mice, there were important species differences. The lesions in rats were generally more extensive and more severe than those in mice at similar exposure concentrations. In rats, foreign body giant cells were occasionally observed and some of the alveolar macrophages developed the morphologi cal characteristics of epithelioid macrophages. More importantly, the inflammatory lesions in rats were accompanied by interstitial fibrosis, hyperplasia of alveolar type II epithelial cells, and, infrequently, squamous metaplasia of the alveolar epithelium. The differences in pulmonary response cannot be attributed to differences in lung talc burden, since fibrosis and alveolar epithelial hyperplasia were observed in rats exposed to 6 mg/m3, which had lung talc burdens less than that of mice exposed to 18 mg/m3. Saffiotti and Stinson (1988) have reported similar differences in pulmonary response between rats and mice following intratracheal instillation of silica. These authors found that silica-induced alveolar epithelial hyperplasia in mice was transient, returning to normal within several months, while that in rats was generally more severe and persisted until the end of the study. Since inhalation studies using both rats and mice are seldom performed, it is uncertain if this species difference might exist for other particulate substances. The difference in pulmonary response between rats and mice may be related, in part, to species differ ences in reactivity of alveolar macrophages following phagocytosis of the talc particles. As the principal phagocytic cell of the lung, the alveolar macrophage is believed to play a major role in the inflammatory and fibrogenic reactions to inhaled particles (Brain, 1980; Brody, 1991). Much of the early work in this area centered on the differential cytotoxicity of phagocytized particles, particularly the various crystal line forms of asbestos and silica, to alveolar macro phages and the subsequent release of lysosomal enzymes which have proteolytic, elastolytic, and inflammatory properties (Brody and Davis, 1982; Nathan, 1987). More recently, alveolar macrophages have been found to produce arachidonic acid metab olites (Kouzan et aL, 1985) and various cytokines that regulate cell proliferation, differentiation, and extra cellular matrix production (Kelley, 1990). Of particu lar interest, rat alveolar macrophages exposed to iron spheres and asbestos fibers have been found to produce increased amounts of a homologue of platelet-derived growth factor (PDGF), the most potent mitogen known for mesenchymal cells (Bonner et aL, 1989, 1990), and TGF-B, a potent inhibitor of mesenchymal cell proliferation and stimulator of matrix production (Kalter et aL, 1989). Little is known about the putative role of PDGF and 52 Talc, NTP TR 421 TGF-B and other macrophage-derived products in the pathogenesis of lung disease, but they are likely to be important mediators of many cellular events. The lesions in the lungs of rats exposed to aerosols of talc are very similar, qualitatively, to those reported to occur following long-term (approximately 2 years) exposure to other inorganic, non-fibrous, particulate substances including titanium dioxide (Lee et al., 1985), chromium dioxide (Lee et al., 1988), antimony trioxide and antimony ore concentrate (predominant ly antimony trisulfide) (Groth et aL, 1986), and volcanic ash (Wehner et aL, 1986). Aerosols of each of these particulate substances were reported to elicit pulmonary inflammation, characterized primarily by the accumulation of alveolar macrophages, hyperpla sia and squamous metaplasia of the alveolar epitheli um, and fibrosis. Since the various components of the pulmonary response were not quantified in these studies, there may be quantitative differences in the degree of inflammation, fibrosis, and cellular degen erative hyperplastic and metaplastic changes to these particulate substances. The lesions in rats exposed to talc are also similar to those observed in rats exposed to silica, but with important differences. Silica generally produces an inflammatory response that is more pronounced and persistent than the response to the relatively more inert particles like titanium dioxide and talc (Saffiotti and Stinson, 1988; Driscoll et al, 1990). Further, while only occasional multinucleated foreign body giant cells and epithelioid macrophages were seen in the cellular response to talc, rats exposed to silica develop discrete nodular aggregates of epithelioid macrophages with multinucleated cells more typical of granulomatous inflammation. The quantitative and qualitative differences in pulmo nary toxicity of inhaled particles are likely related to the particle size, structure (amorphous, crystalline, and/or fibrous), surface chemistry, solubility (or durability), chemistry of soluble components, cyto toxicity, and other factors. While much of the research in this area has focused on asbestos (as well as other fibers) and silica, the same principles are likely to explain the differences in biological activity of other particulate substances. Although a complete discussion of these factors is beyond the scope of this report, some of the evidence is presented here. A number of studies of the various forms of silicon dioxide have found that amorphous silica produces the mildest, slowest developing pulmonary changes followed, in ascending order, by quartz, cristobalite and tridymite (Allison, 1977; Hemenway et a l, 1986). Amorphous silica generally lacks a detectable crystal line X-ray diffraction pattern, while, of the crystalline forms, quartz has a less ordered symmetry than cristobalite and tridymite. Moreover, stishovite, which lacks the tetrahedral structure of other forms of silica, also lacks the fibrogenicity and cytotoxicity of the other forms (Brieger and Gross, 1967). In general, the ability of various forms of silica to elicit pulmonary fibrosis parallels their cytotoxicity in vitro to alveolar macrophages (Reiser and Last, 1979). Further, there is a correlation between cytotoxicity and hemolytic activity in vitro (Allison, 1977). The biochemical basis of macrophage cyto toxicity and hemolytic activity is not fully understood, but the surface of crystalline silica presents highly reactive hydroxyl groups of silicic acid residues (silanol) that act as proton donors and may combine with constituents of cellular membranes (Langer and Nolan, 1986). Kaolinite (aluminum silicate), mica (potassium aluminum silicate), and talc (magnesium silicate) are also hemolytic in vitro (Narang et al, 1977). Dissolution of silicic acid residues from kaolinite, mica, and talc reduces the toxicity of these particulates, supporting the hypothesis that the reactive hydroxyl groups play an important role in cytotoxicity and hemolytic activity. Following phagocytosis of silica (Allison, 1977) or kaolinite (Brody and Davis, 1982) particles by alveo lar macrophages, hydrolytic enzymes are released from secondary lysosomes apparently as a result of the interaction of the particles with the lysosomal membrane. While the release of lysosomal enzymes into the cytoplasm may be directly responsible for cell death, it is less clear to what extent lysosomal enzymes released from the cells contribute to the other pulmonary lesions. Certainly, the ability to kill alveolar macrophages (cytotoxicity) is likely to inhibit or delay removal of the particles from the lung, increase the lung burden, and allow other biological effects to occur. As already mentioned, macrophages secrete a large number of molecules including polypeptide hormones or cytokines, complement components, coagulation factors, arachidonic acid and its metabolites, bioactive lipids (prostaglandins and leukotrienes), binding proteins, enzyme inhibitors, extracellular matrix or cell adhesion proteins, and others (Nathan, 1987). Discussion and Conclusions S3 Some, or perhaps many, of the apparent differences in the pulmonary response of rats to the various particulate substances may be related to the extent to which they cause cytotoxicity and nonspecific release of lysosomal enzymes or cause macrophages to secrete specific effector substances like the cytokines and inflammatory mediators. Exposure of female rats to 18 mg talc/m3was associ ated with increased incidences of alveolar/bronchiolar adenoma (0 mg/m3, 1/50; 6 mg/m3, 0/48; 18 mg/m3, 9/50), alveolar/bronchiolar carcinoma (0/50, 0/48, 5/50), and squamous cell carcinoma (0/50,0/48,1/50). The overall incidence of alveolar/bronchiolar adenoma or carcinoma (combined) in female rats of the 18 mg/m3was significantly (P<0.001) greater than that of controls (1/50, 0/48, 13/50). The incidence of pulmonary neoplasms in female rats exposed to 18 mg/m3also greatly exceeds that of control females (8/529,1.5%) in the NTP lifetime studies reported by Solleveld et al (1984). While comparison with the historical controls from NTP lifetime studies has some limitations (e.g., the studies were conducted about a decade earlier and are not contemporary), such a comparison provides some perspective. The increased incidence of pulmonary neoplasms in the 18 mg/m3 female rats was considered clear evidence of carcinogenic activity based on a) the strength of the statistical evidence (P<0.001), b) the increase in malignant as well as benign neoplasms, and c) comparison with lifetime historical controls. In contrast to female rats, there was no increase in the incidence of pulmonary neoplasms in male rats or in male or female mice exposed to talc aerosols. While precise comparisons between studies of talc and other particulate substances cannot be made because of differences in route of administration (intratracheal versus inhalation), strain of rat used, and exposure duration, such comparison provides some perspective (Table 12). In 2-year inhalation studies of titanium dioxide (Lee et al, 1985), chromium dioxide (Lee et a l, 1988), antimony trioxide and antimony ore concentrate (predomi nantly antimony trisulfide) (Groth et al, 1986), volcanic ash (Wehner et al, 1986), and quartz (Dagle et al, 1986), female rats had greater incidences of pulmonary neoplasms than male rats. Chromium dioxide, volcanic ash, antimony trioxide, and antimony ore concentrate induced pulmonary neoplasms only in female rats, whereas titanium dioxide and quartz induced pulmonary neoplasms in male and female rats with a preponderance of neoplasms in females. The morphological types of neoplasms induced by the particulates in the studies cited above also vary somewhat. The neoplasms in female rats exposed to talc were primarily alveolar/bronchiolar adenomas and carcinomas, although one squamous cell carcino ma also occurred. In female rats exposed to antimo ny trioxide or antimony ore concentrate (Groth et al, 1986), there were sim ilar numbers of alveolar/bronchiolar neoplasms and squamous cell carcinomas (Table 12). Further, several scirrhous carcinomas were observed in antimony exposed rats. In female rats exposed to titanium dioxide (Lee et al, 1985), the incidences of alveolar/bronchiolar neo plasms and squamous cell carcinoma were also similar, whereas all but one of the neoplasms in males were alveolar/bronchiolar neoplasms. In contrast, nearly all the pulmonary neoplasms induced by quartz (Dagle et al, 1986), volcanic ash (Wehner et al, 1986) or chromium dioxide (Lee et al, 1988) were squamous cell (epidermoid) carcinomas. The pathogenesis of pulmonary neoplasms induced by relatively insoluble particulate substances, such as talc, is currently unknown. Although a genotoxic mechanism cannot be ruled out, there are several lines of evidence to suggest that a direct effect of the particulate on the target cell genome is not involved. First, the insoluble nature of these particulates makes it unlikely that any chemical constituents will reach sufficient concentration to affect the target cells within the relatively short period between the time they are deposited on the alveolar surface and the time they are phagocytized. Further, although occasional alveolar epithelial cells have been observed to contain particles following intratracheal or inhalation exposure (Sorokin and Brian, 1975; Lee et al, 1979), the majority of particles are rapidly phagocytized by alveolar macrophages, some within minutes of deposition in the lung (Lauweryns and Baert, 1974). It is also clear that physical character istics (crystalline structure, fiber dimension) and surface chemistry (presence of reactive groups on the particle surface), rather than soluble chemical compo nents, are principal determinants of tissue reaction, and perhaps of carcinogenicity. The carcinogenicity of many fibrous materials (fiberglass, attapulgite, silicon carbide, mineral wool, and potassium titanate) decreases as fiber diameter exceeds 2.5 /um and as fiber length decreases below 10 gm (Stanton and Wrench, 1972; Stanton et al, 1977). 54 Talc, NTP TR 421 T a b le 12 Results of Selected Whole Body Inhalation Carcinogenicity Studies of Particulate Materials Compound and Dose Study D u ratio n Species Effects on Lungs2 Talc at 0, 6, or 18 mg/m3 (this study) Male: 113 weeks Female: 122 weeks Titanium dioxide at 0, 10, 50, or 250 mg/m3 (Lee e t a l., 1985) Titanium tetrachloride. at 0, 0.1, 1.0, or 10 mg/m3 (Lee e t a l., 1986) Chromium dioxide at 0, 0.5, 0.5b, or 25 mg/m3 (Lee e t a !., 1988) Antimony trioxide at 0 or 45 mg/m3 (Groth e t a h , 1986) Antimony trisulfide at 0 or 40 mg/m3 (Groth e t a h , 1986) Volcanic ash at 0, 5, or 50 mg/m3 (Wehner e t a h , 1986) Quartz at 0 or 50 mg/m3 (W ehner e t a h , 1986) 104 weeks 104 weeks 104 weeks 73 weeks 72 weeks up to 104 weeks up to 104 weeks F344/N rats CD rats Females: alveolar/bronchiolar adenoma (1/50, 0/48, 9/50); atveolar/bronchiolar carcinoma (0/50, 0/48, 5/50); squamous cell carcinoma (0/50, 0/48, 1/50) Females: alveolar/bronchiolar adenoma (0/77, 0/75, 0/74, 13/74); squamous cell carcinoma (0/77, 0/75, 0/74, 13/74) Crl:CD rats Females: squamous cell carcinoma (0/77, 0/75, 0/79, 3/75); Males: squamous cell carcinoma (0/79, 0/77, 0/78, 2/75) SpragueDawley rats Females: squamous cell carcinoma (0/106, 0/103, 0/108, 2/108); keratin cyst (0/106, 0/103, 0/108, 6/108) Wistar rats Wistar rats F344 rats F344 rats Females: alveolar/bronchiolar neoplasms (0/90, 11/90); squamous cell carcinoma (0/90, 9/90); scirrhous carcinoma (0/90, 5/90) Females: alveolar/bronchiolar neoplasms (0/90, 6/90); squamous cell carcinoma (0/90, 9/90); scirrhous carcinoma (0/90, 4/90) Females: several' squamous cell carcinomas in the 50 mg/m3 group. Male: one squamous cell carcinoma in the 50 mg/m3 group. Females: moderate' numbers of squamous cell carcinomas in the 50 mg/m3 group. Males: one squamous cell carcinoma in the 50 mg/m3 group. ` Neoplasm incidences are given as the number of animals with neoplasm per number of animals examined. The incidences are given in the order of increasing exposure concentration. b This dose represents unstabilized chromium dioxide; the other doses represent stabilized chromium dioxide. c Precise numbers not available in journal article. A potential mechanism for the development of pulmonary neoplasms associated with insoluble particulate substances is that the prolonged stimulus for cell replication, due not only to cell injury but to the release of mitogenic growth factors from alveolar macrophages, provides a favorable environment for the promotion and progression of spontaneously initiated cells. The interim evaluations in the NTP talc study clearly demonstrate a progressive impair ment of homeostatic growth regulation in the areas of chronic inflammation and fibrosis associated with talc deposition in rats. Hyperplasia of the alveolar epithelium was evident at 6 months and became more extensive and severe with duration of exposure. Not only were there increased numbers of cells (hyperpla sia), but some cells assumed morphologic features atypical of regenerating or differentiated type II cells (epithelial dysplasia). The altered or dysplastic epithelium was particularly evident in areas of fibro sis. The squamous metaplasia observed in female rats also represents altered differentiation of popula tions of alveolar epithelial cells and is notable in light Discussion and Conclusions 55 of the development of squamous cysts and squamous cell carcinomas. The lack of a carcinogenic effect in male rats or in mice exposed to talc aerosols does not negate the possibility of a mechanism as described above. First, the difference between male and female rats may be one of magnitude rather than an absolute difference in effect. The influence of the length of exposure on the development of these late appearing lung neo plasms cannot be discounted; the length of exposure was 113 weeks for males and 122 weeks for females. Further, the promotion and progression of neoplasia involve a complex series of molecular events that are likely to differ qualitatively or quantitatively in males and females. Clearly, there are sex differences in the incidence of spontaneous and chemically induced neoplasms. As for mice exposed to talc, there was no histologic evidence of impaired growth regulation or fibrosis, consistent with the mechanism proposed above. Pheochromocytomas (benign, malignant, or complex) of the adrenal medulla occurred with significant positive trends in both male and female rats exposed to talc (males: 26/49, 32/48, 37/47; females: 13/48, 14/47, 23/49). Further, the numbers of male and female rats with bilateral pheochromocytomas were also increased in the exposed groups. The overall incidences of this neoplasm in the 18 mg/m3 groups were significantly greater than those of the controls. Comparison with historical controls of NTP lifetime studies is not considered relevant, since there has been a pronounced increase in the spontaneous occurrence of pheochromocytomas in male rats in studies conducted by the NTP over the last 10 years (Rao et aL, 1990). In contrast to the pheochromocytomas, the incidences of adrenal medulla hyperplasia in exposed male rats were lower than in controls, and the incidences were similar in all female groups. Because of the small size of the adrenal medulla, pheochromocytomas tend to obscure much or all of the remaining tissue. Therefore, the lower incidences of hyperplasia in groups of exposed males can be attributed, in part, to the larger number of pheochromocytomas. While the increased incidences of pheochromocytomas in male rats were exposure related, the increase was considered to represent some, rather than clear, evidence of carcinogenic activity because a) the increase was associated primarily with benign neo plasms and b) there was no supporting increase in the incidence of hyperplasia. The increased incidence of pheochromocytomas in female rats was also exposure related. Although the strength of the statistical association indicates that the pheochromocytomas are exposure related, a plausible mechanism for their increased occurrence in rats exposed to talc aerosols is not readily apparent. Since talc is relatively insoluble, it is extremely unlikely that any soluble components could have reached concentrations high enough in the blood to affect the adrenal medulla cells. Al though purely speculative, there are two general hypotheses that might be considered. First, the increased incidence of adrenal pheochromocytomas may be a nonspecific effect of stress as a result of the chronic pulmonary inflammation. The body is known to respond to an exogenous challenge such as injury, inflammation, or infection by a set of distinct physio logic, metabolic, and endocrine changes including increases in serum adrenocorticotrophic hormone and cortisone levels, growth hormone, and catecholamine synthesis. Further, the adrenal medulla, as a modi fied sympathetic ganglia, reacts to neural as well as hormonal stimuli in the secretion of catecholamines. While prolonged stimulus of secretion is coupled with cellular hypertrophy and hyperplasia (cell proliferation) in many endocrine tissues, it is un known if this occurs in the adrenal medulla. More over, if prolonged stress were to increase the rate of occurrence or growth of medullary proliferative lesions, similar exposure-related increases in pheochromocytoma incidence might be expected in other chronic toxicity/carcinogenicity studies. This has not generally been the case. Exposure-related increased incidences of pheochromocytoma were not observed or not reported in the 2-year inhalation studies of other particulate substances reported above. A second hypothesis to consider is that cytokines (growth factors), released from macrophages or other cells in the lung, might be responsible for increasing the rate of growth of pheochromocytomas. Although alveolar macrophages have been found to secrete a number of cytokines known to stimulate proliferation of a variety of cell types, cytokines are generally believed to affect cells only in close proximity within the same organ. However, it has recently been found 56 Talc, NTP TR 421 that measurable levels of hepatocyte growth factor are present in the plasma after two-thirds hepatectomy (Lindroos et aL, 1992). Thus, some cytokines or growth factors may have wider effects than currently known. C o n c lu sio n s Under the conditions of these inhalation studies, there was some evidence o f carcinogenic activity* of talc in male F344/N rats based on an increased inci dence of benign or malignant pheochromocytomas of the adrenal gland. There was clear evidence o f carcinogenic activity of talc in female F344/N rats based on increased incidences of alveolar/bronchiolar adenomas and carcinomas of the lung and benign or malignant pheochromocytomas of the adrenal gland. There was no evidence o f carcinogenic activity of talc in male or female B6C3FJ mice exposed to 6 or 18 mg/m3. The principal toxic lesions associated with inhalation exposure to the same concentrations of talc in rats included chronic granulomatous inflammation, alveolar epithelial hyperplasia, squamous metaplasia and squamous cysts, and interstitial fibrosis of the lung. These lesions were accompanied by impaired pulmonary function characterized primarily by reduced lung volumes, reduced dynamic and/or quasistatic lung compliance, reduced gas exchange efficiency, and nonuniform intrapulmonary gas distribution. In mice, inhalation exposure to talc produced chronic inflammation of the lung with the accumulation of alveolar macrophages. Explanation of Levels of Evidence of Carcinogenic Activity is on page 9. A summary of Technical Reports Review Subcommittee comments and the public discussion on this Technical Report appears on page 11. 57 REFERENCES Allison, AC. (1977). 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Steril 22, 32-40. 63 APPENDIX A SUMMARY OF LESIONS IN MALE RATS IN THE LIFETIME INHALATION STUDY OF TALC Table A1 Table A2 Table A3 Table A4 Summary of the Incidence of Neoplasms in Male Rats in the Lifetime Inhalation Study of T a lc ................................................................................... Individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of T a lc ................................................................................... Statistical Analysis of Primary Neoplasms in Male Rats in the Lifetime Inhalation Study of T a lc ................................................................................... Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the Lifetime Inhalation Study of T a lc ................................................................................... 64 68 86 90 64 Talc, NTP TR 421 Table A1 Summary of the Incidence of Neoplasms in Male Rats in the Lifetime Inhalation Study of Talc* 0 mg/m3 9 6 mg/m 18 mg/m3 D isposition Summ ary Animals initially in study Early deaths Moribund Natural deaths Survivors Died last week of study Terminal sacrifice Animals examined microscopically Alim entary System Intestine large, cecum Intestine large, colon Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Neoplastic nodule Neoplastic nodule, multiple Osteosarcoma, metastatic, multiple, bone Hepatocyte, adenoma Mesentery Pancreas Salivary glands Fibroma Stomach, forestomach Fibrosarcoma Stomach, glandular Fibrosarcoma Cardiovascular System Heart Endocrine System Adrenal gland, cortex Adrenal gland, medulla Osteosarcoma, metastatic, uncertain primary site Pheochromocytoma malignant Pheochromocytoma complex Pheochromocytoma benign Bilateral, pheochromocytoma malignant Bilateral, pheochromocytoma benign Islets, pancreatic Adenoma Carcinoma Parathyroid gland Adenoma Pituitary gland Pars distalis, adenoma Pars distalis, carcinoma Pars intermedia, adenoma 49 23 17 1 8 49 (42) (43) (48) (39) (40) (49) 2 (4%) 1 (2%) (2) (48) (49) (49) (49) (49) (49) (49) 2 (4%) 13 (27%) 1 (2%) 12 (24%) (47) 1 (2%) 1 (2%) (45) (47) 12 (26%) 50 19 17 2 12 50 (38) (43) (44) (34) (38) (50) 1 (2%) 1 (2%) (46) (50) 1 (2%) (47) (47) (50) (49) (48) 3 (6%) 2 (4%) 9 (19%) 21 (44%) (41) (45) 1 (2%) (50) 11 (22%) 1 (2%) 50 20 14 3 13 50 (37) (46) (46) (35) (40) (48) 1 (2%) 3 (6%) (1) (47) (50) (47) 1 (2%) (47) 1 (2%) (50) (48) (47) 1 (2%) 6 (13%) 1 (2%) 20 (43%) 1 (2%) 16 (34%) (43) 2 (5%) (46) (49) 10 (20%) 2 (4%) Lesions in Male Rats Table A1 luminary of the Incidence of Neoplasms in Male Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 IS mg/m3 E ndocrine System (continued) Thyroid gland C-cell, adenoma C-cell, carcinoma Follicular cell, adenoma (45) 3 (7%) (46) 4 (9%) 1 (2%) (46) 3 (7%) 1 (2%) G eneral Body System Tissue NOS Pheochromocytoma malignant, metastatic, adrenal gland (1) 0) 1 (100%) Genital System Epididymis Preputial gland Adenoma Carcinoma Prostate Seminal vesicle Testes Bilateral, interstitial cell, adenoma Interstitial cell, adenoma (49) (48) 1 (2%) 1 (2%) (49) (49) (49) 18 (37%) 13 (27%) (50) (49) 1 (2%) 6 (12%) (45) (48) (50) 24 (48%) 15 (30%) (49) (48) 1 (2%) 1 (2%) (48) (47) (50) 24 (48%) 12 (24%) lem atopoietic System Bone marrow Lymph node Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Fibrosarcoma Fibrous histiocytoma Osteosarcoma, metastatic, bone Thymus Thymoma malignant (48) (49) (41) (46) (48) (49) (49) 1 (2%) 1 (2%) (48) 1 (2%) (48) (50) (48) (48) (49) (48) (50) 1 (2%) (40) (47) (50) (49) (47) (47) (47) (48) (43) Integum entary System Mammary gland Adenocarcinoma Skin Basosquamous tumor malignant Fibroma Fibrous histiocytoma Keratoacanthoma Neurofibroma Squamous cell carcinoma Subcutaneous tissue, fibroma Subcutaneous tissue, fibrosarcoma Subcutaneous tissue, schwannoma malignant (45) 1 (2%) (48) 1 (2%) (48) (50) 2 (4%) 2 (4%) 1 (2%) 1 (2%) 1 (2%) 1 (2%) (50) (50) 1 (2%) 1 (2%) 2 (4%) 65 66 Talc, NTP TR 421 Table Al Summary of the Incidence of Neoplasms in Male Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 m g/m 3 M usculoskeletal System Bone Pelvis, osteosarcoma Scapula, osteosarcoma Vertebra, osteosarcoma Skeletal muscle Nervous System Brain Astrocytoma malignant Respiratory System Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma, multiple Fibrosarcoma, metastatic, salivary glands Osteosarcoma, metastatic Osteosarcoma, metastatic, uncertain primary site Osteosarcoma, metastatic, multiple, bone Nose Chondroma Sarcoma Special Senses System None Urinary System Kidney Renal tubule, carcinoma Urinary bladder Papilloma System ic Lesions Multiple organsb Leukemia mononuclear Lymphoma malignant lymphocytic Mesothelioma benign Mesothelioma malignant . (49) 1 (2%) (1) (49) 1 (2%) (49) 1 (2%) 1 (2%) (49) 1 (2%) (49) 2 (4%) (49) 1 (2%) (49) 24 (49%) 1 (2%) 1 (2%) (50) 1 (2%) (50) (50) 1 (2%) 1 (2%) (48) 1 (2%) (49) (48) (50) 21 (42%) (50) 1 (2%) (50) (50) 1 (2%) 1 (2%) 1 (2%) (47) (48) (47) (50) 23 ( 4 6 % ) 1 (2%) Lesions in Male Rats Table A1 Summary of the Incidence of Neoplasms in Male Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 m g/m 3 18 m g/m3 N eoplasm Sum m ary Total animals with primary neop!asmsc Total primary neoplasms Total animals with benign neoplasms Total benign neoplasms Total animals with malignant neoplasms Total malignant neoplasms Total animals with metastatic neoplasms Total metastatic neoplasms Total animals with malignant neoplasms, uncertain primary site 48 116 42 78 34 38 2 4 49 135 45 96 33 39 2 2 a Number of animals examined microscopically at site and number of animals with lesion Number of animals with any tissue examined microscopically c Primary neoplasms: all neoplasms except metastatic neoplasms 50 137 45 98 ->33 39 1 2 1 67 68 Talc, NTP TR 421 Table A2 Individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 0 mg/m3 Number of Days on Study 3345 55556 66 6666 6 667 77 77 7 362 56 8 99 2 23 3 5 5 78 8 9 0 00 0 23 40 9 18 60328 15060 228 00 49 49 Carcass ID Number 3 33 3 42 2 33 433 3 43 3 3 3 3 33 3 2 4 606 4 19 9 18 2346 1444 118 9 19 1 10 80 34 58 70 923 853 87 6 50 3 6 4 11111 1111111111111111111 Alim entary System Esophagus Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Neoplastic nodule, multiple Osteosarcoma, metastatic, multiple, bone Mesentery Pancreas Salivary glands Stomach Stomach, forestomach Stomach, glandular ++++++++++++++++++++++++ +++ ++++ +++++ +A+ ++ ++ + ++ ++ +++++ ++++ +++ +A+ ++ ++ ++ + ++ + + + M+ + + + + + + + + A + + + + + + + + + + M+ + + + + + + + M+ + + A M+ + + + + + + + + ++++++++++++++++++++++++ ++++ + ++ ++++ ++++ + +++ + ++ ++ A++ ++++ +++ +++A++A+ ++ +A+A A++ + ++ + ++ ++ + +A+ +A+ + + ++ +A ++++++++++++++++++++++++ X X + + + + + + + + + + M+ + + + + + + + + + + + + + ++ +++ +++ ++ + ++ +++ + ++ ++ ++ + +++ ++ ++ +++ +++ ++ + ++ + ++ ++ + + ++ +++ + +++ +++ +++ ++ + +++ + + + ++ + +++ ++++ +++ ++ ++ + ++ ++ + Cardiovascular System Blood vessel Heart ++ ++ +++++ +++ ++ + ++ +++ ++ ++ ++ Endocrine System Adrenal gland Adrenal gland, cortex Adrenal gland, medulla Pheochromocytoma malignant Pheochromocytoma benign Bilateral, pheochromocytoma malignant Bilateral, pheochromocytoma benign Islets, pancreatic Adenoma Carcinoma Parathyroid gland Pituitary gland Pars distaiis, adenoma Thyroid gland C-cell, adenoma ++++++++++++++++++++++++ + ++ ++ ++ +++ +++ ++ ++ ++ ++ + ++ ++++++++++++++++++++++++ X X XX XX XX + + + + + + + + + M+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + M+ + + + +++++++++++++I ++++I +++++ X XX X XX +++++++++++++A++++++ ++++ X +: Tissue examined microscopically A: Autolysis precludes examination M: Missing tissue I: Insufficient tissue X: Lesion present Blank: Not examined Lesions in Male Rats 69 Table A2 Individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 0 mg/m1 (continued) Number of Days on Study 7777777777777777777788888 4444456668888899999900000 0 15679 14624567059999000 00 Carcass ID Number 3232344333334333333322334 6969 11194288 12932678 9924 1 78 2 19 10 173 98 5 16 749 16 37 249 1 1 1 11 111 1 11 111 1 11 1 11 1 1 11 1 Total T issu es/ Tumors Alim entary System Esophagus Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Neoplastic nodule, multiple Osteosarcoma, metastatic, multiple, bone Mesentery Pancreas Salivary glands Stomach Stomach, forestomach Stomach, glandular ++ ++++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ + +++ +++A++A+A++ ++ ++ ++ ++ ++ + A + + + + MA + + A + A + + + + + + A + + + + + +A+ ++ + +A+ +A+A+ + + ++ + ++ + +++ M+ + M+ + + A + + A MA M+ + + + + + + + + + + +A++++++ +++++++ ++++++++++ +A+++ +++ +++ +++ ++ ++ ++ ++ + ++ +A+ ++ + +A+AA+A+ + + ++ + + + ++ ++ +A+ ++ + +A++A+A++ ++ + +A+ +++ + +++++++++++++++++++++++++ X + +++++++++++++++++++++++++ +++++++++++++++++++++++++ +++++++++++++++++++++++++ +++ ++ +++ + ++ +++ ++ ++ ++ + +++ + ++ ++++ +++ ++ ++ + ++ ++ ++ ++ + ++ Cardiovascular System Blood vessel Heart + +4 +++++++++++++++++++++++++ 49 45 42 43 38 48 48 39 40 49 2 1 2 48 49 49 49 49 49 Endocrine System Adrenal gland Adrenal gland, cortex Adrenal gland, medulla Pheochromocytoma malignant Pheochromocytoma benign Bilateral, pheochromocytoma malignant Bilateral, pheochromocytoma benign Islets, pancreatic Adenoma Carcinoma Parathyroid gland Pituitary gland Pars distalis, adenoma Thyroid gland C-cell, adenoma +++++++++++++++++++++++++ +++++++++++++++++++++++++ +++++++++++++++++++++++++ X X X XX X X XX X XXX X X XX X XX ++++++++++++++++++++I ++++ X X M+ + + + + + + + + + M+ + + + + + + + M+ + + + +++++++++++++++++++++++++ X XX X XX + + + + + + + A + + + + A + + M+ + + + + + + + + XX 49 49 49 2 13 1 12 47 1 1 45 47 12 45 3 70 Talc, NTP TR 421 Table A2 Individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 0 mg/m3 (continued) Number o f Days on Study 334555556666666666777777 362568992233557889000023 4 0 9 18 603 28 150 6 0 22 8 00 49 4 9 Carcass ID Number 333 34 2233 433 343 33 333 33 24 60 64 199 18 23 46 14 44 1189 19 1 108 0 34 58 70 9 23 853 8 76 50 36 4 111 11 11 11111 111 11 11 11 11 1 General Body System Tissue NOS + Genital System Epididymis Preputial gland Adenoma Carcinoma Prostate Seminal vesicle Testes Bilateral, interstitial cell, adenoma Interstitial cell, adenoma H e m a to p o ie tic S y stem Bone marrow Lymph node Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Fibrosarcoma Osteosarcoma, metastatic, bone Thymus Thymoma malignant + + + + 4- + + + + + + + 4- + 4- 4- + + + 4- 4- + + + + + + 4- 4- 4- + + + + 4- + + + + + + + + 4- 4- + + + X X + + 4- + 4- + 4- + 4- + 4- 4- 4- 4- 4- 4- + 4- 4- 4- + + + + + + + + 4- 4- 4- 4- + + 4- + 4- 4- + 4- 4- 4- + 4- + + + + + 4- 4- 4- + 4- 4- 4- + 4- 4- 4- + 4- + 4- 4- 4- 4- 4- + + + + XX XX XX XX X + + 4- 4- + + 4- 4- 4- 4- 4- 4- 4- A 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ 4- + 4- M M 4- 4- 4- + 4- 4- 4- 4- 4- + 4- + 4- 4- 4- 4- 4- 4+ + 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4- 4- 4+ + 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 44- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- X X 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- X In te g u m e n ta ry S ystem Mammary gland Adenocarcinoma Skin Subcutaneous tissue, schwannoma malignant M + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M M 4- 4- 4- 4- + 4- + 4M 4- + 4- + 4- 4- 4- 4- + + 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- X M u sc u lo sk e le ta l S ystem Bone Scapula, osteosarcoma Skeletal muscle 4- 4- 4- 4- 4- 4- 4- 4- + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4X 4- Lesions in Male Rats 71 Table A2 individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 0 mg/m3 (continued) N um ber of Days on Study 7777777777777777777788888 4444456668888899999900000 0 15679 1462456 70 5999900000 C arcass ID Num ber 32 323 4433 333 433 3 3 33 3 22 33 4 6969 111942 88 1293267899 241 782 19 10 17398 5 16 7 49 16 37 2 49 1111111111111111111111111 Total T issu e s/ T um ors General Body System Tissue NOS 1 Genital System Epididymis Preputial gland Adenoma Carcinoma Prostate Seminal vesicle Testes Bilateral, interstitial cell, adenoma Interstitial ceil, adenoma +++++++++++++++++++++++++ + + + + + + + + + + + + + + M+ + + + + + + + + + +++++++++++++++++++++++++ ++++++++++++++++ +++++++++ + ++ ++++ +++ ++ +++ ++ + ++ ++ + ++ XX XXXXXX XX X XX X X XXX XX XX 49 48 1 1 49 49 49 18 13 H em atopoietic System Bone marrow Lymph node Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Fibrosarcoma Osteosarcoma, metastatic, bone Thymus Thymoma malignant + ++ ++ + ++++ ++ ++ +++ + ++ ++ ++ + + + ++ +++ +++ ++ ++ ++ ++ ++ ++ + ++ + + + + + + + + + + MI + + M+ + + M+ + + + MM + + + + + + + M+ + + + + + M+ + + + + + + + + + + + + + + + + + + + + M+ + + + + + + + + + + + + +++++++++++++++++++++++++ + ++ ++++ ++ + ++ ++ + + ++ ++ ++ + ++ + + + + + + + + + + + M+ + + + + + + + + + + + + 48 49 41 46 48 49 49 1 1 48 1 Integum entary System Mammary gland Adenocarcinoma Skin Subcutaneous tissue, schwannoma malignant + + + + + + M+ + + + + + + + + + + + + + + + + + X + ++ + ++ ++ ++ + +++ ++ + +++ +++ ++ 45 1 48 1 M usculoskeletal System Bone Scapula, osteosarcoma Skeletal muscle +++++++++++++++++++++++++ 49 1 1 72 Talc, NTP TR 421 Table A2 Individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 0 mg/m3 (continued) Number o f Days on Study 334555556666666666777777 362568992233557889000023 409186032815060228004949 Carcass ID Number 333342233433343333333324 6 0 6 4 1 9 9 1 8 2 3 4 6 1 4 4 4 1 189191 108034587092385387650364 111111111111111111111111 Nervous System Brain Astrocytoma malignant ++ ++++ +++++ +++ +++ ++ ++ ++ + Respiratory System Larynx Lung Fibrosarcoma, metastatic, salivary glands Osteosarcoma, metastatic, multiple, bone Nose Chondroma Trachea +I ++++++++++++++++++++++ ++++++++++++++++++++++++ X X ++++++++++++++++++++++++ X +++ +++ +++++ +++ +++ + ++ ++ + + Special Senses System Eye ++ Urinary System Kidney Renal tubule, carcinoma Urinary bladder Papilloma + + + '+ + + + + + + + + + + + + + + + + + + + + ++++++ ++++++++++++++++++ System ic Lesions Multiple organs Leukemia mononuclear Lymphoma malignant lymphocytic Mesothelioma benign + + + + + + + + ++ + + + + + ++ + + + + + + + XX XX X X X X XX X X X X X Lesions in Male Rats 73 Table A2 Individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 0 mg/m1 (continued) Number o f Days on Study 7777777777777777777788888 4444456668888899999900000 0156791462456705999900000 Carcass ID Number 3232344333334333333322334 6969111942881293267899241 7821910173985167491637249 1111111111111111111111111 Total T issu es/ T um ors Nervous System Brain Astrocytoma malignant +++++++++++++++++++++++++ X 49 1 Respiratory System Larynx Lung Fibrosarcoma, metastatic, salivary glands Osteosarcoma, metastatic, multiple, bone Nose Chondroma Trachea +++++++++++++++++++++++++ + ++++++++++++++++++++++++ +++++++++++++++++++++++++ +++++++++++++++++++++++++ 48 49 1 1 49 1 49 Special Senses System Eye +3 Urinary System Kidney Renal tubule, carcinoma Urinary bladder Papilloma ++ +++++ + ++ +++ ++ ++ + ++ ++ ++ + XX +++++++++++++++++++++++++ X 49 2 49 1 System ic Lesions Multiple organs Leukemia mononuclear Lymphoma malignant lymphocytic Mesothelioma benign +++++++++++++++++++++++++ XXXXX X X XXX X 49 24 1 1 74 Talc, NTP TR 421 Table A2 Individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 6 mg/m3 Number o f Days on Study 1555555566666666667777777 822457990134556779 1223344 679483334 138073370528 490 1 Carcass ID Number 00 10 10 0 10 00 100 0 100 10 0 00 0 1 020500407 1726350050 189852 697954933 1980 170332237464 111 1111 1 11 1111 111 1 11 11 111 A lim entary System Esophagus Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Neoplastic nodule, multiple Hepatocyte, adenoma Pancreas Salivary glands Fibroma Stomach Stomach, forestomach Stomach, glandular 4 +4 4 4 +4 4 +++4 ++4 4 4 4 4 4 4 4 4 4 4 + + +A+4 4 4 4 +A++ +A 4 4 4 4 A4 4 4 4 4 + + 4 A + + 4 + 4 A A A + 4- A 4 4 4 4 A 4 4 4 4 A +++A+4 + 4 4 4 AA+4 A4 4 4 4 A4 4 4 4 4 4 4 4 A 4 M4 4 M+ A + + 4 A 4 4 4 4 A 4 4 4 4 4 4 4 4 A4 + 4 4 4 + AA+ 4 A+ 4 4 4 A4 4 4 4 4 4 + 4 A+ + 4 4 4 4 AA+ 4 A4 4 4 4 A4 4 4 + 4 + + + A4 4 4 + + AAA+ 4 A4 4 4 AA4 4 4 AA + + + A+ 4 + + 4 AAA + 4 A 4 4 4 AA4 4 4 4 A +4 4 ++4 ++4 4 4 4 +4 4 4 4 4 4 4 4 4 4 4 4 4 + 4 A + + 4 4 + + AA+ 4 4 4 4 4 4 A4 4 4 4 4 4 4 4 4 4 4 +4 4 ++++4 4 4 4 4 4 4 4 4 4 4 4 4 + + A 4 4 4 4 4 +AA + 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 A4 4 4 4 4 4 AA + 4 4 4 4 4 4 4 4 + 4 4 4 4 4 +A4 + + + 4 4 AA+ 4 4 4 4 4 4 4 4 + + 4 4 Cardiovascular System Blood vessel Heart 44 ++4 +++4 +4 4 4 ++4 4 4 4 4 4 4 4 4 4 4 4 E n d o c rin e S y stem Adrenal gland Adrenal gland, cortex Adrenal gland, medulla Pheochromocytoma malignant Pheochromocytoma complex Pheochromocytoma benign Bilateral, pheochromocytoma benign Islets, pancreatic Parathyroid gland Adenoma Pituitary gland Pars distalis, adenoma Pars distalis, carcinoma Thyroid gland C-cell, adenoma C-cell, carcinoma 4 4 4 4 4 4 4 4 4 4 A+ 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 A+ 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 A+4 I 4 4 4 4 4 4 4 4 4 4 4 X X X X XX X XX XXX 4 4 4 A 4 4 4 4 4 4 A A 4 4 4 M4 4 4 A 4 4 M4 4 4 4 4 4 4 4 M 4 4 4 + MM4 4 4 4 4 4 4 4 4 4 4 4 X 44 4444 44 4 4++44 4 44 4 4 4 444 4 4 X X XX 4 4 4 A 4 4 4 4 4 4 AA4 4 A4 4 4 4 4 4 4 4 4 4 Lesions in Maie Rats 75 Table A2 individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 6 mg/nr1(continued) Number of Days on Study 7777777777777777778888888 4455668888999999990000000 6779 120 137 199999990000000 Carcass ID Number 0 100 11000000000000000 111 1 2393205520303335770792223 628264 10854 1056245889 1590 1111111111111111111111111 Total T issu es/ Tumors Alim entary System Esophagus Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Neoplastic nodule, multiple Hepatocyte, adenoma Pancreas Salivary glands Fibroma Stomach Stomach, forestomach Stomach, glandular 4 + + + 4 4 4 4 + + + + 4- 4- 4- 4 4 4 4 4 4 4 4 4 4 + + + + 4 + 4 4 4 + A 4 4 4- 4- 4 4 4 4 4 4 4 4 4 4 + 4 A A + A + + + + A 4- + 4- 4- 4 4 A 4 4 4 4 4 4 4 4 + + 4 + + + + + + A + 4- 4- 4- 4 4 A 4 4 4 4 4 4 4 4 4 M M + + + + + + A + 4- 4- 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 + 4 + + A 4 + 4- 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 + + + + 4 4 4 A 4 4 4- 4 4 4 4 4 4 4 4 4 4 4 + A A A + A + + A 4 A + 4- 4 4 4 4 A 4 4 4 4 4 4 4 4 + 4 A 4 + + 4 A 4 A + + 4 4- 4 4 A 4 4 4 4 4 4 4 4 + + + + 4 + 4 + 4 + 4- 4- 4 4- 4 4 4 4 4 4 4 4 4 4 X X + + + + + + + + + + + 4- 4 4- 4 4 4 4 4 4 4 4 4 4 4 + + + 4 + + + + + + + 4- 4 4- 4 4 4 4 4 4 4 4 4 4 4 X 4 4 + + 4 4 4 4 4 + + 4- 4- 4- 4 4 4 4 4 4 4 4 4 4 4 + 4 + + + 4 + 4 + + + 4 4- 4 4- 4 4 4 4 4 4 4 4 4 4 + + + + + + + + + + 4 4- 4- 4 4 4 4 4 4 4 4 4 4 4 4 50 45 38 43 41 44 44 34 38 50 1 1 46 50 1 47 47 47 Cardiovascular System Blood vessel Heart 4 44 44 +44444 ++4 444444 44444444 5 50 Endocrine System Adrenal gland Adrenal gland, cortex Adrenal gland, medulla Pheochromocytoma malignant Pheochromocytoma complex Pheochromocytoma benign Bilateral, pheochromocytoma benign Islets, pancreatic Parathyroid gland Adenoma Pituitary gland Pars distalis, adenoma Pars distalis, carcinoma Thyroid gland C-cell, adenoma C-cell, carcinoma 4444444444444444444444444 4444444444444444444444444 4444444444444444444444444 XX X X X XX X XX XXXXX XXX X XX XX 4 4 M4 4 M4 4 4 4 4 4 4 4 4 4 4 M4 4 4 4 4 4 4 4 4 4 4 4 4 M4 4 4 4 4 4 M4 4 4 4 4 4 4 4 4 4 4 4444444444444444444444444 XX XX XX X X 444444444444444 4444444444 XX XX X 49 49 48 3 2 9 21 41 45 1 50 11 1 46 4 1 76 Talc, NTP TR 421 Table A2 Individual Animal Tumor Pathology of Male Rats In the Lifetime Inhalation Study of Talc: 6 mg/ro3 (continued) Number o f Days on Study 155555556666666666 7777777 8224579901345567791223344 679 48 3334 1380 7 3370 5 28 49 0 1 Carcass ID Number 00 10 10 0 10 00 10 0 0 10 0 10 0 0 00 1 020500 407 1726350050 18 9852 6 9 79549 33 1980 170 3 32 23 746 4 1 111 1111 111 11 111 1 111 1 1111 General Body System Tissue NOS Pheochromocytoma malignant, metastatic, adrenal gland + X Genital System Epididymis Preputial gland Adenoma Carcinoma Prostate Seminal vesicle Testes Bilateral, interstitial cell, adenoma Interstitial cell, adenoma +4+4 +++++++++++++++++++++ 4 + + + + + + + + + M+ + + + + + + + + + + + 4 + XX XX + + + M+ + + + M+ M+ M+ + + + 4 + + 4 + + + A + + + M + + + + +- M 4 + + + + + + + + + + + + + + +4 +++++++++++++++++++++++ X XX X XX XX XX X XX X H em atopoietic System Bone marrow Lymph node Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Fibrous histiocytoma Thymus + + + + + 4 4 4- + 4- + A A + 4- 4- 4- + + + + 4- + 4- 4+ + + 4- 4- + + + + + + + + 4- 4- + + 4- + 4- + + 4- + + + 4- 4- 4- 4- 4- 4- + M + + + 4 4 4 4 4 4 4 A 4 4 + + + + 4 4- 4 4 4 4 4 4 4 4 1 + + 4- 4- + + 4- + 4- i + + + 4 4 + 4- + 4- + M + + + 4- + + + 4- 4- 4- 4- + 4- + 4- 4- + + + + 4- + + + 4 4 4- + A 4 4 4 4- 4- + 4- + + + + + 4+ + 4 + + + 4- + 4- + + + + 4- 4- 4- 4- + + + + 4- + + + 4 4- 4- M 4 4 4 4 4- 4 4 M 4- 4 4- 4- M 4- 4- 4- 4- 4- i + 4 Integumentary System Mammary gland Skin Fibroma Keratoacanthoma Neurofibroma Squamous cell carcinoma Subcutaneous tissue, fibroma Subcutaneous tissue, fibrosarcoma + + + + + + + + M 4- 4- + + 4- 4- + 4- 4- 4- 4- 4 + + + + + + + + + + + + 4- 4- + + + + + + 4 4- 4- 4 4- 4 4 4 4- X X Lesions in Maie Rats 77 Table A2 Individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 6 mg/m3 (continued) N um ber of Days on Study 7777777777777777778888888 4455668888999999990000000 6 779 120 137 19 99 99 99 0 00 00 00 C arcass ID N um ber 0 10 0 110000 00 00 00 00 0 00 1 111 2393205520303335770792223 62 826 4 10 8 54 10 56 2 458 8 9 1590 1111111111111111111111111 Total Tissues/ T um ors G eneral Body System Tissue NOS Pheochromocytoma malignant, metastatic, adrenal gland 1 1 G enital System Epididymis Preputial gland Adenoma Carcinoma Prostate Seminal vesicle Testes Bilateral, interstitial cell, adenoma Interstitial cell, adenoma + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + + 4- X XX + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4* 4- 4- 4- + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4* + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + + + XXX XX X XXXX XXXXXX XXXXX X XX X 50 49 1 6 45 48 50 24 15 H ematopoietic System Bone marrow Lymph node Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Fibrous histiocytoma Thymus + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- + + + 4+ + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + + + + I + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- X + M + + + + + 4- 4- M i 4- 4- 4- M 4- 4- 4- 4- 4- 4- M 4- M 4- 48 50 48 48 49 48 50 1 40 Integum entary System Mammary gland Skin Fibroma Keratoacanlhoma Neurofibroma Squamous cell carcinoma Subcutaneous tissue, fibroma Subcutaneous tissue, fibrosarcoma M + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + + + + + + + 4- 4* 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 48 50 78 Talc, NTP TR 421 Table A2 Individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Tala 6 mg/ro3 (continued) Num ber of Days on Study 15 55555566666666667777777 8 22457990 134556779 1223344 6 7948 33 34 1380 733 70 5 28 4 9 0 1 C arcass ID N um ber 00 10 10 0 100 0 100 0 100 10 00 00 1 0 2 0 5 0 0 4 0 7 17 2 6 3 5 0 0 50 18 9 8 5 2 6 9 79 549 33 198 0 17 03 322 3 74 64 1 111111111111111111111111 M usculoskeletal System Bone Pelvis, osteosarcoma +++++++++++++++++++++++++ X Nervous System Brain +++ ++++ ++++ +++ ++ ++++ ++ + ++ R espiratory System Larynx Lung Alveola r/bronchiolar adenoma Osteosarcoma, metastatic Nose Sarcoma Trachea +++++++++++++++++++++++++ ++ ++ +++++ ++++ ++ ++ ++ ++ + +++ X + + ++ + ++ ++ +A+ + ++ ++ + ++ ++ + ++ X ++++ +++ ++++ +++ ++ ++ ++ ++ + ++ Special Senses System Eye + Urinary System Kidney Urinary bladder +++A+++++ ++++++ ++++ ++++++ + + + ++ + + ++ +A + + + + + + + + + + + + + + Systemic Lesions Multiple organs Leukemia mononuclear + ++ +++++ +++ + ++ ++ ++++ ++ +++ XX XXX XXX XX XX Lesions in Male Rats 79 Table A2 Individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 6 mg/m3 (continued) Num ber of Days on Study 7777777777777777778888888 4455668888999999990000000 6779 120 137 199999990000000 C arcass ID Num ber 0 100 11000000000000000 111 1 2393205520303335770792223 6 28 26 4 10 85 4 10 56 2 4588 9 15 90 11 111 1 1111 11 111 1 111 11 1 111 Total T issu es/ Tumors M usculoskeletal System Bone Pelvis, osteosarcoma + + + + + + + + + + + 4- + + + + + + + + + + + + + 50 1 Nervous System Brain + + + 4* + + + + + + + + + + + + + + + + + + + + + 50 Respiratory System Larynx Lung Aiveolar/bronchiolar adenoma Osteosarcoma, metastatic Nose Sarcoma Trachea +++++++++ +++++++++++++++ ++ ++ ++ ++ +++ ++ ++ +++ + +++++ + X + ++ ++A+++ +++ ++ +++ + ++ ++ ++ + +++++++++++++++++++++++++ 49 50 1 1 48 1 50 Special Senses System Eye 4- 2 Urinary System Kidney Urinary bladder 4- + + 4 + + 4- 4 4 - 4 - 4 4- + + + 4 4- 4 4- 4* 4 4- 4- 4- 4 4- 4- + 4* M + + 4- 4 - 4 - 4 - 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 49 48 System ic Lesions Multiple organs Leukemia mononuclear 4- + + + + 4- 4- 4- 4 - 4 - 4 - 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- X XX X X X XX X 50 21 80 Talc, NTP TR 421 Table A2 Individual Animal Tumor Pathology or Male Rats in the Lifetime Inhalation Study of Talc: 18 mg/m1 N um ber of Days on Study 2455666666666666666677777 49 0900 1 1 1 1234 55 5789 90 0 12 3 8 20 4794 5578 45 1136 3 78 159 27 C arcass ID N um ber 2 12 12 11122 12222222 112 12 11 14074774269 15026727525557 953744594657 127706625 1202 1111111111111111111111111 Alim entary System Esophagus Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Neoplastic nodule Neoplastic nodule, multiple Mesentery Pancreas Salivary glands Stomach Stomach, forestomach Fibrosarcoma Stomach, glandular Fibrosarcoma Tongue 444444444444444444+4444 44 4 4+4444 444 44A444 44 44 4 44 4 4 +A4AA4 4 4 4 4 4 4 A4 4 4 4 + +AA4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4A4 4 4 +44 44 4 4 4 4 4 MMA 4 4 4 4 4 4 M4 A 4 4 4 4 4 MMMM 4 M4 4 +4A4 4 4 4 4 4 4 4A4 4 4 4 4 4 + 4 4 4 4 4 ++4A+4 44 4 4 4 4A4 4 4 4 4 4 4 4 4 4 4 4 MA A A 4 4 4 4 M4 4 4 A 4 4 4 + 4 4 A A A 4 4 4 +AAA4 4 4 4 4 4 4 4 A4 4 4 4 4 4 AA 4 4 4 4 4 4- + 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 X XX 4 + 44A4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 44 +44444444444444 44444+4 444 4 4 4A4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 +4 4 4 4 4 4A4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 44 X +44A44 4 4444 444 4 444 44 +4 444 X Cardiovascular System Blood vessel Heart 4 44 4444444444444444444444444 Endocrine System Adrenal gland Adrenal gland, cortex Adrenal gland, medulla Osteosarcoma, metastatic, uncertain primary site Pheochromocytoma malignant Pheochromocytoma complex Pheochromocytoma benign Bilateral, pheochromocytoma malignant Bilateral, pheochromocytoma benign Islets, pancreatic Adenoma Parathyroid gland Pituitary gland Pars distalis, adenoma Pars intermedia, adenoma Thyroid gland C-cell, adenoma Follicular cell, adenoma 4444444444444444444444444 4444444444444444444444444 4 4 4 M4 M4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 X X XXXXXX XX X XX XX 4 4 4A4 4 4 4 4 4 4 4 4I 4 4 4 4 4 4 4 4 4 4 4 X M4 4 4 4 4 4 I 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 M 4 4 4 M4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 XX X XX 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4A4 4 4 4 X Lesions in Male Rats 81 Table A 2 Individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 18 mg/mJ (continued) Number of Days on Study 7777777777777777788888888 4788999999999999900000000 3838113489999999900000000 Carcass ID Num ber 2221222111111111122222222 2049477945567899922444677 21 2 4 5 4 1 7 6 5 6 9 8 0 3 6 8 0 8 3 6 7 8 5 6 1111111111111111111111111 Total T issu es/ Tumors Alim entary System Esophagus Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine smalt, ileum Intestine small, jejunum Liver Neoplastic nodule Neoplastic nodule, multiple Mesentery Pancreas Salivary glands Stomach Stomach, forestomach Fibrosarcoma Stomach, glandular Fibrosarcoma Tongue + + + + + + + ++ + + + + + + + + ++ + I + + + + A A + + + + + ++ + + + + + + + + ++ + + + + + + A A + + A + A +A A + + + + + + + ++ + + + + A + A A + + + + + ++ + + + + + + + + ++ + + + + + A A A M + +M + ++ + + + + + + + + +M + + M + + + A A + + + + + ++ + + + + + + + + ++ + + + + + + A A + + + + + ++ + + + + + + + + ++ + + + + + + A A + + + A + +A + + + + + + + + ++ + + + + A A A A + + + + + ++ + + + + + + + + ++ + + + + A A A A + + + + + ++ + + + + + + + + ++ + + + + + + X AA ++ AA AA ++ + ++ + ++ + ++ + + + ++ + + ++ + + ++ + + ++ + + + + + + + + ++ + + + + + + + + ++ + + + + + + + + ++ + + + + + + + + ++ +++ +++ +++ +++ +++ +++ +++ +++ AA + + + + + ++ + + + + + + + + ++ + + + + + + + 49 47 37 46 34 46 46 35 40 48 1 3 1 47 50 47 47 1 47 1 1 Cardiovascular System Blood vessel Heart + +5 +++++++++++++++++++++++++ 50 Endocrine System Adrenal gland Adrenal gland, cortex Adrenal gland, medulla Osteosarcoma, metastatic, uncertain primary site Pheochromocytoma malignant Pheochromocytoma complex Pheochromocytoma benign Bilateral, pheochromocytoma malignant Bilateral, pheochromocytoma benign Islets, pancreatic Adenoma Parathyroid gland Pituitary gland Pars distalis, adenoma Pars intermedia, adenoma Thyroid gland C-cell, adenoma Follicular cell, adenoma +A++ ++ + +++ ++ + ++ + ++ + ++ + + ++ AA+ + + + + + + + + + + + + + + + + + + + + + + +A++++++++++++++++++++ +++ XX X XX X XXX XX XX XXXXX X X XXX X XX XX XX A A + + + + + + + MM+ + + + + + + M+ + + + + + X + + + + + M+ + + + + + + + + + + + + + + + + + + + +' + + + + + + + + + + + + + + + + + + + + + + + X XX X XX X A A + + + M+ + + + + + + + + + + + + + + + + + + XX X 49 48 47 1 6 1 20 1 16 43 2 46 49 10 2 46 3 1 82 Talc, NTP TR 421 Table A2 Individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 18 mg/m3 (continued) N um ber of Days on Study 2455666666666666666677777 490900 1111234555789900 123 8 20 4794 5578 45 1 136 3 78 1592 7 C arcass ID N um ber 2 12 12 11122 12222222 112 12 11 140 74774269 15026727 525 557 953744594657 127706625 1202 1111111111111111111111111 General Body System None Genital System Epididymis Penis Preputial gland Adenoma Carcinoma Prostate Seminal vesicle Testes Bilateral, interstitial celt, adenoma Interstitial cell, adenoma + + + + + + + M+ + + + + + + + + + + + + + + + + + + + + + + + + + M+ + + + + + + + M+ + + + + + X +++A+++ +++ ++ + +++ ++ ++ ++ +++ +++A++++ ++++++++++++A++++ +++++++++++++++++++++++++ X XXX XXX X XX XXX X X Hem atopoietic System Bone marrow Lymph node Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Thymus + + +++ ++ ++ + +++A+ + + ++ +++ +++ +++++++++++++++++++++++++ + + + + + + + + + + + + + + + + + M+ + + + + + + +++A+ +++++I + ++ ++ + ++ ++ + ++ + I M+ + + + + + + + + + + + + + + + + + + + + + + +++++ +++ ++++++++++++++ +++ + + +A++ +++ + ++++ ++ ++ ++ ++ + ++ I + + + + + + + + M+ M+ + + + M+ + + + + + + + Integum entary System Mammary gland Skin Basosquamous tumor malignant Fibrous histiocytoma Keratoacanthoma ++++++ +++ +++ +++ + ++ + ++ ++ ++ +++++++++++++++++++++++++ X X M usculoskeletal System Bone Vertebra, osteosarcoma +++++++++++++++++++++++++ X Lesions in Male Rats 83 Table A2 Individual Animal Tumor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 18 mg/m3 (continued) Num ber of Days on Study 7777777777777777788888888 4788999999999999900000000 3838 113489999999900000000 C arcass ID Num ber 222 1222 111111111122222222 20494 7794 556 78999 22 4 44 67 7 2 12454 17656980 3680 8367856 1111111111111111111111111 Total Tissues/ T um ors General Body System None G enital System Epididymis Penis Preputial gland Adenoma Carcinoma Prostate Seminal vesicle Testes Bilateral, interstitial cell, adenoma Interstitial cell, adenoma +++++++++++++++++++++++++ ++ + +++++++++++++++++++++++++ X A++++++++++++ ++++++++++++ A++++ ++++++++++++++++++++ +++++++++++++++++++++++++ XX X X X X XX XXX XXXXX X XX X X 49 3 48 1 1 48 47 50 24 12 Hematopoietic System Bone marrow Lymph node Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Thymus AA+ ++ ++ + + + + ++ + + + + + + + + + + + + + ++++ ++ +++ +++ +++ +++ +++ +++ +++++++++++++++++++++++++ A+ + + + + + + + + + + + + + + + + + + + + + + + +++++++++++++++++++++I +++ AAA+ + + + + + + + + + + + + + + + + + + + + + +A+++ +++++ ++++ ++ +++ +++ +++ A + + + M+ + + + + + + + + + + + + + + + + + + M 47 50 49 47 47 47 48 43 Integum entary System Mammary gland Skin Basosquamous tumor malignant Fibrous histiocytoma Keratoacanthoma +++++++++++++++++++++++++ +++++++++++++++++++++++++ X X 50 50 1 1 2 M usculoskeletal System Bone Vertebra, osteosarcoma +++++++++++++++++++++++++ 50 1 84 Talc, NTP TR 421 Table A2 Individual Animal Tnmor Pathology of Male Rats in the Lifetime Inhalation Study of Talc: 18 mg/m3 (continued) Num ber of Days on Study 2455666666666666666677777 4909001 1 1 1 2 345 55 789 90 012 3 82047945578451 13637815927 C arcass ID Num ber 2121211122122222221121211 1407477426915026727525557 9537445946571277066251202 1111111111111111111111111 Nervous System Brain Spina) cord +++ +++ ++++++++ ++ +++ +++ +++ + Respiratory System Larynx Lung Aiveolar/bronchiolar adenoma AJveolar/bronchiolar carcinoma, multiple Osteosarcoma, metastatic, uncertain primary site Nose Trachea +++++++++++++++++++++++++ +++++++++++++++++++++++++ X +++++++++++++++++++++++++ +++++++++++++++++++++++++ Special Senses System Eye ++ U rinary System Kidney Ureter Urethra Urinary bladder + + + + + + + + + + + + + + + + + + + + ++ + + + + + + +A + + + + + + + + + + + + + + + + + + + + + Systemic Lesions Multiple organs Leukemia mononuclear Mesothelioma malignant +++++++++++++++++++++++++ X XXXXXX XXXX XXX Lesions in Male Rats 85 Table A2 Individual Animal Tumor Pathology or Male Rats In the Lifetime Inhalation Study of Talc 18 mg/m3 (continued) Num ber of Days on Study 7777777777777777788888888 4788999999999999900000000 3838 113489999999900000000 C arcass ID N um ber 222 1222 11111111 1122222222 20 494 77 94 556 78 99 92 24 446 77 2 12454 1765698036808367856 11 11 11111111 111 1111 11 1111 Total Tissues/ T um ors Nervous System Brain Spinal cord ++ ++++ + + ++ + ++ +++ +++ +++ +++ 50 1 R espiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma, multiple Osteosarcoma, metastatic, uncertain primary site Nose Trachea +A ++ + + ++ + + ++ ++ + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + X X A + + + +AA + + + + + + + + + + + + + + + + + + AA + + ++ + + + + + + + + + + + + + + + + + + + 49 50 1 1 1 47 48 Special Senses System Eye 2 U rinary System Kidney Ureter Urethra Urinary bladder AA + + + + + + + + + + + + + + + + + + + + + + + + AA + + + + + + + + + + + + + + + + + + + + + + + 48 1 1 47 Systemic Lesions Multiple organs Leukemia mononuclear Mesothelioma malignant ++++ ++++++++++++++++++++ + XXX XXXX XX X 50 23 1 86 Talc, NTP TR 421 T able A3 Statistical Analysis or Primary Neoplasms in Male Rats in the Lifetime Inhalation Study of Talc Adrenal M edulla: Benign Pheochrom ocytom a Overall rates* Adjusted rates'3 Terminal rates' First incidence (days) Life table tests" Logistic regression tests'1 Cochran-Armitage tesld Fisher exact test 25/49 (51%) 87.6% 6/9 (67%) 429 P =0.434 P-0.007 P =0.007 Adrenal M edulla: M alignant Pheochrom ocytom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 3/49 (6%) 17.2% 1/9 (11%) 670 P =0.242 P =0.096 P =0.083 Adrenal M edulla: Benign, M alignant, or Complex Pheochromocytom a Overall rates 26/49 (53%) Adjusted rates 91.7% Terminal rates 7/9 (78%) First incidence (days) 429 Life table tests P =0.483 Logistic regression tests P=0.007 Cochran-Armitage test P = 0.007 Fisher exact test Liver. H epatocellular Adenom a or N eoplastic Nodule Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 2/49 (4%) 11.2% 0/9 (0%) 698 P =0.359 P =0.248 P-0.237 Pancreatic Islets: Adenoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage lest Fisher exact test 1/47 (2%) 12.5% 1/8 (13%) 799 (T) P =0.387 P =0.308 P = 0.304 30/48 (63%) 90.2% 11/14 (79%) 558 P-0.515N P=0.213 P-0.175 3/48 (6%) 15.2% 1/14 (7%) 544 P-0.552N P =0.662 P =0.651 32/48 (67%) 93.6% 12/14 (86%) 544 P-0.549N P=0.147 P=0.123 2/50 (4%) 14.3% 2/14 (14%) 799 (T) P-0.586N P=0.661 N P=0.684N 0/41 (0%) 0.0% 0/13 (0%) _e P=0.403N P --0.403N P-0.534N 36/47 (77%) 100.0% 16/16 (100%) 614 P --0.499 P = 0.009 P-0.008 7/47 (15%) 31.5% 3/16 (19%) 645 P-0.376 P-0.178 P-0.142 37/47 (79%) 100.0% 16/16 (100%) 614 P =0.539 P =0.006 P =0.007 4/48 (8%) 14.9% 1/16 (6%) 615 P = 0.434 P =0.333 P = 0.329 2/43 (5%) 9.9% 1/13 (8%) 617 P-0.612 P =0.479 P =0.466 Lesions in Male Rats Table A3 Statistical Analysis of Primary Neoplasms in Male Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 Pancreatic Islets: Adenoma or Carcinoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Pituitary Gland (Pars D istalis): Adenoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Pituitary Gland (Pars D istalis): Adenoma or Carcinom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Preputial Gland: Carcinoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Preputial Gland: Adenom a or Carcinoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 2/47 (4%) 25.0% 2/8 (25%) 799 (T) P = 0.650 P =0.544 P =0.531 12/47 (26%) 53.6% 3/9 (33%) 568 P=0.174N P=0.307N p=0.344N 12/47 (26%) 53.6% 3/9 (33%) 568 P=0.159N P=0.287N P=0325N 1/48 (2%) 2.3% 0/9 (0%) 586 P =0.361 N p=0.440N P=0.425N 2/48 (4%) 4.4% 0/9 (0%) 429 P=0.331N P=0.454N P=0.436N 0/41 (0%) 0.0% 0/13 (0%) P=0.135N P=0.135N P=0.282N 11/50 (22%) 42.8% 3/14 (21%) 558 P=0.334N P=0.419N P=0.432N 12/50 (24%) 45.8% 3/14 (21%) 558 P=0.411N P=0.509N P=0.524N 6/49 (12%) 22.5% 1/14 (7%) 527 P =0.090 P =0.058 P =0.059 7/49 (14%) 28.5% 2/14 (14%) 527 P =0.134 P =0.078 P = 0.084 2/43 (5%) 9.9% 1/13 (8%) 617 P=0.560N P = 0.683 P=0.657 10/49 (20%) 42.1% 4/16 (25%) 697 P=0.160N P=0.324N P=0.362N 10/49 (20%) 42.1% 4/16 (25%) 697 P=0.160N P=0.324N P=0.362N 1/48 (2%) 2.5% 0/16 (0%) 628 P=0.753N P=0.750 P=0.753N 2/48 (4%) 8.6% 1/16 (6%) 628 P=0.632N P = 0.673 P=0.692N 87 8 Talc, NTP TR 421 T able A3 Statistical Analysis of Primary Neoplasms in Male Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 Skin; K eratoacanthom a or Squam ous Cell Carcinoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Testes: Adenoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Thyroid Gland (C-cell): Adenoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Thyroid Gland (C-cell): Adenom a or Carcinoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test All Organs: M ononuclear Cell Leukem ia Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 0/49 (0%) 0.0% 0/9 (0%) - P=0.414 P = 0.323 P=0.319 31/49 (63%) 100.0% 9/9 (100%) 551 P=0.198N P = 0.333 P =0.295 3/45 (7%) 24.5% 2/9 (22%) 682 P=0.348N P=0.511N P=0.560N 3/45 (7%) 24.5% 2/9 (22%) 682 P=0.296N P=0.467N P=0.523N 24/49 (49%) 70.3% 3/9 (33%) 334 P=0.298N P = 0.501 N P=0.486N 3/50 (6%) 13.5% 1/14 (7%) 663 P=0.161 P=0.128 P=0.125 39/50 (78%) 100.0% 14/14 (100%) 544 P=0.524 P = 0.056 P =0.082 4/46 (9%) 28.6% 4/14 (29%) 799 (T) P=0.620N P=0.641 P=0.512 5/46 (11%) 33.0% 4/14 (29%) 787 P=0.568 P =0.502 P = 0.369 21/50 (42%) 59.9% 4/14 (29%) 529 P=0.232N P=0.317N P=0.310N 2/50 (4%) 6.6% 0/16 (0%) 594 P =0.331 P =0.239 P=0.253 36/50 (72%) 97.0% 15/16 (94%) 609 P=0.245N P = 0.268 P = 0.238 3/46 (7%) 14.5% 2/16 (13%) 614 P=0.476N P=0.625N P=0.651N 3/46 (7%) 14.5% 2/16 (13%) 614 P=0.476N P=0.625N P=0.651N 23/50 (46%) 62.5% 6/16 (38%) 492 P=0.269N P=0.479N P=0.462N Lesions in Maie Rats 89 Table A3 Statistical Analysis of Primary Neoplasms in Male Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 m g/m 3 18 mg/m3 Ail Organs: Benign N eoplasm s Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact lest All Organs: M alignant N eoplasm s Overall rates Adjusted rates Terminal rales First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test All Organs: Benign or M alignant N eoplasm s Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 42/49 (86%) 100.0% 9 /9 (100%) 429 P=0.161N P = 0.463 P = 0.353 34/49 (69%) 88.4% 6/9 (67%) 334 P=0.222N P=0.534N P=0.505N 48/49 (98%) 100.0% 9 /9 (100%) 334 P=0.154N P =0.337 P=0.348 45/50 (90%) 100.0% 14/14 (100%) 544 P=0.314N P =0.430 P =0.365 34/50 (68%) 80.9% 7/14 (50%) 527 P=0.308N P=0.539N P=0.527N 49/50 (98%) 100.0% 14/14 (100%) 527 P=0.241N P =0.771 P =0.747 45/50 (90%) 100.0% 16/16 (100%) 594 P=0.153N P =0.480 P =0.365 34/50 (68%) 80.0% 9A6 (56%) 248 P=0.216N P=0.571N P=0.527N 50/50 (100%) 100.0% 16A6 (100%) 248 P=0.139N P =0.506 P = 0.495 (T)Terminal sacrifice Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, bone marrow, brain, epididymis, heart, kidney, larynx, liver, lung, nose, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, testes, thyroid gland, and urinary bladder, for other tissues, denominator is number of animals necropsied. b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality c Observed incidence at terminal kill Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests compare directly the overall incidence rales. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. e Not applicable; no neoplasms in animal group 9 Talc, NTP TR 421 Table A4 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the Lifetime Inhalation Study of Talc* 0 mg/m3 6 mg/m3 18 mg/m3 Disposition Sum m ary Animals initially in study Early deaths Moribund Natural deaths Survivors Died last week of study Terminal sacrifice Animals examined microscopically Alimentary System Esophagus Inflammation Intestine large, cecum Hemorrhage Inflammation Parasite metazoan Ulcer Intestine large, colon Hyperplasia, lymphoid Inflammation Mineralization Parasite metazoan Intestine large, rectum Inflammation Metaplasia, squamous, focal Parasite metazoan Intestine small, duodenum Inflammation Mineralization Necrosis, focal Ulcer Intestine small, ileum Hyperplasia, lymphoid Lymphatic, ectasia Liver Angiectasis, focal Atrophy Basophilic focus Clear cell focus Congestion Degeneration, cystic Degeneration, diffuse Eosinophilic focus Fatty change Fibrosis Hematocyst Hyperplasia, focal Inflammation, granulomatous, focal Inflammation, necrotizing, focal Necrosis, focal Thrombosis Bile duct, hyperplasia 49 23 17 1 8 49 (49) (42) 9 (21%) 3 (7%) 1 (2%) (43) 1 (2%) 1 (2%) 2 (5%) (38) 6 (16%) (48) 1 (2%) 1 (2%) 1 (2%) (39) (49) 1 (2%) 1 (2%) 18 (37%) 3 (6%) 9 (18%) 2 (4%) 16 (33%) 1 (2%) 3 (6%) 3 (6%) 1 (2%) 39 (80%) 50 19 17 2 12 50 (50) (38) 1 (3%) 2 (5%) 4 (11%) (43) 1 (2%) (41) 1 (2%) 2 (5%) (44) 1 (2%) (34) 1 (3%) 1 (3%) (50) 18 (36%) 7 (14%) 1 (2%) 17 (34%) 7 (14%) 14 (28%) 1 (2%) 1 (2%) 46 (92%) 50 20 14 3 13 50 (49) 1 (2%) (37) 5 (14%) 4 (11%) (46) 1 (2%) 1 (2%) 1 (2%) (34) 1 (3%) 1 (3%) (46) 1 (2%) (35) 2 (6%) (48) 19 (40%) 4 (8%) 9 (19%) 1 (2%) 7 (15%) 12 (25%) 1 (2%) 1 (2%) 1 (2%) 44 (92%) Lesions in Maie Rats 91 T a b l e A4 Summary of the Incidence of Nonneopiastic Lesions in Male Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 A lim entary System (continued) Liver (continued) Cenlrilobular, atrophy Centrilobular, degeneration Centrilobular, degeneration, fatty Centrilobular, necrosis Mesentery Inflammation Pancreas Lobules, atrophy Salivary glands Inflammation Necrosis Stomach, forestomach Hyperkeratosis Inflammation Mineralization Ulcer Stomach, glandular Mineralization Ulcer Cardiovascular System Blood vessel Aorta, mineralization Mesenteric artery, aneurysm Mesenteric artery, inflammation Mesenteric artery, mineralization Mesenteric artery, thrombosis Heart Cardiomyopathy Atrium, thrombosis Epicardium, hyperplasia Myocardium, inflammation Myocardium, mineralization Endocrine System Adrenal gland, cortex Degeneration Degeneration, fatty Degeneration, focal Hyperplasia, diffuse Hyperplasia, focal Necrosis, focal Adrenal gland, medulla Hyperplasia Bilateral, hyperplasia islets, pancreatic Hyperplasia Parathyroid gland Hyperplasia Bilateral, hyperplasia 9 (18%) 8 (16%) 5 (10%) (2) (48) 11 (23%) (49) 1 (2%) (49) 1 (2%) 1 (2%) 5 (10%) (49) 6 (12%) 3 (6%) () 3 (75%) 3 (75%) 1 (25%) (49) 42 (86%) 9 (18%) 1 (2%) 2 (4%) (49) 1 (2%) 8 (16%) 1 (2%) 11 (22%) 1 (2%) (49) 19 (39%) 1 (2%) . (47) (45) 6 (13%) 1 (2%) 4 (8%) 12 (24%) (46) 7 (15%) (50) (47) 4 (9%) 5 (11%) (47) 6 (13%) 3 (6%) (5) 5 (100%) 5 (100%) 1 (20%) (50) 47 (94%) 5 (10%) 1 (2%) 6 (12%) (49) 4 (8%) (48) 8 (17%) (41) (45) 11 (24%) 7 (15%) 9 (19%) 1 (2%) 2 (4%) (1) 1 (100%) (47) 8 (17%) (50) 1 (2%) (47) 1 (2%) 1 (2%) 8 (17%) (47) 6 (13%) 2 (4%) (5) 4 (80%) 2 (40%) 1 (20%) 3 (60%) 1 (20%) (50) 50 (100%) 11 (22%) 5 (10%) (48) 2 (4%) 2 (4%) 9 (19%) (47) 8 (17%) 1 (2%) (43) 1 (2%) (46) 12 (26%) 92 Talc, NTP TR 421 T able A4 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 E ndocrine System (continued) Pituitary gland Angiectasis, focal Cyst Pars distalis, hyperplasia Pars nervosa, hyperplasia Thyroid gland C-cell, hyperplasia (47) 8 (17%) (45) 5 (11%) (50) 1 (2%) 1 (2%) 8 (16%) 1 (2%) (46) 7 (15%) (49) 1 (2%) 7 (14%) (46) 2 (4%) General Body System None Genital System Epididymis Spermatocele Preputial gland Hyperplasia Inflammation Prostate Atrophy Inflammation Seminal vesicle Atrophy Inflammation Testes Atrophy Hyperplasia, lymphoid Hyperplasia, lymphoid, focal Interstitial cell, hyperplasia Serosa, proliferation (49) (48) 3 (6%) 7 (15%) (49) 1 (2%) 22 (45%) (49) 1 (2%) 1 (2%) (49) 14 (29%) 2 (4%) (50) 1 (2%) (49) 2 (4%) (45) 14 (31%) (48) (50) 11 (22%) 2 (4%) 1 (2%) (49) (48) 1 (2%) 5 (10%) (48) 1 (2%) 19 (40%) (47) (50) 16 (32%) 1 (2%) 3 (6%) 1 (2%) Hematopoietic System Bone marrow Atrophy Atrophy, focal Inflammation Myelofibrosis Myeloid cell, hyperplasia Lymph node Hemorrhage, chronic Pancreatic, atrophy Pancreatic, hyperplasia, lymphoid Lymph node, bronchial Atrophy Hemorrhage Hemorrhage, acute Hemorrhage, chronic Hyperplasia, histiocytic Lymph node, mandibular Hemorrhage Hyperplasia, lymphoid Hyperplasia, plasma cell Inflammation, chronic active (48) 2 (4%) (49) 1 (2%) 1 (2%) (41) 2 (5%) 1 (2%) 4 (10%) (46) (48) 1 (2%) 1 (2%) 1 (2%) 3 (6%) (50) 1 (2%) (48) 1 (2%) 44 (92%) (48) 1 (2%) 2 (4%) 2 (4%) (47) 2 (4%) 1 (2%) 6 (13%) (50) (49) 46 (94%) (47) 5 (11%) 2 (4%) Lesions in Male Rats 93 Table A4 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 1 6 mg/m 18 mg/m3 H em atopoietic System (continued) Lymph node, mediastinal Atrophy Hemorrhage Hemorrhage, acute Hemorrhage, chronic Hyperplasia, histiocytic Pigmentation, hemosiderin Lymph node, mesenteric Atrophy Hemorrhage Hemorrhage, acute Hyperplasia, lymphoid Hyperplasia, plasma cell Inflammation, chronic active Spleen Atrophy Autolysis Congestion, chronic Cyst Fibrosis Fibrosis, focal Hematopoietic cell proliferation Hyperplasia, histiocytic Hyperplasia, lymphoid Infarct inflammation, granulomatous, focal Thymus Atrophy Cyst (48) 1 (2%) 1 (2%) 6 (13%) 1 (2%) (49) 1 (2%) 1 (2%) 1 (2%) (49) 1 (2%) 1 (2%) 1 (2%) 3 (6%) 1 (2%) (48) 1 (2%) (49) 3 (6%) 40 (82%) (48) 2 (4%) 2 (4%) (50) 1 (2%) 5 (10%) 2 (4%) 1 (2%) 1 (2%) (40) 2 (5%) (47) 43 (91%) (47) 3 (6%) 1 (2%) 1 (2%) (48) 2 (4%) 1 (2%) 1 (2%) 2 (4%) 3 (6%) 1 (2%) 1 (2%) (43) Integumentary System Mammary gland Galactocele Skin Cyst epithelial inclusion Subcutaneous tissue, inflammation Tail, necrosis (45) 1 (2%) (48) * 1 (2%) (48) (50) 1 (2%) (50) (50) 1 (2%) M usculoskeletal System Bone Fibrous osteodystrophy Coccygeal, necrosis Pelvis, fracture (49) 3 (6%) 1 (2%) (50) 4 (8%) 1 (2%) (50) 5 (10%) Nervous System Brain Compression Hemorrhage Infarct Necrosis, focal Spinal cord Degeneration (49) 5 (10%) 1 (2%) (50) 2 (4%) 1 (2%) 2 (4%) (50) 2 (4%) 1 (2%) 0) 1 (100%) 94 Talc, NTP TR 421 Table A4 Summary of the Incidence of Nonneopiastic Lesions in Male Rats in the Lifetime Inhalation Study of Talc (continued) 1 0 mg/m 6 m g/m 3 18 m g/m 3 Respiratory System Larynx Inflammation, suppurative Lung Congestion Crystals, focal Cyst Hemorrhage, chronic Infarct Inflammation, granulomatous Inflammation, suppurative Mineralization Alveolar epithelium, hyperplasia Alveolus, hemorrhage, focal Alveolus, metaplasia, squamous Artery, thrombosis lnterstitium, fibrosis, focal lnterstitium, mineralization Peribronchial, hyperplasia, histiocytic Nose Inflammation, suppurative Lumen, foreign body Lumen, hemorrhage Mucosa, inflammation, suppurative Nasolacrimal duct, inflammation, suppurative Respiratory epithelium, hyperplasia Trachea Inflammation, suppurative (48) 6 (13%) (49) 1 (2%) 1 (2%) 2 (4%) 1 (2%) 2 (4%) 5 (10%) 1 (2%) 1 (2%) 1 (2%) 2 (4%) (49) 2 (4%) 1 (2%) 4 (8%) (49) 3 (6%) (49) (50) 50 (100%) 2 (4%) 4 (8%) 26 (52%) 16 (32%) 1 (2%) 12 (24%) (48) 1 (2%) 5 (10%) 1 (2%) 3 (6%) (50) Special Senses System Eye Cataract Inflammation, chronic Cornea, inflammation, necrotizing Cornea, necrosis Lens, cataract Retina, degeneration (3) 1 (33%) 1 (33%) 1 (33%) 2 (67%) (2) 1 (50%) 1 (50%) Urinary System KidneyCalculus micro observation only Cyst Hydronephrosis Nephropathy Medulla, inflammation Renal tubule, necrosis Ureter Calculus micro observation only Urethra Fibrosis Urinary bladder Calculus gross observation Inflammation Mucosa, hyperplasia (49) 3 (6%) 45 (92%) (49) 1 (2%) (49) 1 (2%) 47 (96%) 1 (2%) 1 (2%) (48) Number of animals examined microscopically at site and number of animals with lesion (49) (50) 3 (6%) 49 (98%) 38 (76%) 2 (4%) 33 (66%) 4 (8%) 8 (16%) (47) 1 (2%) 2 (4%) 14 (30%) (48) 1 (2%) (2) 2 (100%) 1 (50%) 1 (50%) 1 (50%) (48) 1 (2%) 1 (2%) 1 (2%) 43 (90%) 1 (2%) 0) 1 (100%) (1) 1 (100%) (47) 1 (2%) 1 (2%) 95 APPENDIX B SUMMARY OF LESIONS IN FEMALE RATS IN THE LIFETIME INHALATION STUDY OF TALC T a b l e B1 T a b l e B2 T a b l e B3 T a b l e B4 Summary of the Incidence of Neoplasms in Female Rats in the Lifetime Inhalation Study of T a lc ................................................................................... Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of T a lc ................................................................................... Statistical Analysis of Primary Neoplasms in Female Rats in the Lifetime Inhalation Study of T a lc ................................................................................... S u m m a r y of the Incidence of Nonneoplastic Lesions in Female Rats in the Lifetime Inhalation Study of T a lc ................................................................................... 96 100 118 123 96 Talc, NTP TR 421 Table B1 Summary of the Incidence o i Neoplasms in Female Rats in the Lifetime Inhalation Study of Talc* 0 mg/m3 6 mg/m3 18 mg/m3 Disposition Sum m ary Animals initially in study Early deaths Moribund Natural deaths Survivors Terminal sacrifice Missing Animals examined microscopically A lim entary System Intestine small, ileum Liver Granulosa-theca tumor malignant, metastatic, ovary Hepatocellular carcinoma Neoplastic nodule Pancreas Pharynx Squamous cell carcinoma Salivary glands Fibrosarcoma Sarcoma Stomach, forestomach Stomach, glandular Tongue Sarcoma, metastatic Squamous cell papilloma Tooth Adamantinoma benign C ardiovascular System Heart Granulosa-theca tumor malignant, metastatic, ovary Endocrine System Adrenal gland, cortex Granulosa-theca tumor malignant, metastatic, ovary Adrenal gland, medulla Granulosa-theca tumor malignant, metastatic, ovary Pheochromocytoma malignant Pheochromocytoma benign Bilateral, pheochromocytoma malignant Bilateral, pheochromocytoma benign Islets, pancreatic Adenoma Parathyroid gland Pituitary gland Pars distalis, adenoma Pars distalis, carcinoma 50 28 11 11 50 (44) (50) (50) (50) (50) (50) (50) (50) (48) 13 (27%) (50) 1 (2%) (43) (50) 19 (38%) 3 (6%) 50 17 19 13 1 49 (32) (48) 1 (2%) 3 (6%) (46) (48) 1 (2%) (45) (47) (2) 1 (50%) 1 (50%) (1) 1 (100%) (48) (47) (47) 1 (2%) 10 (21%) 4 (9%) (45) 1 (2%) (42) (47) 18 (38%) 3 (6%) 50 27 14 9 50 (38) (50) 1 (2%) 1 (2%) (49) (1) 1 (100%) (50) 1 (2%) (49) (50) (50) 1 (2%) (49) 1 (2%) (49) 1 (2%) 7 (14%) 11 (22%) 3 (6%) 7 (14%) (49) 1 (2%) (47) (50) 21 (42%) 2 (4%) Lesions in Female Rats ABLE B1 Summary of the Incidence of Neoplasms in Female Rats in the Lifetime Inhalation Study of Talc (continued) 0 rag/m3 6 mg/m3 18 mg/m3 E ndocrine System (continued) Thyroid gland Bilateral, C-ceil, carcinoma C-cell, adenoma C-cell, carcinoma Follicular cell, adenoma G eneral Body System None Genital System Clitoral gland Adenoma Carcinoma Ovary Granulosa cell tumor malignant Granulosa cell tumor benign Granulosa-theca tumor benign Bilateral, granulosa-theca tumor malignant Uterus Polyp stromal Sarcoma stromal Hematopoietic System Bone marrow Lymph node Lymph node, bronchial Adenocarcinoma, metastatic, thyroid gland Squamous cell carcinoma, metastatic, lung Lymph node, mandibular Sarcoma, metastatic Lymph node, mediastinal Adenocarcinoma, metastatic, thyroid gland Carcinoma, metastatic, uncertain primary site Fibrosarcoma, metastatic, skin Granulosa-theca tumor malignant, metastatic, ovary . Lymph node, mesenteric Spleen Thymus Mixed tumor malignant Myxoma Schwannoma benign Thymoma benign Integum entary System Mammary gland Adenocarcinoma Adenoma Fibroadenoma Fibroma Fibrosarcoma (50) 1 (2%) 5 (10%) 2 (4%) (47) 2 (4%) (50) 1 (2%) (50) 5 (10%) (50) (50) (46) 1 (2%) (47) (47) 1 (2%) (49) (50) (47) 1 (2%) (50) 2 (4%) 1 (2%) 11 (22%) 1 (2%) (47) 2 (4%) 1 (2%) (44) (47) 2 (4%) 1 (2%) (48) 7 (15%) 1 (2%) (43) (48) (47) (46) 1 (2%) (44) (47) (48) (44) 1 (2%) 1 (2%) (48) 2 (4%) 10 (21%) 1 (2%) (49) 6 (12%) 2 (4%) (46) 1 (2%) 1 (2%) (50) 1 (2%) (50) 4 (8%) (49) (50) (47) 1 (2%) (47) (47) 1 (2%) 1 (2%) 1 (2%) (47) (50) (47) 1 (2%) (50) 2 (4%) 2 (4%) 13 (26%) 1 (2%) 97 98 Talc, NTP TR 421 Table Bl Summary or the Incidence of Neoplasms in Female Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 m g/m 3 18 mg/in3 Integum entary System (continued) Skin Keratoacanthoma Subcutaneous tissue, fibrosarcoma M usculoskeletal System Bone Mandible, sarcoma Mandible, sarcoma, metastatic Skeletal muscle Liposarcoma (50) (50) 1 (2%) (1) (49) 1 (2%) 1 (2%) (48) 1 (2%) 0) 1 (100%) (50) 1 (2%) 1 (2%) (50) Nervous System Brain Astrocytoma benign Carcinoma, metastatic, pituitary gland Ependymoma malignant Respiratory System Larynx Adenocarcinoma, metastatic, thyroid gland Lung Adenocarcinoma, metastatic, multiple, mammary gland Alveolar/bronchiolar adenoma Alveolar/bronchiolar adenoma, multiple Alveolar/bronchiolar carcinoma Alveolar/bronchiolar carcinoma, multiple Granulosa-theca tumor malignant, metastatic, ovary Squamous cell carcinoma (50) 1 (2%) 2 (4%) 1 (2%) (50) 1 (2%) (50) 1 (2%) 1 (2%) (48) 1 (2%) (48) (48) (50) 1 (2%) (48) (50) 8 (16%) 1 (2%) 4 (8%) 1 (2%) 1 (2%) 1 (2%) Special Senses System None Urinary System Kidney Lipoma Urinary bladder System ic Lesions Multiple organs1" Leukemia mononuclear Lymphoma malignant lymphocytic Lymphoma malignant mixed (49) (50) (50) . 13 (26%) (47) 1 (2%) (45) (49) 20 (41%) 2 (4%) 1 (2%) (49) (50) (50) 18 (36%) Lesions in Female Rats T a b l e B1 Summary of the Incidence of Neoplasms in Female Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 m g/m 3 18 m g/m 31 Neoplasm Summ ary Total animals with primary neoplasms0 Total primary neoplasms Total animals with benign neoplasms Total benign neoplasms Total animals with malignant neoplasms Total malignant neoplasms Total animals with metastatic neoplasms Total metastatic neoplasms Total animals with malignant neoplasms, uncertain primary site 44 85 38 59 23 26 4 6 47 100 35 65 31 35 3 8 1 Number of animals examined microscopically at site and number of animals with lesion Number of animals with any tissue examined microscopically c Primary neoplasms: all neoplasms except metastatic neoplasms 49 124 39 78 35 46 4 10 1 99 100 Talc, NTP TR 421 Table B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 0 mg/m3 Number o f Days on Study 3 3 3 5 5 5 5 56 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 9956 88 92347 789 112 36 666 77 00 888 46964 778 86 6 9 7 1267 829 Carcass ID Number 3433334433334333343333334 33 70 5 53 25 80 70 05 2 00 9 8 52 2 30 50 76 07 298 4 26 28 9 7 567 2 18 6 18 1 11 111 11 111 11 1 1 11 111 111 11 Alim entary System E sop h agu s Intestine large In testin e large, cecu m Intestine large, colon Intestine large, rectum Intestine sm all Intestine sm all, duodenum Intestine sm all, ileum Intestine sm all, jejunum L iver M esentery Pancreas Salivary glands Stom ach Stom ach, forestom ach S tom ach , glandular M 4- + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- + 4- 4- 4- 4- 4- 4- 4- A 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ A + 4- 4- 4- 4- 4- 4- 4- A 4- 4- 4- A A 4- 4- 4- 4- 4- 4- 4- 4- 4+ + + 4- 4- 4- 4- 4- 4- 4- A 4- 4- 4- 4- A 4- 4- 4- 4- 4- + 4- 4- 4+ A M 4- 4- 4- A + M M M 4- 4- 4- 4- 4- 4- M 4- 4- M 4- 4- 4- 4+ A + 4- 4- 4- 4- 4- 4- 4- A 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4+ A + 4- 4- 4- 4- 4- 4- 4- A 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ A + 4- 4- 4- 4- 4- 4- 4- A 4- 4- 4- + A 4- A A A 4- 4- 4- 4- .4+ A + 4- 4- 4- 4- A 4- 4- A 4- 4- 4- + A 4- 4- 4- A 4- 4- 4- 4- 44- + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + + + 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + + 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- Cardiovascular System B lood vessel H eart 4- 4- + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- + 4- 4- 4- 4- Endocrine System A dren al gland A dren al gland, cortex A dren al gland, m edulla P h eoch rom ocytom a benign Islets, pancreatic Adenom a P arathyroid gland P ituitary gland Pars distalis, aden om a Pars d istalis, carcinom a T hyroid gland B ilateral, C -cell, carcinom a C-cell, adenoma C-cell, carcinoma General Body System None +: Tissue examined microscopically A: Autolysis precludes examination 4- 4- 4- 4- 4- 4- 4- 4- 4- 4* 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4* 4- 4- 4- 44- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 44- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- I 4- X X XX 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M M 4- 4- 4- 4- 4- 4- 4- 4- M 4- 4- 4- 4- 4- M 4- 4- 4- 4- 4- 4- M 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- X XXX XX X XX X 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- X M: Missing tissue I: Insufficient tissue X: Lesion present Blank: Not examined Lesions in Female Rats 101 Table B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 0 mg/m3 (continued) Number of Days on Study 7788888888888888888888888 8800011122344566 666666666 69058367475 16922222333333 C arcass ID N um ber 4333343433334433333333444 2 15 302728575020 1233889002 5 156 1437324632 329020 19078 1111111111111111111111111 Total Tissues/ T um ors Alim entary System Esophagus Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Mesentery Pancreas Salivary glands Stomach Stomach, forestomach Stomach, glandular +++ ++++++++ ++++ + +++ +++ ++ + +++++++++++++++++++++++++ +++++++++++++++++++++++++ ++++++ +++ +++ + +++ +++ +++ +++ M+ + + M+ + + + + + + + + + + + + + + M+ + M+ + ++ ++++ ++++++ ++++ ++ +++ ++ + +++++++++++++++++++++++++ ++ +++++++ +++++ +++ +++ +++ ++ +++++++++++++++++++++++++ +++++++++++++++++++++++++ +++++++++++++++++++++++++ + ++ + ++ + ++ +++ +++ ++ + +++ + + ++ +++++++++++++++++++++++++ +++++++++++++++++++++++++ +++++++++++++++++++++++++ 49 49 46 48 38 48 48 44 45 50 1 50 50 50 50 50 Cardiovascular System Blood vessel Heart ++ ++ +++ +++++ + +++ +++ ++ +++ +++ 3 50 Endocrine System Adrenal gland Adrenal gland, cortex Adrenal gland, medulla Pheochromocytoma benign Islets, pancreatic Adenoma Parathyroid gland Pituitary gland Pars distalis, adenoma Pars distalis, carcinoma Thyroid gland Bilateral, C-cell, carcinoma C-cell, adenoma C-cell, carcinoma Genera) Body System None + + ++ + ++ + +++ + + ++ + ++ + ++ + + ++ +++++++++++++++++++++++++ + + + + + + + + + + + M+ + + + + + + + + + + + + XX XX XX X XX +++++++++++++++++++++++++ X + + + + + + + + M+ + + + + + + + + + + M+ + + + +++++++++++++++++++++++++ X XX X X XX XX X XX +++++++++++++++++++++++++ X XX X XX X 50 50 48 13 50 1 43 50 19 3 50 1 5 2 102 Talc, NTP TR 421 Table B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 0 mg/m3 (continued) Number o f Days on Study 3 33 55 5 55 66 666 667 7 77 7 77 7 77 799 56889 2347789 112366 6677 0088846964778866971267829 Carcass ID Number 34 3 33 344 33 3 34 3 33 343 3 33 334 3370553258070052009852230 50760 72984262897567 2 186 18 1111111111111111111111111 Genital System Clitoral gland Carcinoma Ovary Granulosa cell tumor malignant Uterus Polyp stromal H em atopoietic System Bone marrow Lymph node Lymph node, bronchial Adenocarcinoma, metastatic, thyroid gland Lymph node, mandibular Lymph node, mediastinal Adenocarcinoma, metastatic, thyroid gland Lymph node, mesenteric Spleen Thymus Thymoma benign Integumentary System Mammary gland Adenocarcinoma Adenoma Fibroadenoma Fibroma Skin M + + + 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- M 4- 4- 4- + + + + 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- X + + + + 4- 4- + + + 4- + 4- 4- 4- + + 4- 4- + 4- + 4- 4- 4- 4- X + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + + + + 4- 4- 4- 4- 4- 4- 4- 4- M 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + M+ M+ ++ 4- 4- 44- 4- 4- X 4- + 4- 4- 4- 4- + 4- 4- 4- 4- 4- M 4- ++ 4- 4- 4- 4- 4- M 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- X M + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- + + 4- 4- + + + + 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M M M + 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- + + 4- 4- 4- 4- 4- 4- 4- 4XX XX X X + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4* 4- 4- 4- 4- M usculoskeletal System Bone M andible, sarcom a Skeletal m uscle Nervous System B ra in Astrocytoma benign Carcinoma, metastatic, pituitary gland Ependymoma malignant 4*4-4-4-4-4-4-4-4-4-4-4-44- ++ + ++ + + ++ + ++ X 4' 4 - 4 - ' 4 - 4 - 4 - 4 - 4 - 4 - 4 - 4 - 4 - 4 - 4-4-4-4- X ++ 4- + 4" + X + 4- Lesions in Female Rats 103 Fable B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 0 mg/m3 (continued) Number of Days on Study 7788888888888888888888888 88000 11122344566666666 666 6 9 0 5 8 36 7 47 5 16 9 2 2 2 2 2 3 3 3 3 3 3 Carcass ID Num ber 4333343433334433333333444 2 153027 28 5750 20 123 388 900 2 5 156 14373246323 29020 19078 1111111111111111111111111 Total T issu es/ Tumors Genital System Clitoral gland Carcinoma Ovary Granulosa cell tumor malignant Uterus Polyp stromal + + + 4 4 + + + 4 4 4 4 M4 4 4 4 4 4 4 4 4 4 4 4 XX 4 ++4 4 ++4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 44 + + + 4 4 4 + 4- 4 4 4 4 4 4 4 4 4 4 4 4 ' 4 4 4 4 4 XX X X 47 2 50 1 50 5 H em atopoietic System Bone marrow Lymph node Lymph node, bronchial Adenocarcinoma, metastatic, thyroid gland Lymph node, mandibular Lymph node, mediastinal Adenocarcinoma, metastatic, thyroid gland Lymph node, mesenteric Spleen Thymus Thymoma benign ++++444 444444444444 444444 4++++4 +4 44 44 4 444 444 444 444 + 4 M 4 -f 4 + -f 4 4 4 4 4 4 4 4 M 4 4 M 4 4 4 4 4 4 4 + 4 4 4 4 4 4 4 4 4 4 4 4 4 M4 4 4 4 4 4 4 4 + + + 4 4 + 4 4 4 4 4 4 4 4 4 4 4 M4 4 4 4 4 4 4 +4 +4 4+4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 ++4 4 +4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 ++4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 X 50 50 46 1 47 47 1 49 50 47 1 Integumentary System Mammary gland Adenocarcinoma Adenoma Fibroadenoma Fibroma Skin 444 44 44 444444444444444 444 X XX XXX X X X 4 +4 +4 ++4 4 4 4 4 4 4 4 +4 4 4 4 4 4 4 +4 50 2 1 11 1 50 M usculoskeletal System Bone Mandible, sarcoma Skeletal muscle +44 4 44 44 4 444 444 444 44 4 44 4 4 50 1 1 Nervous System Brain Astrocytoma benign Carcinoma, metastatic, pituitary giand Ependymoma malignant ++4 4 +4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 X X 50 1 2 1 104 Talc, NTP TR 421 Table B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 0 mg/m3 (continued) Number o f Days on Study 3335555566666667777777777 7995688923477891 123666677 0088846964778866971267829 Carcass ID Number 3433334433334333343333334 3370553258070052009852230 5076072984262897567218618 1111111111111111111111111 Respiratory System Larynx Adenocarcinoma, metastatic, thyroid gland Lung Adenocarcinoma, metastatic, multiple, mammary gland Alveolar/bronchiolar adenoma Nose Trachea +++++++++++++++++++++++++ X + +++ +++ ++++++ ++++ ++ + ++ +++ X + A + + + + + + + +A + + + + + + + + + + + + + + ++++++ +++ +++ ++++ ++++ + +++ + Special Senses System Eye Harderian gland + + ++ Urinary System Kidney Urinary bladder + + + + + -f- + + + + A + + + + + + + + + + 4- + *+- + +++ +++ +++++ +++ ++++ ++ ++ +++ System ic Lesions Multiple organs Leukemia mononuclear + ++++ ++++++ ++ +++ +++ ++ ++ ++ XXXX X X X Lesions in Female Rats 105 T a b l e B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 0 mg/m' (continued) Number o f Days on Study 7788888888888888888888888 880 0 0 11 12 2344 56 666 66 666 66 69058367475 169 22222333333 C arcass ID Num ber 4333343433334433333333444 2 15302 728575020 12338 89002 5 156 143732 4632 3290 20 190 78 1111111111111111111111111 Total T issu es/ Tumors Respiratory System Larynx Adenocarcinoma, metastatic, thyroid gland Lung Adenocarcinoma, metastatic, multiple, mammary gland Alveoiar/bronchiolar adenoma Nose Trachea ++ + + + + + + + + + + + + + + + + ++ ++++ + ++ + ++++ + ++ +++ ++ + + ++ + ++ + ++ X +++++++++++++++++++ ++++++ ++ ++++ +++++ +++++ ++ ++ +++ ++ 50 1 50 1 1 48 50 Special Senses System Eye Harderian gland ++ + 2 5 Urinary System Kidney Urinary bladder + ++ +++++++ + ++ +++ ++ +++ ++ ++ +++++++++++++++++++++++++ 49 50 System ic Lesions Multiple organs Leukemia mononuclear +++++++++++++++++++++++++ X XXX X X 50 13 106 Talc, NTP TR 421 Table B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 6 mg/m3 N um ber of Days on Study 4555555666666777777777777 80 2 56 6 64 56 7 990 22 2 34 4 566 66 28 67 166 7 1880 750 49 807 502 6 8 C arcass ID N um ber 000 1000 10 110000 1 10 10 10 00 0 346 3649 194 1497 40 18 13 162 16 88 8793 50 6 18 52 009 69 59 27 373 1111111111111111111111111 Alim entary System Esophagus Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Hepatocellular carcinoma Neoplastic nodule Mesentery Pancreas Salivary glands Lymphoma malignant lymphocytic, metastatic Sarcoma Stomach Stomach, forestomach Stomach, glandular Tongue Sarcoma, metastatic Squamous cell papilloma Tooth Adamantinoma benign +A+4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 +A4 +4 4 4 A4 4 4 4 4 4 A4 A4 4 4 4 A4 A4 4 A 4- 4- 4 4 4 A A 4 4 4 A 4 A 4 A 4 4 A A A 4 A A 4 A 4 4- 4 4 4 A 4 4 4 4 4 4 A 4 A 4 4 A 4 A 4 A 4 + A + 4- 4 4 4 A M 4 M M M 4 M 4 A 4 4 4 4 M 4 A 4 4 A4 4 4 4 4 AA4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 A4 4 4 4 4 AA4 4 4 4 4 4 4 A4 4 4 4 4 4 4 4 + AA4 4 4 4 AA4 A 4 A 4 AAA 4 4 A 4 A4 A4 4 A4 4 4 4 4 AA4 4 4 4 4 A4 A4 4 A4 A4 A4 4A4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 X 44 +A+ 4 4 4 4 A4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 + 4 4 A4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 X 4 A4 4 4 4 4 4 4 4 4 4 4 + 4 4A4 4 4 4 4 4 4 4 4 A+ 4 4 4 4 4 4 4 4 4 4 4 4 4A4 4 4 4 4 4 4 4 4A+4 4 4 4 4 4 4 4 4 4 4 4 4A4 +4 4 4 4 4 4 4 X 4 X Cardiovascular System Blood vessel Heart 4 +A+ 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 E ndocrine System Adrenal gland Adrenal gland, cortex Adrenal gland, medulla Pheochromocytoma malignant Pheochromocytoma benign Bilateral, pheochromocytoma benign Islets, pancreatic Adenoma Parathyroid gland Pituitary gland Pars distalis, adenoma Pars distalis, carcinoma 4A4 4 4 4 4A4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4A4 4 4 4 4A4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4A4 4 44 4A4 4 4 4 44 4 4 4 4 4 4 4 4 44 4 X XX X 4A4 4 4 4 4A4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 M 4 A 4 4 4 4 M+ 4 4 M4 4 4 4 4 4 4 4 4 4 I 4 4 4 4A4 4 4 4 4 I 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 XX XXXX X XX Lesions in Female Rats 107 Table B2 individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 6 mg/mJ (continued) Num ber o f Days on Study 777788888888888888888888 899 90 111 144 66666 666 666 66 68890 2589 19 222 222 233 3333 C arcass ID N um ber 10 1100 00 100000 000 0 00 111 1 42438 136 146 112 2467 89 1334 4438547674468017216 11360 1 111111 1 11111 1 111 111 11 11 Total Tissues/ T um ors Alim entary System Esophagus Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Hepatocellular carcinoma Neoplastic nodule Mesentery Pancreas Salivary glands Lymphoma malignant lymphocytic, metastatic Sarcoma Stomach Stomach, foiestomach Stomach, glandular Tongue Sarcoma, metastatic Squamous cell papilloma Tooth Adamantinoma benign ++4++4444444444444444444 + 4 + 4- 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 AAAA 44444444444444444444 + A 4 4- 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 M+ 4 4 4 4 4 4 M4 4 4 4 4 4 4 4 4 4 4 4 4 4 444+44444444444444444444 + 4 4 4 4 4 4 4 M4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 AAAA 4A 444444444444444444 +A +4444444444444 444 44444 + 4+ 4 4 44 44 4 444 44 4 44 4 4 44 4 4 X XX 4 M4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 + + 4 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 X ++4444444444444444444444 + 4 + 4- 4 4 4 4 4 4 4 4 4 4 M 4 M 4 4 4 4 4 4 4 4- + + 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 X 48 43 34 41 37 46 44 32 40 48 1 3 2 46 48 1 1 47 45 47 2 1 1 1 1 Cardiovascular System Blood vessel Heart 44 + + + 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 48 Endocrine System Adrenal gland Adrenal gland, cortex Adrenal gland, medulla Pheochroinocytoma malignant Pheochromocytoma benign Bilateral, pheochromocytoma benign Islets, pancieatic Adenoma Parathyroid gland Pituitary gland Pars distqlis, adenoma Pars distalis, carcinoma 444444444444444444444444 4444444444444444444444 44 444444444444444444444444 X X X XX X XX XX X 4 M4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 X 4 MMI 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 444444444444444444444444 X XXXXX X X X XX X 47 47 47 1 10 4 45 1 42 47 18 3 108 Talc, NTP TR 421 Table B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 6 mg/m3 (continued) N um ber of Days on Stndy 4555555666666777777777777 8025666456799022234456666 2 86 7 1667 188075 0498 07 50 268 C arcass ID N um ber 0 0 0 10 00 10 1 10 0 0 0 1 10 10 10 0 0 0 34 6 36 49 19 4 1497 40 18 13 16 2 16 888793506 18 5200969 5927373 1 1 1 1 1 1 1 11 1 1 11 1 1 1 1 1 1 1 1 1 1 1 1 E ndocrine System (continued) Thyroid gland C-cell, carcinoma Follicular cell, adenoma + A + + + + 4* + + + + + + + + + + + + A + + + + + General Body System None Genital System Clitoral gland Ovary Granulosa cell tumor benign Granulosa-theca tumor benign Uterus Lymphoma malignant lymphocytic, metastatic Polyp stroma! Sarcoma stromal + A 4- 4- 4- 4- 4* 4- 4- 4- 4- 4* 4- 4- 4- 4- 4- 4- M M 4- 4- 4- M M + A 4- 4- 4- 4- + 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- X + + 4- 4* 4- 4- + 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4* XX X X H em atopoietic System Bone marrow Lymph node Lymph node, bronchial Lymph node, mandibular Sarcoma, metastatic Lymph node, mediastinal Lymph node, mesenteric Spleen Thymus Mixed tumor malignant Myxoma + A 4- 4- 4- 4- A A 4- 4- 4- 4- A 4- 4- 4- 4- 4- 4- 4- 4- A 4- 4- 4- + A 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + A 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4- 4- 4- 4- 4- 4- 4- 4- 4- 44- A 4* M 4- 4- 4- A 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- X + A 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- M 4- 44- A 4- 4- 4- 4- 4- A 4- 4- 4* 4- + 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 44- A 4* 4- 4- 4- + 4- 4* 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4+ A 4- 4- 4- 4- 4- 4- 4- 4- 4- M M 4- 4- 4- 4- i 4- 4* 4- 4- 4* 4- 4- X X Integumentary System Mammary gland Adenoma Fibroadenoma Fibroma Lymphoma malignant lymphocytic, metastatic Skin Keraloacanlhoma Subcutaneous tissue, fibrosarcoma 4- + + 4- 4- 4- 4- 4- 4- 4* M 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- XX XX X + 4- 4* 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4* 4X Lesions in Female Rats 109 T a b l e B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 6 mg/m3 (continued) Num ber of Days on Study 777788888888888888888888 89990 1111446666666666666 6 8 8 9 0 2 5 89 19 2 2 2 2 2 2 2 3 3 3 3 3 3 C arcass ID N um ber 10 1 10 0 0 0 10 0 0 0 0 0 0 0 0 0 0 1 1 1 1 42438 136 146 112246789 1334 4438 5476 74468 0 172 16 11360 111111111111111111111111 Total T issu e s/ T um ors E ndocrine System (continued) Thyroid gland C-cell, carcinoma Follicular ceil, adenoma + + + + + + + + + + + 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4XX X 47 2 1 General Body System None Genital System Clitoral gland Ovary Granulosa cell tumor benign Granulosa-theca tumor benign Uterus Lymphoma malignant lymphocytic, metastatic Polyp stromal Sarcoma stromal + + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + + + + + + + + + 4- 4* 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4- XX + + + + + + + + + + 4- 4- 4- 4- 4* 4- 4- 4- 4- 4- 4- 4- M 4- X X X XX 44 47 2 1 48 1 7 1 Hematopoietic System Bone marrow Lymph node Lymph node, bronchial Lymph node, mandibular Sarcoma, metastatic Lymph node, mediastinal Lymph node, mesenteric Spleen Thymus Mixed tumor malignant Myxoma + + A 4- + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4* + + + + + + + + 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + + + + + + + + + + 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- + 4- + + + 4- + + + + M M 4- 4- 4- 4- 4- 4- M 4- 4- 4- + 4- 4- 4- + + + + + + + + + + + 4- 4- + 4- 4- + 4- 4- 4- 4- 4- 4- 4+ + + + + + + + + + 4- 4- + 4- 4- + 4- 4- + 4- 4- 4- 4- 4- + + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- + M 4- 4- 4- 4- 43 48 47 46 1 44 47 48 44 1 1 Integum entary System Mammary gland Adenoma Fibroadenoma Fibroma Lymphoma malignant lymphocytic, metastatic Skin Keratoacanthoma Subcutaneous tissue, fibrosarcoma ++ + +++ +++ X 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + XX X XX X X X + + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 48 2 10 1 1 49 110 Talc, NTP TR 421 Table B2 Individual Animal Tumor Pathology of Female Rats In the Lifetime Inhalation Study of Talc: 6 mg/nr' (continued) Num ber o f Days o d Study 4555555666666777777777777 8025666456799022234456666 2867166718807504980750268 Carcass ID Number 0001000101100001101010000 3463649194149740181316216 8887935061852009695927373 1111111111111111111111111 M usculoskeletal System Bone Mandible, sarcoma, metastatic Skeletal muscle Liposarcoma +A +++++++++++++ + + + + ++++++ X Nervous System Brain Carcinoma, metastatic, pituitary gland +A+++++++++++ ++ + + + + +++ +++ X Respiratory System Larynx Lung Nose Trachea +A + ++++++ +++++ +++ ++ ++ ++ ++ +A + + ++ + + +++ ++ + ++ ++ +++ + + + + + A + + + + +A + + + + + + + +A + + +A + + + + +A + ++++++ + ++ ++ ++ +++ ++ ++ ++ Special Senses System Harderian gland ++ Urinary System Kidney Lipoma Lymphoma malignant lymphocytic, metastatic Urinary bladder + A + + + + +A + + + + + + + + + + + + + + + + + + A + M+ + + A + + + + + + + + + + + + + + + + + System ic Lesions Multiple organs Leukemia mononuclear Lymphoma malignant lymphocytic Lymphoma malignant mixed +++++++++++++++++++++++++ X xxxxx xxxxx X X Lesions in Female Rats 111 T a b l e B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 6 mg/nr' (continued) Number of Days on Study 777788888888888888888888 89990 11I 1446666666666666 6889 0 2589 192 2 222 22 333 333 Carcass ID Number 10 110000 100000000000 1111 42 438 136 14 6 1 122 46 789 133 4 44385 476 744680 172 16 1 1360 11 11 11 1 11 11 1111 111 11 1 111 Total Tissues/ Tumors M usculoskeletal System Bone Mandible, sarcoma, metastatic Skeletal muscle Liposarcoma + + + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4X 48 1 1 1 Nervous System Brain Carcinoma, metastatic, pituitary gland + + + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 48 1 Respiratory System Larynx Lung Nose Trachea + + + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4* + + + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + + + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 48 48 45 48 Special Senses System Harderian gland ++ 4- 4- 4- 7 Urinary System Kidney Lipoma Lymphoma malignant lymphocytic, metastatic Urinary bladder + + + + + + + + + 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4X X + + + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4- 47 1 1 45 Systemic Lesions Multiple organs Leukemia mononuclear Lymphoma malignant lymphocytic Lymphoma malignant mixed + 4* + + + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- X XXX XXXXX 49 20 1X2 Talc, NTP TR 421 Table B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 18 mg/m1 Num ber of Days on Study 5555666666666667777777777 35 89 134667 778990 11 123 4447 68645360 15684376067490673 C arcass ID Num ber 12221222 122222 12 1222 12 122 6 8 11883888050 48068 3 58 6 93 0 706 23 50 168758 096 02 6 6 13 299 1111111111111111111111111 Alimentary System Esophagus Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum intestine small, jejunum Liver Granulosa-theca tumor malignant, metastatic, ovary Neoplastic nodule Pancreas Pharynx Squamous cell carcinoma Salivary glands Fibrosarcoma Stomach Stomach, forestomach Stomach, glandular + + + + + + + + + + + + + + + + + + + + 4- 4- 4- 4- 4+ + A + + A + + + + + + + + + + + + + + + + 4- 4- 4+ + A + + A + + + + M + + + + + + + 4- + + + A 4- 4+ + A + + A + + + + + + + + + + + + + 4- + A 4- 4- 4+ M A M + A + + + + + + M + + + + + 4- M 4- 4- 4- 4- 4+ + A + + + + + + + + + + + + + + + + 4- 4- 4- 4- 4- 4+ + A + + + + + + + + + + + + + + + M 4- 4- + 4- 4- 4+ + A + + A A + + + + + A + A + + 4- A 4- A 4- A 4- 4+ + A + + A + + + + + + + + + + + + 4- + A 4- 4- 4- 4+ + + + + + + + + + + + + + + + + + + + 4- 4- 4- 4- 4- X + + A + + + + + + + + + + + + + + + + + + + 4- 4* + + X + + + + + + + + + + + + + + + + + + + + + + 4- 4* + + + + + + + + + + + + + + + + + + 4- 4- 4- + 4- 4- 4- 4+ + + + + + + + + + + + + + + + + 4- + 4- + 4- 4- 4* 4* + + + + + + + + + + + + + + + + + + 4- 4- 4- 4- 4- 4* 4- Cardiovascular System Blood vessel Heart Granulosa-theca tumor malignant, metastatic, ovary + + + + + + + + + + + + + + + + + 4- + + + 4- 4- 4- 4X Endocrine System Adrenal gland Adrenal gland, cortex Granulosa-theca tumor malignant, metastatic, ovary Adrenal gland, medulla Granulosa-theca tumor malignant, metastatic, ovary Pheochromocytoma malignant Pheochromocyloma benign Bilateral, pheochromocytoma malignant Bilateral, pheochromocytoma benign Islets, pancreatic Adenoma Parathyroid gland + + A + + + ++ + + + + + + + + + ++ + + + + + + + + A + + + ++ + + + + + + + + + ++ + + + + + + X + +A + + + + + + + + + + + + + + + + + + + + + + X X . XX + + A + + + + + + + + + + + + + ++ + + + + + + + X M+ + + + + + + + + + + + + + + ++ + + + + + + + Lesions in Female Rats 113 Table B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 18 mg/m1 (continued) Num ber of Days on Study 7 7 78 8 88 88 88 8 8 88 888 888 888 8 8890222234444555666666666 466323895 1128047222223333 C arcass ID N um ber 2222 11122112222 1111122222 58356657 19905636566833558 7733247750 1584 15836228496 111 11 11 111 1111 1 111 1111 111 Total Tissues/ T um ors A lim entary System Esophagus Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine stpall Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Granulosa-theca tumor malignant, metastatic, ovary Neoplastic nodule Pancreas Pharynx Squamous cell carcinoma Salivary glands Fibrosarcoma Stomach Stomach, forestomach Stomach, glandular + + + + 4- 4- + 4- + 4- + 4- 4- + 4- 4- 4- + + 4- + 4- + + 4+ + + + + A 4- 4- 4- 4- + 4- 4- 4- A + 4- 4- 4- + 4- + + 4- + A + + + + A 4- 4- + 4- + 4- 4- 4- A + 4- + + 4- 4- 4- + 4- + + + + + + A + + 4- + 4- 4- + + A 4- 4- + + 4- + 4- + 4- 4+ + + 4- + A 4- 4- M 4- 4- 4- 4- 4- A + + 4- 4- 4- 4- 4- + + 4+ + + + + A 4- 4- + 4- + + 4- 4- 4- + 4- + + + + 4- 4- + + + + + + + A 4- + + 4- + + 4- 4- 4- + + + + + + 4- 4- 4- 4A + + 4- + A 4- 4- + + A 4- 4- 4- A 4- + + 4- 4- 4- + + 4- + + + + + + A 4- 4- 4- + 4- + 4- 4- 4- + 4- + + 4- 4- 4* + + + + + + 4- + + + + 4- + + + + 4- 4- + + + 4- 4- + + + 4- 4- X + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- + + + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- X + + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- + + + + 4- 4- 4- 4- 4- M 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + + + 4- 4- 4- 4- 4- 4- 4- + 4- 4- + 4- + + + 4- 4- + 4- + 4- 4- 50 46 43 45 41 48 47 38 46 50 1 1 49 1 1 50 1 50 49 50 Cardiovascular System Blood vessel Heart Granulosa-theca tumor malignant, metastatic, ovary 4+ + + + 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 1 50 1 Endocrine System Adrenal gland Adrenal gland, cortex Granuiosa-theca tumor malignant, metastatic, ovary Adrenal gland, medulla Granulosa-theca tumor malignant, metastatic, ovary Pheochromocytoma malignant PheochrOmocytoma benign Bilateral, pheochromocytoma malignant Bilateral, pheochromocytoma benign Islets, pancreatic Adenoma Parathyroid gland 4- + 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- + 4- + 4- 44- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- X X XXXX X X X XXXX X X XX XX X X X X XX 4- 4- 4- + 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4- 4- 4- 4- 4- 4- 4- 4- 49 49 1 49 1 7 11 3 7 49 1 47 114 Talc, NTP TR 421 Table B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 18 mg/m3 (continued) Num ber of Days on Study 5555666666666667777777777 3589 134667778990 111234447 68645360 1S6 84376067490673 Carcass ID Num ber 1222 1222 122222 12 1222 12 122 6 8 1 18 8 3 8 8 8 0 5 0 4 80 6 8 35 8 6 9 30 70623501687580960266 13299 111 111111 1 11 1111 11 11 11111 E ndocrine System (continued) Pituitary gland Pars distalis, adenoma Pars distalis, carcinoma Thyroid gland C-cell, adenoma C-cell, carcinoma ++ +++++++ ++++ +++ +++ ++++ ++ XXXXX X XX ++A++++++++ +++++++++++ +++ XX XX General Body System None Genital System Chloral gland Adenoma Carcinoma Ovary Bilateral, granulosa-theca tumor malignant Uterus Polyp stromal + + + + + + + + + + + + + + + + + + + + + M+ M+ +++++++++++++++++++++++++ X ++ +++++ ++++ ++++ ++ ++ ++ ++ ++ XX Hem atopoietic System Bone marrow Lymph node Lymph node, bronchial Squamous cell carcinoma, metastatic, lung Lymph node, mandibular Lymph node, mediastinal Carcinoma, metastatic, uncertain primary site Fibrosarcoma, metastatic, skin Granulosa-theca tumor malignant, metastatic, ovary Lymph node, mesenteric Spleen Thymus Schwannoma benign ++A+ + ++ ++++++ ++ ++ ++ ++ + +++ +++++++++++++++++++++++++ + + + + + + + + + + + M+ + M+ + + + M+ + + + + X + + + + + + + M+ + + + + + + + + + + + + + M+ + +++ +++++ +++ ++++ +++ + ++ +++ + X X ++A+ +++ ++++ +++ ++ ++ ++ +++ + + ++ + +++ +++ +++ +++ ++++ ++ ++ ++ + + + MM+ + + + + + + + + + + + + + + + + + + + Lesions in Female Rats 115 Table B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 18 mg/m3 (continued) Num ber of Days on Study 7778888888888888888888888 88 902 222 34 444 5 55 66 6 6 66 6 66 466 323895 1 12804 72 222 23 3 33 C arcass ID Num ber 2222 11122 112222 1111122222 58 356657 19905636566 833558 7 73324 7750 158 4 158 36 2284 96 1111111111 111 111111111111 Total Tissues/ T um ors E ndocrine System (continued) Pituitary gland Pars distalis, adenoma Pars distalis, carcinoma Thyroid gland C-cell, adenoma C-cell, carcinoma + + ++ ++ + ++ +++ +++ + ++ ++ + + ++ + X XX XXXXXXXX X XX X + + ++++ +++ ++ ++++ +++ ++ +++ ++ XX XX 50 21 2 49 6 2 G eneral Body System None Genital System Clitoral gland Adenoma Carcinoma Ovary Bilateral, granulosa-theca tumor malignant Uterus Polyp stromal + + + + + M+ + + + + + + + M+ + + + + + + + + + X X + + ++ ++++ ++++ +++ ++ +++ ++++ + +++++++++++++++++++++++++ XX 46 1 1 50 1 50 4 Hematopoietic System Bone marrow Lymph node Lymph node, bronchial Squamous cell carcinoma, metastatic, lung Lymph node, mandibular Lymph node, mediastinal Carcinoma, metastatic, uncertain primary site Fibrosarcoma, metastatic, sltin Granulosa-theca tumor malignant, metastatic, ovary Lymph node, mesenteric Spleen Thymus Schwannoma benign +++++++++++++++++++++++++ +++++++++++++++++++++++++ + +++ +++ ++++ ++++ + ++++ +++ ++ 49 50 47 '1 + + + + + + + + + + + + + + M+ + + + + + + + + + 47 + + + + +. + + + + + + + + + M + + + + + M + + + M 47 X1 1 + + + + + + + + + + + + + + M+ + M+ + + + + + + + + +++++ +++ ++ +++ + ++++ ++ ++ + M + + + + + + + + + + + + + + + + + + + '+ + + + + X 1 47 50 47 1 1 1 6 Talc, NTP TR 421 Table B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 18 mg/m3 (continued) Number of Days on Study 55556666666 6666 7777 777 77 7 3589 134667778990 1112 34447 68645360 15684376067490673 Carcass ID Number 12221222 122222 12 1222 12 122 68 11883888 05048 06 8 3 5 86 9 30 70623 50 1687580960266 13299 11111111111111111111 11111 integum entary System Mammary gland Adenocarcinoma Adenoma Fibroadenoma Fibrosarcoma Skin Keratoacanthoma Subcutaneous tissue, fibrosarcoma M usculoskeletal System Bone Nervous System Brain Carcinoma, metastatic, pituitary gland +++++++++++++++++++++++++ XX XXX +++++++++++++++++++++++++ X X +++++++++++++++++++++++++ +++ ++++ +++++ ++ ++ ++ ++ ++ ++ + X Respiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar adenoma, multiple Alveolar/bronchiolar carcinoma Alveolar/bronchiolar carcinoma, multiple Granulosa-theca tumor malignant, metastatic, ovary Squamous cell carcinoma Nose Trachea +++++I +I +++++++++++++++++ + +++ ++ ++++ ++ ++ + ++ ++ +++ + ++ XX X X +++++++++++++++ +A++++++++ + +++ +++ + ++++ ++ +++ +++ ++ +++ Special Senses System Eye Harderian gland Lacrimal gland +++ + ++ + + Urinary System Kidney Urinary bladder ++A+ ++++++ +++ +++ ++ ++ ++ ++ + +++ +++++++++++++++++++++ System ic Lesions Multiple organs Leukemia mononuclear +++++++++++++++++++++++++ X X XX X X X X X XX Lesions in Female Rats 117 Table B2 Individual Animal Tumor Pathology of Female Rats in the Lifetime Inhalation Study of Talc: 18 mg/m1(continued) Number of Days on Study 77 788 888 88 888 8 88 88 8 888 888 8890222234444555666666666 4663238951 128047222223333 Carcass ID Number 2222 11122112222 1111122222 5835665719905636566833558 77332477501584 15836228496 1111111111111111111111111 Total Tissues,/ Tumors Integumentary System Mammary gland Adenocarcinoma Adenoma Fibroadenoma Fibrosarcoma Skin Keratoacanthoma Subcutaneous tissue, fibrosarcoma +++++++++++++++++++++++++ XX XX X X X X XXX X X ++ ++++++ ++ +++ ++++++++++++ 50 2 2 13 1 50 1 1 M usculoskeletal System Bone +++++++++++++++++++++++++ 50 Nervous System Brain Carcinoma, metastatic, pituitary gland + + ++ + +++ ++ + ++ + ++ + ++ + ++ + ++ 50 1 Respiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar adenoma, multiple Alveolar/bronchiolar carcinoma Alveolar/bronchiolar carcinoma, multiple Granulosa-theca tumor malignant, metastatic, ovary Squamous cell carcinoma Nose Trachea +++ ++++ +++++ ++ ++ ++ ++ ++++ + +++++++++++++++++++++++++ X XX X X X X XX XX X A+++ ++++ ++ +++ ++++ ++ + + ++++ ++++ + + + + + + + + + + + + ++ + ++ + + + + 48 50 8 1 4 1 1 1 48 50 Special Senses System Eye Harderian gland Lacrimal gland ++ + +++ + + +2 + 15 1 Urinary System Kidney Urinary bladder System ic Lesions Multiple organs Leukemia mononuclear +++++++++++++++++++++++++ + + + + + + + + + + + + + + + + + + + + + + + + + 49 50 +++++++++++++++++++++++++ XX XX X XX 50 18 118 Talc, NTP TR 421 Table B3 Statistical Analysis of Primary Neoplasms in Female Rats in the Lifetime Inhalation Study of Talc 0 mg/m3 6 mg/m3 18 mg/m3 Adrenal M edulla: Benign Pheochrom ocytom a Overall rates* Adjusted rates*" Terminal rates* First incidence (days) Life table tests*" Logistic regression tests'1 Cochran-Armitage lestd Fisher exact test3 13/48 (27%) 61.3% 5A1 (45%) 678 P =0.135 P=0.185 P=0.180 Adrenal M edulla: M alignant Pheochrom ocytom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 0/48 (0%) 0.0% 0/11 (0%) _ P< 0.001 P <0.001 P< 0.001 Adrenal M edulla: Benign or M alignant Pheochrom ocytom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 13/48 (27%) 61.3% 5/11 (45%) 678 P=0.016 P=0.014 P = 0.021 L iven N eoplastic Nodule Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 0/50 (0%) 0.0% 0/11 (0%) - P=0J50 P = 0.561 P = 0.556 L iven N eoplastic N odule or H epatocellular Carcinoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 0/50 (0%) 0.0% 0/11 (0%) P=0.575 P =0.602 P =0.599 14/47 (30%) 59.7% 5A3 (38%) 705 P =0.529 P =0.541 P =0.474 1/47 (2%) 7.1% 0A3 (0%) 849 P=0,531 P =0.509 P =0.495 14/47 (30%) 59.7% 5A3 (38%) 705 P =0-529 P=0.541 P = 0.474 3/48 (6%) 13.6% 0A3 (0%) 724 P=0.114 P=0.117 P=0.114 4/48 (8%) 20.2% 1A3 (8%) 724 P=0.066 P =0.060 P = 0.054 18/49 (37%) 82-5% 6/9 (67%) 697 P=0.183 P=0.225 P=0.212 10/49 (20%) 56.9% 3/9 (33%) 784 P =0.002 P=0.001 P<0,001 23/49 (47%) 95.2% 8/9 (89%) 697 P =0.033 P = 0.024 P=0.034 1/50 (2%) 10.0% 0/9 (0%) 857 P =0.464 P =0.496 P=0.500 1/50 (2%) 10.0% 0/9 (0%) 857 P=0.464 P =0.496 P=0.500 Lesions in Female Rats Table B3 Statistical Analysis of Primary Neoplasms in Female Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m 6 mg/m3 18 mg/m3 Lung: Alveolar/bronchiolar Adenoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Annitage lest Fisher exact test Lung: Alveolar/bronchiolar Carcinom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Annitage test Fisher exact test Lung: Alveolar/bronchiolar Adenom a or Carcinoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test M amm ary Gland: Fibroadenom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test M amm ary Gland: Fibroma, Fibroadenom a, or Adenoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 1/50 (2%) 4.5% 0/11 (0%) 805 P<0.001 P < 0.001 P <0.001 0/50 (0%) 0.0% 0/11 (0%) - P=0.002 P=0.003 P = 0.004 1/50 (2%) 4.5% 0/11 (0%) 805 P <0.001 P<0.001 P<0.001 11/50 (22%) 47.6% 2/11 (18%) 634 P =0.304 P=0.363 P =0.343 13/50 (26%) 54.7% 3/11 (27%) 634 P=0.314 P=0.394 P=0.371 0/48 (0%) 0.0% 0/13 (0%) - P=0.529N P=0.503N P=0.510N 0/48 (0%) 0.0% 0/13 (0%) - 0/48 (0%) 0.0% 0/13 (0%) - P=0.529N P=0.503N P=0.510N 10/49 (20%) 41.4% 3/13 (23%) 482 P=0.489N P=0.508N P=0.521N 13/49 (27%) 59.0% 6/13 (46%) 482 P=0.544N P=0.585 P=0.567 9/50 (18%) 40.8% 1/9 (11%) 716 P=0.015 P=0.010 P =0.008 5/50 (10%) 41.7% 3/9 (33%) 828 P = 0.027 P = 0.028 P =0.028 13/50 (26%) 65.8% 4/9 (44%) 716 P =0.001 P < 0.001 P<0.001 13/50 (26%) 64.0% 4/9 (44%) 678 P =0.394 P =0.428 P =0.408 15/50 (30%) 68.6% 4/9 (44%) 678 P = 0.404 P =0.434 P=0.412 119 120 Talc, NTP TR 421 Table B3 Statistical Analysis or Primary Neoplasms in Female Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 IS mg/m3 M ammary Gland: Fibroma, Fibroadenom a, Adenoma, or Adenocarcinoma Overall rates 15/50 (30%) Adjusted rates 56.6% Terminal rates 3/11 (27%) First incidence (days) 370 Life table tests P=0.378 Logistic regression tests P=0.457 Cochran-Armitage test P = 0.430 Fisher exact test Pituitary Gland (Pars D istalis): Adenoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 19/50 (38%) 62.1% 3/11 (27%) 568 P=0.360 P=0.409 P = 0.380 Pituitary Gland (Pars D istalis): Carcinoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 3/50 (6%) 17.1% 1/11 (9%) 6% P=0.438N P=0.427N P=0.418N Pituitary Gland (Pars D istalis): Adenoma or Carcinoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 22/50 (44%) 69.8% 4/11 (36%) 568 P = 0.429 P = 0.506 P=0.471 Thyroid Gland (C-cell): Adenoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 5/50 (10%) 33.5% 2/11 (18%) 805 P=0.253 P=0.283 P = 0.276 13/49 (27%) 59.0% 6/13 (46%) 482 P=0.386N P=0.425N P=0.437N 18/47 (38%) 60.5% 3/13 (23%) 697 P=0.512N P= 0.557N P=0.571 3/47 (6%) 12.2% 1/13 (8%) 566 P=0.636N P =0.634 P = 0.631 21/47 (45%) 66.2% 4/13 (31%) 566 P=0.502N P=0.570N P = 0.554 0/47 (0%) 0.0% 0/13 (0%) - P=0.030N P=0.029N P=0.033N 16/50 (32%) 70.1% 4/9 (44%) 678 P =0.494 P = 0.531 P=0.500 21/50 (42%) 78.3% 4/9 (44%) 633 P =0.425 P=0.457 P=0.419 2/50 (4%) 5.6% 0/9 (0%) 676 P=0.506N P=0.497N P=0.500N 23/50 (46%) 79.5% 4/9 (44%) 633 P =0.488 P=0.545 P = 0.500 6/49 (12%) 34.0% 2/9 (22%) 678 P=0.467 P = 0.505 P =0.486 Lesions in Female Rats Table B3 Statistical Analysis of Primary Neoplasms in Female Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/nr 6 mg/m3 18 mg/m3 Thyroid Gland (C-cell): Carcinom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Thyroid Gland (C-cell): Adenoma or Carcinom a Overall rates Adjusted rales Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Uterus: Strom al Polyp Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Uterus: Strom al Polyp or Strom al Sarcom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test All Organs: M ononuclear C ell L eukem ia Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 3/50 (6%) 11.1% 0A1 (0%) 677 P=0.430N P=0.462N P=0.463N 8/50 (16%) 40.9% 2A1 (18%) 677 P=0.418 P= 0.435 P=0.414 5/50 (10%) 22.3% 1A1 (9%) 398 P=0.439N P=0.376N P=0.386N 5/50 (10%) 22.3% 1A1 (9%) 398 P=0.412N P=0.36ON P=0.363N 13/50 (26%) 45.7% 1A1 (9%) 390 P=0.234 P=0.226 P=0.250 2/47 (4%) 12.2% 0A3 (0%) 818 P=0.507N P=0.516N P=0.530N 2/47 (4%) 12.2% 0A3 (0%) 818 P=0.051N P=0.048N P=0.056N 7/49 (14%) 34.4% 3/13 (23%) 678 P=0.400 P =0.372 P =0.365 8/49 (16%) 35.8% 3A3 (23%) 557 P=0.298 P=0.265 P = 0.264 20/49 (41%) 73.3% 8A3 (62%) 526 P =0.164 P =0.084 P = 0.088 2/49 (4%) 4.9% 0/9 (0%) 675 P=0.493N P=0.533N P=0.510N 8/49 (16%) 37.2% 2/9 (22%) 675 P=0.579 P=0.599N P =0.590 4/50 (8%) 19.5% 1/9 (11%) 678 P=0.532N P=0.505N P=0.500N 4/50 (8%) 19.5% 1/9 (11%) 678 P=0.532N P = 0.505 N P=0.500N 18/50 (36%) 60.1% 3/9 (33%) 536 P =0.232 P=0.152 P=0.194 121 122 Talc, NTP TR 421 Table B3 Statistical Analysis of Primary Neoplasms in Female Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 All O rgans: M alig n an t L ym phom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage lest Fisher exact lest All O rgans: B enign N eoplasm s Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test All O rgans: M alig n an t N eoplasm s Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test All O rgans: B enign o r M alig n a n t N eoplasm s Overall rates Adjusted rales Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact lest 0/50 (0%) 0.0% 0/11 (0%) P=0.525N P=0.497N P=0.499N 38/50 (76%) 97.2% 10/11 (91%) 398 P=0.338 P=0.544 P =0.415 23/50 (46%) 69.3% 4/11 (36%) 370 P = 0.054 P=0.013 P=0.016 44/50 (88%) 97.6% 10/11 (91%) 370 P=0.248 P =0.053 P = 0.049 3/49 (6%) 10.3% 0/13 (0%) 724 P=0.124 P=0.118 P=0.117 35/49 (71%) 96.9% 12/13 (92%) 482 P=0.350N P=0.312N P=0.387N 31/49 (63%) 85.8% 9/13 (69%) 526 P=0.189 P = 0.056 P = 0.064 47/49 (96%) 97.9% 12/13 (92%) 482 P=0.447 P =0.145 P=0.141 0/50 (0%) 0.0% 0/9 (0%) - - 39/50 (78%) 97.4% 8/9 (89%) 558 P =0.440 P=0.562N P = 0.500 35/50 (70%) 90.5% 6/9 (67%) 536 P =0.061 P =0.010 P=0.013 49/50 (98%) 100.0% 9/9 (100%) 536 P =0.279 P = 0.060 P =0.056 a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, bone marrow, brain, clitoral gland, heart, iddney, larynx, liver, lung, nose, ovary, pancreas, parathyroid gland, pituitary gland, salivary gland, spleen, thyroid gland, and urinary bladder, for other tissues, denominator is number of animals necropsied. b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality c Observed incidence at terminal kill d Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. e Not applicable; no neoplasms in animal group Lesions in Female Rats 123 T able B4 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the Lifetime Inhalation Study of Talc1 "I 0 mg/m 6 mg/m 9 18 mg/m Disposition Sum m ary Animals initially in study Early deaths Moribund Natural deaths Survivors Terminal sacrifice Missing Animals examined microscopically Alim entary System Intestine large, cecum Hemorrhage, focal Inflammation Parasite metazoan Ulcer Intestine large, colon Inflammation Parasite metazoan Intestine large, rectum Inflammation Parasite metazoan Intestine small, duodenum Necrosis, focal Intestine small, ileum Hyperplasia, lymphoid Liver Atrophy Basophilic focus Clear cell focus Cyst multilocular Degeneration, cystic Eosinophilic focus Fatty change Hematopoietic cell proliferation Infiltration cellular, mononuclear cell Inflammation, granulomatous, focal Inflammation, necrotizing, focal Inflammation, suppurative Necrosis, focal Pigmentation, hemosiderin Thrombosis Bile duct, hyperplasia Centrilobuiar, atrophy Centrilobular, degeneration Centrilobuiar, necrosis Hepatocyte, atrophy, focal Serosa, thrombosis Mesentery Granuloma Inflammation, chronic active 50 28 11 11 50 (46) 11 (24%) 7 (15%) 1 (2%) (48) 2 (4%) (38) 4 (11%) 2 (5%) (48) 1 (2%) (44) 2 (5%) (50) 27 (54%) 1 (2%) 1 (2%) 2 (4%) 18 (36%) 1 (2%) 13 (26%) 1 (2%) 5 (10%) 1 (2%) 36 (72%) 10 (20%) 2 (4%) 0) 1 (100%) 50 17 19 13 1 49 (34) 1 (3%) 1 (3%) 3 (9%) 1 (3%) (41) 1 (2%) 3 (7%) (37) 1 (3%) (44) (32) (48) 1 (2%) 17 (35%) 2 (4%) 2 (4%) 5 (10%) 18 (38%) 3 (6%) 1 (2%) 1 (2%) 38 (79%) 2 (4%) 14 (29%) 2 (4%) 3 (6%) (2) 1 (50%) 1 (50%) 50 27 14 9 50 (43) 6 (14%) 6 (14%) 1 (2%) (45) 2 (4%) 3 (7%) (41) 1 (2%) (47) (38) (50) 1 (2%) 21 (42%) 1 (2%) 1 (2%) 4 (8%) 14 (28%) 1 (2%) 4 (8%) 2 (4%) 1 (2%) 36 (72%) 6 (12%) 10 (20%) 2 (4%) 1 (2%) 124 Talc, NTP TR 421 Table B4 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/nr' 6 mg/m3 18 m g/m 3 A lim entary System (continued) Pancreas Hyperplasia, nodular Inflammation Lobules, atrophy Salivary glands Inflammation Stomach, foreslomach Hyperkeratosis Inflammation Mineralization Ulcer Stomach, glandular Erosion Inflammation Mineralization Ulcer Ulcer, multiple Arteriole, muscularis, lamina propria, mineralization C ardiovascular System Blood vessel Aorta, mineralization Mesenteric artery, aneurysm Mesenteric artery, inflammation Mesenteric artery, mineralization Mesenteric artery, thrombosis Heart Cardiomyopathy Inflammation, focal Atrium, thrombosis Myocardium, embolus Myocardium, inflammation, focal Myocardium, mineralization (50) 1 (2%) 7 (14%) (50) 2 (4%) (50) 1 (2%) 1 (2%) 9 (18%) (50) 1 (2%) 2 (4%) 3 (6%) 1 (2%) (3) 1 (33%) 3 (100%) 1 (33%) (50) 35 (70%) 1 (2%) 5 (10%) 1 (2%) (46) 1 (2%) 7 (15%) (48) (45) 1 (2%) 4 (9%) (47) 1 (2%) 2 (4%) 2 (4%) 1 (2%) (3) 3 (100%) 1 (33%) 1 (33%) (48) 40 (83%) 8 (17%) 2 (4%) 1 (2%) 4 (8%) (49) 9 (18%) (50) (49) 1 (2%) 2 (4%) 3 (6%) (50) 1 (2%) 2 (4%) 3 (6%) 1 (2%) (1) 1 (100%) 1 (100%) (50) 36 (72%) 1 (2%) 5 (10%) 3 (6%) Endocrine System Adrenal gland, cortex Degeneration, cystic Degeneration, fatty Degeneration, focal Hyperplasia, diffuse Hyperplasia, focal Necrosis Necrosis, focal Pigmentation, hemosiderin Adrenal gland, medulla Cyst Hyperplasia Bilateral, hyperplasia (50) 1 (2%) 3 (6%) 1 (2%) 9 (18%) 1 (2%) 1 (2%) (48) 1 (2%) 20 (42%) 2 (4%) (47) 1 (2%) 1 (2%) 12 (26%) 1 (2%) (47) 18 (38%) 2 (4%) (49) 1 (2%) 13 (27%) 2 (4%) (49) 14 (29%) 2 (4%) Lesions in Female Rats Table B4 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 E ndocrine System (continued) Parathyroid gland Hyperplasia Bilateral, hyperplasia Pituitary gland Cyst Pars distalis, hyperplasia Pars distalis, necrosis Thyroid gland C-cell, hyperplasia General Body System None Genital System Clitoral gland Hyperplasia Inflammation Ovary Cyst Uterus Cyst Inflammation Endometrium, hyperplasia Lamina propria, fibrosis Hematopoietic System Bone marrow Atrophy Hyperplasia, histiocytic Inflammation, granulomatous, focal Myelofibrosis Necrosis, focal Myeloid cell, hyperplasia Lymph node Axillary, hemorrhage, chronic Lymph node, bronchial cyst Fibrosis Hemorrhage, chronic Hyperplasia, histiocytic Inflammation, suppurative Pigmentation, hemosiderin Lymph node, mandibular Hyperplasia, lymphoid Hyperplasia, plasma cell Inflammation, chronic active Inflammation, suppurative (43) 3 (7%) 1 (2%) (50) 2 (4%) 10 (20%) (50) 10 (20%) (42) 4 (10%) (47) 6 (13%) (47) 8 (17%) (47) 2 (4%) (50) 1 (2%) 4 (8%) 1 (2%) (49) 4 (8%) (47) 2 (4%) 1 (2%) (50) 5 (10%) (50) 1 (2%) 1 (2%) 3 (6%) 20 (40%) (50) 1 (2%) 1 (2%) 1 (2%) 1 (2%) 2 (4%) (50) (46) 1 (2%) 1 (2%) 1 (2%) (47) . 2 (4%) 1 (2%) (44) 1 (2%) (47) (48) 1 (2%) 1 (2%) 39 (81%) (43) 2 (5%) 3 (7%) 1 (2%) 2 (5%) (48) (47) 1 (2%) 1 (2%) 40 (85%) (4b) 1 (2%) 1 (2%) (4b) 1 (2%) 1 (2%) (50) 1 (2%) (50) 1 (2%) 19 (38%) (49) 1 (2%) 1 (2%) 3 (b%) 3 (b%) (50) 1 (2%) (47) 45 (9b%) (47) 1 (2%) 1 (2%) 1 (2%) 1 (2%) 125 126 Talc, NTP TR 421 Table B4 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 H em atopoietic System (continued) Lymph node, mediastinal Hemorrhage, chronic Hyperplasia, histiocytic Hyperplasia, lymphoid Inflammation, chronic active Inflammation, suppurative Lymph node, mesenteric Hemorrhage Hyperplasia, lymphoid Hyperplasia, plasma cell Inflammation, chronic active Inflammation, granulomatous Spleen Atrophy Fibrosis, focal Hematopoietic cell proliferation Hyperplasia, lymphoid Inflammation, granulomatous, focal Pigmentation, hemosiderin Capsule, hemorrhage Thymus Inflammation (47) 1 (2%) 1 (2%) 1 (2%) (49) 2 (4%) 1 (2%) 4 (8%) (50) 2 (4%) 3 (6%) 4 (8%) 1 (2%) 2 (4%) (47) 1 (2%) (44) 33 (75%) 1 (2%) (47) 1 (2%) 1 (2%) 1 (2%) (48) 2 (4%) 1 (2%) 6 (13%) (44) (47) 40 (85%) 1 (2%) 1 (2%) (47) 2 (4%) 1 (2%) (50) 2 (4%) 1 (2%) 7 (14%) 1 (2%) 1 (2%) (47) Integumentary System Mammary gland Galactocele Hyperplasia, cystic Lobules, hyperplasia Skin Inflammation, focal (50) (50) 1 (2%) (48) 1 (2%) 2 (4%) (49) (50) 1 (2%) (50) M usculoskeletal System Bone Fibrous osteodystrophy Hyperostosis Pelvis, fracture Vertebra, cyst (50) 4 (8%) 4 (8%) 1 (2%) 1 (2%) (48) 3 (6%) 1 (2%) (50) 4 (8%) 3 (6%) Nervous System Brain Compression Hemorrhage Hydrocephalus Inflammation, focal White matter, necrosis, focal (50) 8 (16%) (48) 7 (15%) 1 (2%) 1 (2%) (50) 9 (18%) 1 (2%) 1 (2%) 2 (4%) Lesions in Female Rats Table B4 Summary or the Incidence of Nonneoplastic Lesions in Female Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 R esp irato ry System Larynx Inflammation, necrotizing Inflammation, suppurative Lung Crystals, focal Cyst Cyst, multiple Edema Hemorrhage Hyperplasia, adenomatous, diffuse Inflammation, granulomatous Inflammation, suppurative Mineralization Alveolar epithelium, hyperplasia Alveolus, metaplasia, squamous Bronchus, epithelium, degeneration, focal Interstitium, fibrosis Interstitium, fibrosis, focal Interstitium, mineralization Peribronchial, hyperplasia, histiocytic Nose Inflammation, suppurative Lumen, foreign body Mucosa, inflammation, suppurative Nasolacrimal duct, inflammation, suppurative Nerve, developmental malformation Olfactory epithelium, metaplasia Respiratory epithelium, hyperplasia Respiratory epithelium, metaplasia, squamous Trachea Inflammation, necrotizing Inflammation, suppurative Special Senses System Eye Cataract Retina, degeneration Harderian gland Inflammation (50) 2 (4%) (50) 1 (2%) 1 (2%) 2 (4%) 2 (4%) 2 (4%) 1 (2%) 1 (2%) (48) 1 (2%) 1 (2%) 1 (2%) (50) 3 (6%) (2) 2 (100%) 2 (100%) (5) 4 (80%) (48) 1 (2%) (48) I (2%) 1 (2%) 47 (98%) 1 (2%) 2 (4%) 27 (56%) 24 (50%) 1 (2%) 8 (17%) (45) 1 (2%) 3(7% ) 1 (2%) 1 (2%) 1 (2%) (48) 1 (2%) (2) 3 (43%) (48) I (2%) I (2%) (50) 5 (10%) 2 (4%) 1 (2%) 2 (4%) 50 (100%) 47 (94%) 8 (16%) 1 (2%) 44 (88%) 1 (2%) 9 (18%) (48) 1 (2%) 5 (10%) 2 (4%) (50) 1 (2%) 2 (4%) (2) 2 (100%) 2 (100%) (15) 3 (20%) 127 128 Talc, NTP TR 421 Table B4 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the Lifetime Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 m g/m 3 Urinary System Kidney Abscess Cyst Cyst, multiple Embolus, multiple Infarct Infarct, multiple Inflammation Nephropathy Capsule, inflammation Medulla, inflammation Renal tubule, necrosis Urinary bladder Inflammation (49) 1 (2%) 1 (2%) 1 (2%) 1 (2%) 44 (90%) 1 (2%) (50) (47) 1 (2%) 1 (2%) 1 (2%) 43 (91%) 1 (2%) 1 (2%) (45) 2 Number of animals examined microscopically at site and number of animals with lesion (49) 1 (2%) 1 (2%) 42 (86%) 1 (2%) 2 (4%) (50) 1 (2%) 129 APPENDIX C SUMMARY OF LESIONS IN MALE MICE IN THE 2-YEAR INHALATION STUDY OF TALC Table Cl T a b l e C2 T a b l e C3 T a b l e C4 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of T a l c ...................................................................................... Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of T a l c ...................................................................................... Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of T a l c ...................................................................................... Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of T a l c ...................................................................................... 131 134 152 156 13C Talc, NTP TR 421 Lesions in Mate Mice Table C l Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Talc* 0 mg/m 6 mg/m IS mg/m3 D isposition Summary Animals initially in study Early deaths Moribund . Natural deaths Survivors Terminal sacrifice Missexed Missing Animals examined microscopically Alimentary System Gallbladder Intestine large, colon Intestine small, duodenum Intestine small, ileum Adenocarcinoma Liver Hemangiosarcoma Hemangiosarcoma, metastatic, spleen Hepatocellular carcinoma Hepatocellular adenoma Hepatocellular adenoma, multiple Pancreas Hepatocellular carcinoma, metastatic, liver Salivary glands Stomach, glandular Cardiovascular System Heart Alveolar/bronchiolar carcinoma, metastatic, lung Endocrine System Adrenal gland Spindle cell, adenoma Adrenal gland, cortex Adenoma Adrenal gland, medulla Pheochromocytoma malignant Pituitary gland Adenoma Pars intermedia, adenoma Thyroid gland Follicular cell, adenoma General Body System Tissue NOS Hemangioma Hemangiosarcoma, metastatic, spleen 50 1 16 30 1 2 46 (31) (36) (32) (33) (45) 1 (2%) 1 (2%) 6 (13%) 1 (2%) 2 (4%) (42) 1 (2%) (45) (39) (45) (43) 1 (2%) (43) (39) 1 (3%) (44) 1 (2%) (45) 50 2 18 28 1 1 47 (29) (38) (30) (32) 1 (3%) (47) 5 (11%) 8 (17%) 1 (2%) (39) (46) (43) (46) (46) 1 (2%) (46) 1 (2%) (39) (44) 2 (5%) (46) (3) 50 3 14 32 1 49 (35) (39) (34) (35) (48) 1 (2%) 11 (23%) 4 (8%) (42) (47) (43) (49) 1 (2%) (47) 1 (2%) (47) 1 (2%) (42) (46) (45) 2 (4%) (2) 1 (50%) 1 (50%) 131 132 Talc, NTP TR 421 Table C l Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 Genital System Epididymis Prostate Seminal vesicle Testes Hemangiosarcoma Hematopoietic System Bone marrow Hemangiosarcoma, metastatic, spleen Lymph node Lymph node, bronchial Alveolar/bronchiolar carcinoma, metastatic, lung Lymph node, mandibular Hemangiosarcoma, metastatic, spleen Lymph node, mediastinal Lymph node, mesenteric Hemangiosarcoma, metastatic, spleen Spleen Hemangiosarcoma Thymus Alveolar/bronchiolar carcinoma, metastatic, lung (39) (40) (41) (43) 1 (2%) (40) 1 (3%) (45) (32) (23) (9) (36) (44) 2 (5%) (34) (39) (43) (43) (44) (42) (46) (39) (23) (10) (39) (44) (33) (42) (44) (39) (45) (43) (48) (44) 1 (2%) (19) 1 (5%) (7) (40) 1 (3%) (47) 2 (4%) (40) 1 (3%) Integumentary System None M usculoskeletal System Bone Hemangiosarcoma, metastatic, spleen Skeletal muscle Thoracic, alveolar/bronchiolar carcinoma, metastatic, lung (46) (47) (49) 1 (2%) (1) 1 (100%) Nervous System None Respiratory System Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar adenoma, multiple Alveolar/bronchiolar carcinoma Alveolar/bronchiolar carcinoma, multiple Hemangiosarcoma, metastatic, liver Hemangiosarcoma, metastatic, spleen Hepatocellular carcinoma, metastatic, liver (45) 6 (13%) 6 (13%) 1 (2%) 1 (2%) (47) 4 (9%) 2 (4%) 1 (2%) (48) 7 (15%) 2 (4%) 2 (4%) 1 (2%) 2 (4%) Lesions in Male Mice Table Cl Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Talc (continued) 0 mg/nr 6 m g/n r 18 mg/m3 Special Senses System Harderian gland Adenoma (1) 1 (100%) Urinary System Kidney Carcinoma, metastatic, uncertain primary site Urinary bladder Sarcoma (45) 1 (2%) (43) 1 (2%) (46) (38) System ic L esions Multiple organs*1 Lymphoma malignant lymphocytic Lymphoma malignant mixed Lymphoma malignant undifferentiated cell (46) 2 (4%) 3(7% ) (47) 1 (2%) Neoplasm Sum m ary Total animals with primary neoplasmsc Total primary neoplasms Total animals with benign neoplasms Total benign neoplasms Total animals with malignant neoplasms Total malignant neoplasms Total animals with metastatic neoplasms Total metastatic neoplasms Total animals with malignant neoplasms, uncertain primary site 26 36 11 12 20 24 4 5 1 20 26 16 17 8 9 1 1 ` Number of animals examined microscopically at site and number of animals with lesion Number of animals with any tissue examined microscopically c Primary neoplasms: all neoplasms except metastatic neoplasms () 4 (100%) (48) (43) (49) 28 38 18 22 15 16 4 11 133 134 Talc, NTP TR 421 Table C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Talc: 0 mg/m* Number o f Days on Study 0 4 44 45 5 55 55 5 666 77 7 7 77 7 033 88 147 7888 278 133 33 3 3 8 27 468 3 1957797 4066 66 6 6 C arcass ID Number 4345334453455445333333 3 95 17 7 90 19 8 129 5 16 66 7 7 7 5 14 24245559833 57 1470 15 1) 11111111111111111111 Alim entary System Esophagus Gallbladder Intestine large Intestine large, cecum Intestine latge, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Hemangiosarcoma Hemangiosarcoma, metastatic, spleen Hepatocellular carcinoma Hepatocellular adenoma Hepatocellular adenoma, multiple Pancreas Hepatocellular carcinoma, metastatic, liver Salivary glands Stomach Stomach, forestomach Stomach, glandular MM+ M+ + + + + M+ M+ + + + + + + + + + MMMMA A A A M+ + A A + + A M + M + + + A +AAAAAAA + +A+ + + A+ + + + + + A+ AAAAAAA+AAA+ + A+ + + + + + A+ AAAAAAA+ + A+ + + A+ + + + + + A + A A A A A A A + A A MM+ A + + + + + + A+ AAAAAAA +AAA+ +A + + + + + + AAAAAAAAA + AAA + +A+ + + + + + A +AAAAAAA + AAA A + A + + + + + + A +AAAAAAA + AAAA+ A + + + + + + A++ ++ + +++ ++ + ++ + ++ + + ++ + X XX X XXX M+ + + + A + A + + + + + + + A + + + + + + X A + + + + + + + + + + + + + + + + -1- + + + + A+++++++++++++ +++ +++++ A + + + + + + + + 1 + + M+ + + + + + + + + A + A + A M+ A + + + A + + + + + + + + + + Cardiovascular System Heart A+++++++++ +++++++++ +++ Endocrine System Adrenal gland Spindle cell, adenoma Adrenal gland, cortex Adrenal gland, medulla Pheochromocytoma malignant Islets, pancreatic Parathyroid gland Pituitary gland Adenoma Thyroid gland +: Tissue examined microscopically A: Autolysis precludes examination A + + + M+ + + + + + + + + + + I + + + + + A + + + M+ + + + + + + + + + + I + + + + + A + + + MM + + + + + + 1 + + + M+ + + + M M+ I + + M+ A + M+ A + I MI + + + + + M MMM+ MMM+ + + + + + M+ + + MMM+ M M+ + + + + + + + + + + + + + + + + + + + + X A++++ +++++ ++++++++ ++++ M: Missing tissue I: Insufficient tissue n s " ' Lcs*00 present Blank: Not examined A IL a B L C O P Y or athology or Male Mice in Ihe 2-Year Inhalation Study of Talc: 0 mg/mJ (continued; 7 77 777 777 777 7777 7 777 7 77 7 dy 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 67777 778 88 8889 99 90 00 0 00 0 3 344 4444 4 4 44 4 44 4 5 34 44 4 55 99 00 222 33 5 5568 89 19 228 8 2 2 28 23 134 14 12845823 36 7 24 0 1 111 11 111 111 11 1 1 11 111 11 1 1 Total T issu es/ T um ors cecum , colon , rectum 1 <11, duodenum all, ileum all, jejunum osarcoma iosarcoma, metastatic, spleen cellular carcinoma cellular adenoma acellular adenoma, multiple s c r carcinoma, metastatic, y glands .ch ach, forestomaclt ach, glandular vascular System art crine System Adrenal gland Spindle cell, adenoma Adrenal gland, cortex Adrenal gland, medulla Pheochromocytoma malignant Islets, pancreatic Parathyroid gland Pituitary gland Adenoma Thyroid gland ++++++++++++++++++++++++ +++M +++++++++++++++++++ ++ + + ++++ +++ +++ + ++ + ++ + ++ + + ++ +++ +++ +++ ++ ++ + ++ + +++ + ++ ++++++ ++ ++ +++ + ++ +++ + ++ +++++++++I ++++++++++++++ + + + + + + + + + + + + + + + + + + + + + + f + + + + + + + + + + + + + + M+ + + + + + + + + + ++++++++++++++++ +++++++ + + + + + + M+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +' + + + + + + + X XX +++++++++++++++++++++++ + ++ + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + +- + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + ++ + ++ + + + + + + + + + + + + + + + + + + + +I ++++++++++ ++++++++++++++ + + + + ++ + + + + + + + + + + + + + + + ++ + X + + + + ++ + + + + + + + + + + + + + + + ++ + + ++ + ++ + + + + M+ + + + + + + + + + ++ + X + + + + +MI I + + + + MI 1 M + I + + I +M + + + M + ++ M + + + M M + + + M M M M M + ++ M + ++ + ++ + + + + + + + + + + + I + + + ++ + + + + + ++ + + + + + + + + + + + + + + + ++ + 41 31 36 34 36 32 34 32 33 32 45 1 1 6 1 2 42 1 45 45 43 39 45 43 1 43 39 1 26 25 44 1 45 "5T T r ...... ' . 136 Talc, NTP TR 421 Table C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Talc: 0 mg/m1 (continued) Number o f Days on Study 0444455555556667777777 033881 4778882781 333333 8274683195779740666666 Carcass ID Number 4345334453455445333333 3951779019812951666777 5142424555983357147015 1111111111111111111111 General Body System None Genital System Epididymis Preputial gland Prostate Seminal vesicle Testes Hemangiosarcoma H em atopoietic System Bone marrow Hemangiosarcoma, metastatic, spleen Lymph node Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Hemangiosarcoma Thymus + + + M+ + + + + + + + + + M+ + M+ + MI + ++ + + M+ MM+ 1 + + + + + + + + I + + + + + + + + + + A + M+ + + + + A A + + A + + + + + + A + + M+ + + A + + + + + + + + + + + + + + A+A+ +A +A +A+A+ + + + + + + + + + M+ + + + MM+ I + MMMM + MMMMI M+ A M+ A++++ +++++++++++++++++ + M+ + + + I I + + + + + + + + + M + MMMMMM + MMM + MM + + M+ MM+ MMMMMMMMMMM+ M+ M+ + MA + + + + + + + + I M +++++++++++++++++ MI MM+ MMA + M+ + + I + + M + + + + + Integumentary System Mammary gland Skin M usculoskeletal System Bone MMMMMMM+ M+ MI MMMMMMMMMM + + + + + + + + + + + + + M+ + + + + + + + ++++++++++++++++++++++ Nervous System Brain ++++++++++++++++++++++ Lesions in Male Mice 137 Table C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Talc: 0 mg/m3(continued) Num ber of Days on Study 777777777777777777777777 333333333333333334444444 677777788888899990000000 Carcass ID Number 334 4444 44 4444 44 453 44 44 55 990 0 2223 355 56 88 9 192 288 2 2 28 23 13 4 14 128 45 82 336 72 40 1 11 1111 1111 1 111 11 111 1111 1 Total Tissues/ Tumors General Body System None G enital System Epididymis Preputial gland Prostate Semina) vesicle Testes Heroangiosarcoroa + + + + + + + 4 4 4 4 4 4 4 4 M4 4 4 4 M4 4 4 444 4 + 4 4 + + 4 + 4 M 4 + 4 + 4 + 4 + 4- + + 4- + 4 + 4 4 + + + + + + + + 4 + + 4 + 4 + + 4 + + 4- 4+ + 4 + + + + 4 + + + 4 + + 4 4- 4 + + 4 + 4 4 4 X 39 8 40 41 43 1 Hematopoietic System Bone marrow Hemangiosarcoma, metastatic, spleen Lymph node Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Hemangiosa rcoma Thymus +4 +44 +++++++++++4 ++44 44 4 X +4++++++++++++++4 +4 44 444 I 4 M4 + + + M+ + + + 4 4 M4 M+ + M4 4 MM M4 MM+ + + + + + + + + + + 4 MM4 MM4 4 4 MMMM4 MMMM+ + MMM+ MM4 MMMM4 M +++44 +++4+4 ++I ++4 4 44 4 4 44 4 + + + + + 4- + + + + + 4- 4 M 4 4 4 4 4 4 4 4 4 XX I +++++44 4I ++++4 44 +44 4 4 44 40 1 45 32 23 9 36 44 2 34 Integumentary System Mammary gland Skin MMMMMMMMMMMM + MMMM + M + Mi MM + 4 4 + + + + + + + I + + + + + + + + 4 4- 4- + 4- 5 44 M usculoskeletal System Bone + 4- + + + -f + + + + + + + + + 4 - 4 - + + + + + + + 46 Nervous System Brain 4 - 4 - 4 4 - + 4- + + + + 4 - 4 - 4 - 4 - 4 4 4 - 4 4 - 4 - 4 4 4 4 46 13 Talc, NTP TR 421 Table C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Talc; 0 mg/m3 (continued) Number of Days on Study 0444455555556667777777 0338814778882781333333 82746831 95779740666666 Carcass ID Number 4345334453455445333333 3951779019812951666777 5142424555983357147015 1111111111111111111111 Respiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma Alveolar/bronchiolar carcinoma, multiple Hemangiosarcoma, metastatic, liver Nose Trachea A + + + + + + A + + + + + + + + + + +I + + A + + + + + ++ + + + + + + + + + + + + + + X X X + ++ + ++ +A+ + ++ ++ + ++ + + ++ + A + A + + + + + + + + + + + +A + + + + + + Special Senses System Ear Harderian gland Adenoma Urinary System Kidney Carcinoma, metastatic, uncertain primary site Urinary bladder Sarcoma A+++++++++++++++++ ++ ++ X A++A+++A++++ + +++ ++ +++ + X Systemic Lesions Multiple organs Lymphoma malignant mixed Lymphoma malignant undifferentiated cell type ++++++++++++++++++++++ Lesions in Male Mice 139 Table C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Talc: 0 mg/m1 (continued) Number of Days on Study 777777777777777777777777 333333333333333334444444 677777788888899990000000 Carcass ID Number 33 44444444 4444 44 534 444 55 99 00 2223 35556889 19 2288 22 2823 134 14 1284 58 23 36 7 24 0 1 1 11 1111 11111 111 1111 11111 Total Tissues/ Tumors Respiratory System Larynx Lung Aiveolar/bronchiolar adenoma Aheolar/bronchiolar carcinoma Aiveolar/bronchiolar carcinoma, multiple Hemangiosarcoma, metastatic, liver Nose Trachea ] +++++++++++++++++++++++ ++++++++++++++++++++++++ XX X XX X XX XX X ++++++++++++++++++++++++ + + M+ + + + + + + + + + + + + + + + + + + + + 42 45 6 6 1 1 45 42 Special Senses System Ear Harderian gland Adenoma '+ + X 1 1 1 Urinary System Kidney Carcinoma, metastatic, uncertain primary site Urinary bladder Sarcoma ++++++++++++++++++++++++ ++++++++++++++++++++++++ 45 1 43 1 Systemic Lesions Multiple organs Lymphoma malignant mixed Lymphoma malignant undifferentiated cell type + + + + + + + + + + + + + + + + + + + + + + + -1XX X XX 46 2 3 140 Talc, NTP TR 421 Table C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Talc: 6 mg/m3 Num ber of Days on Study 2234555555666666777777 7 5542455899223888 1123333 33436 0840 146 3 158 09 266 66 C arcass ID N um ber 0 100 110 111000 1 100 110 00 0 3340 264552473506 1530000 5 3 2 7 14 0 16 9 14 8 7 0 15 4 2 14 5 8 11111111111111111111111 Alimentary System Esophagus Gallbladder Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Adenocarcinoma Intestine small, jejunum Liver Hepatocellular carcinoma Hepatocellular adenoma Hepatocellular adenoma, multiple Pancreas Salivary glands Stomach Stomach, forestomach Stomach, glandular Tooth + + + M+ + + + + + + + + + + + + + + + + + + A M1 MA A A A + A + I A A A + A A M + + + + A + + AAAAA+ A+ +A + A + + + + + + + + AA + AAAAA+ A+ AA+ A + A+ + + + + + A + + AAAAA+ A+ + A + A+ + + + + + + + A + + A A A MA + A + A A + A + + M+ + + + + AA+ AAAAA+ A + AA + A+ A + A + + + + AA+ AAAAAAA + AAAA+ A + A + + + M AAAAAAAA+ A +AAAA+ A + A + + + + AA+ AAAAA+ A+ AA+ A + AAA + + + + +++++++++++++++++++++++ XX XX XX X MA + A MA M + + + + + M + + + + + + + + + A++ +++++ ++++++ ++ ++ +++ + + + + + A + AA+ + + + + A+ + + + + + + + + + + + + A + AA+ + + + I A+ + + + + i + + + + + + + A+ AA+ + + + + A+ + + + + + + + + + + C ardiovascular System Heart + + ++ + ++ + ++ +++ +++ ++ + ++ + + Endocrine System Adrenal gland Spindle cell, adenoma Adrenal gland, cortex Adenoma Adrenal gland, medulla Islets, pancreatic Parathyroid gland Pituitary gland Pars intermedia, adenoma Thyroid gland A + + + + + + + + + + + + + + + + 4- 4- 4- 4- 4- 4- A + + + + + + + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- A + + + + + + + + + + I + + M + 4- 4- 4- M M 4- 4M A M A M A M M + + + + M + I M 4- 4- 4- 4- M 4- M M M M + + I M M M M + + M M + + 4- 4- M M 4- M M + + I + M + + + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- X + + + + + + + I + + + + + + + + 4- 4- 4- 4- 4- 4- 4- L esions in M aie M ice 141 Table C2 Individual Animal Tumor Pathology or Male Mice in the 2-Year Inhalation Study of Talc: 6 mg/m' (continued) Num ber of Days on Study 7 77 77 777777 7777 77 77 777 77 333333333333333333444444 667 77 77778888 889 99 00 00 00 Carcass ID Num ber 0000000000000 1111100 1111 11336667799 9900235392256 0 4364 56 123 569 157 55 224 58 2 1 11 11111 11 1 111 11 111 11 111 Total Tissues/ T um ors Alimentary System Esophagus Gallbladder Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Adenocarcinoma Intestine small, jejunum Liver Hepatocellular carcinoma Hepatocellular adenoma Hepatocellular adenoma, multiple Pancreas Salivary glands Stomach Stomach, forestomach Stomach, glandular Tooth + + 4 4 4 4- 4- 4- 4- 4- 4- 4- + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + 4 4 + 4- i 4- 4- 4- 4- 4- + 4- M 4- 4- 4- 4- 4- 4- 4- 4- 44 4 4 + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + 4 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ 4 4 + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 44 4 4 I 4- 4- 4- M 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4- 4- 4- M 4 4 + 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4 4 M 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 44 4 4 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- X + + + + 4- M 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4- + + + + 4- 4- 4- 4- 4- 4* 4- 4- 4- 4- 4- + + 4- 4- 4- 4- 4- 4- 4- X XX XXX X + 4 4 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4 i 4- 4- 4- 4- + + 4 4 4- 4- 4- 4- 4- 4- 4- 4- 4- + + 4- 4- 4- 4- 4- 4- 4- 4- 4+ + + 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4+ + 4 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4* 4- 4- 4- 4- 4- 4- 4 + + 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 46 29 38 35 38 32 34 30 32 1 31 47 5 8 1 39 46 43 41 43 3 Cardiovascular System Heart + + 4 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4- 4- 4- 4- 4- 46 Endocrine System Adrenal gland Spindle cell, adenoma Adrenal gland, cortex Adenoma Adrenal gland, medulla Islets, pancreatic Parathyroid gland Pituitary gland Pars intermedia, adenoma Thyroid gland 4 + 4 4 4 + 4 4 4 4 4 4 4- 4- + 4- 4- 4- + 4- 4- 4- 4- 4 X 4 + 4 4 + 4 4 4 4 4 4 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4 X 4 + + 4 4 4 4 4 4 4 I 4- 4- M + 4- 4* 4- 4- 4- 4- 4- M 4 I M + I 4 I 4 I 4 I I M I I i 4- 4- 4- M I 4- I 4- 4M M M 4 4 I M I M 4 M 4- 4- M M 4- + M 4- M 4- 4- 4- 44 + 4 4 4 4- 4 4 4 4 4 4- 4- 4- + 4- M 4- 4- 4- 4* 4- 4- 4- X 4- + 4- 4- 4- 4- 4- 4* 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4* 4- 46 1 46 1 39 20 21 44 2 46 142 Talc, N TP TR 421 Table C2 Individual Animal Tumor Pathology or Male Mice in the 2-Year Inhalation Study of Talc: 6 mg/m3 (continued) N um ber of Days on Study 22345555556 6 6 66 67777777 5542455899 2 2 3888 1123333 334360840 14 6 3 1580926666 C arcass ID Number 0 100 1 10 111000 110 0 1 10 000 3340 26 4552 473506 15 300 00 5 327 140 16 9 148 70 15 42 1458 111 1111 111111 111 111 11 11 General Body System Tissue NOS +++ Genital System Epididymis Preputial gland Prostate Seminal vesicle Testes A+ + + +++ + ++ ++ ++A+++ + + ++ + ++ + + + + MA I A + + + + + + + + + + + + + + + + + A++A+A+ ++ +++ ++A+ ++ ++ + + + A+ ++ +A+ ++ +++ ++A+++ ++ + + + Hematopoietic System Bone marrow Lymph node Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Thymus + + + + AA + AAA + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + M+ + + + + M + I f + + + + + + + M + + + + + M + MM+ MM+ + + + M+ MMM+ M+ + + + MMM MMMMM+ MMM+ MMMMMMMM+ + MMM M M M M + M + + .+ + + + M + + + M + + + + M + A+ + + + AA+ + + + + + + + + + + + + + + + A MMM+ + + I + + M+ M+ I M+ + + + + + + Integum entary System Mammary gland Skin M usculoskeletal System Bone Nervous System Brain R espiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma Hepatocellular carcinoma, metastatic. liver Nose Trachea MMMMMA MMMMMMMMM+ M+ + M+ MM +++++++++++++++++++++++ +++++++++++++++++++++++ +++++++++++++++++++++++ A + + A+AA+ + + + +A + + + + + + + 1 + + +++++ ++++ ++++ ++ ++ ++ + +++ X X X + + + + +A++ + + + + + + + + + + + + + + + + ++++AI +++ ++A+++ ++ ++ ++ + Lesions in Maie Mice 143 Table C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Talc: 6 mg/nr' (continued) Num ber of Days on Study 777 7777777 7777 77 77 777 77 7 333333333333333333444444 6 6 7777777888888999000000 C arcass ID N um ber 0000000000000 1111100 1111 1 13 366 67 79 9990 02 35 392 256 0436456 123569 15755224582 1 11 11 1 1 11 1 11 11 1 1 11 11 11 11 Total Tissues/ T um ors G eneral Body System Tissue NOS 3 Genital System Epididymis Preputial gland Prostate Seminai vesicle Testes M + + + + + + M + + 4- + I + + + + I I + + + I + ++ + + + + + 4* 4- + + + + + + + + + 4- + + + + + + 4+ + + + + 4- 4- + + + + 4- + + + + 4- + + + + + 4- + + + 4- 4- 4- 4- + + + + 4- 4- + + + + 4- + + + 4- + + + 39 6 43 43 44 Hematopoietic System Bone marrow Lymph node Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Thymus + + + + + + + + + + + + + + + + + + 4- 4- 4- 4- 4- 4+ + + + + + + + + + + + + + + + + + + 4- 4- 4- 4- 4+ + + + M + + + + + + + M + + + M + 4- 4- 4- 4- 4- M + M + + M + M M M + M + M + + + M M 4- M 4- 4- M M M M M + M M + M M M M + M M + M M M 4- M M M M 4+ + + + + + + 4- + + + + + + + + + + 4- 4- 4- 4- 4- 4+ + + + + + + + + + + + + + + + + + 4- 4- 4- 4- 4- 4+ + + + I + + + M + I + + + + M + + 4- 4- 4- 4- 4- i 42 4 39 23 10 39 44 33 Integum entary System Mammary gland Skin M M M M M M M M M M M M M M M M M M 4- M M M M M + I + + + + + + + + + + + + + + + + M 4- 4- 4- 4- 4- 5 45 M usculoskeletal System Bone + + + + + + + + + + + + + + + + + 4- 4- 4- 4- 4- 4- 4- 47 Nervous System Brain 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 47 R espiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma Hepatocellular carcinoma, metastatic, liver Nose Trachea 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4XXX X 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 41 47 4 2 1 48 43 144 Talc, NTP TR 421 Table C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Talc: 6 mg/m3 (continued) Number o f Days on Study 22345555556666667777777 5542455899223888 1123333 334 360840 1463 1580926666 Carcass ID Number 0 100 1 10 1 1 100 0 1 10 0 1 100 0 0 334 0264552473506 1530000 5 3 2 7 140 16 9 148 70 15 4 2 14 5 8 11111111111111111111111 Special Senses System None Urinary System Kidney Urinary bladder +++++A+++++ +++ +++ ++ + +++ A+ + A+ AAA+ + + +A+ A+ A+ + + + + + System ic L esions Multiple organs Lymphoma malignant lymphocytic ++++ ++++++++++++++++++ X Lesions in Male Mice 145 Table C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study or Talc: 6 mg/m3 (continued) Num ber of Days on Study 7777 77 777 7777 77 77 777 77 77 333333333333333333444444 66 7777 777888 888 99 90 000 00 C arcass ID N um ber 0000000000000 I 111100 1111 113 3666 7799 9900 2 35 39 225 6 0436456 123569 15755224582 1 1 111 111 1111 1111 11 11 11 11 Total Tissues/ T um ors Special Senses System None U rinary System Kidney Urinary bladder +++ ++ ++++ + +++ ++ ++ +++ ++ ++ + + + + + + + + + + + + + + + + + + +M + + + + 46 38 Systemic Lesions Multiple organs Lymphoma malignant lymphocytic ++++++++++++++++++++++++ 47 1 146 Talc, NTP TR 421 T able C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study or Talc: 18 mg/m1 N um ber or Days on Study 0 11144445556677777777777 2 11523573455722223333333 845928788 14 82 1457 6666666 C arcass ID N um ber 11132 122 122222233 1111222 889 188 21874848 104 8899 111 735056 19 5793 125634923 134 1 111 1111111 1111 11 11 111 11 Alimentary System Esophagus Gallbladder Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Hemangiosarcoma Hepatocellular carcinoma Hepatocellular adenoma Pancreas Salivary glands Stomach Stomach, forestomach Stomach, glandular M 4 4 + 4 M + 4 4 4- 4- 4- 4- 4- M 4 4 4 4 4 4 4 4 + M A A A M A + A + A A M A 4- A 4 A 4 4 4 4 4 4 + A + + + A + + A + A A A 4- A A 4 A 4 4 4 4 4 4 4 A+ 4 AAA4 A+ AAAAAA4 A4 + 4 4 4 4 4 A+ 4 AA4 4 A+ AAA+ AA4 A4 4 4 4 4 4 4 A 4 M + A M 4 M I A A A 4- A A 4 A 4 M 4 4 4 4 4 AA4 AAA 4 A 4 AAAAAA 4 A 4 4 4 4 4 4 4 A A 4 A A M + A MA A A A A A 4 A 4 4 4 4 4 4 4 AAAAAA + A + AAAAAA 4 A 4 4 4 4 4 4 4 AA+ AAA4 A 4 AAAAAA4 A4 4 4 4 4 4 4 + 4 4 + A 4 4 4 4 4- 4- 4- 4- 4- 4 4 4 4 4 4 4 4 4 4 X XX XXXX X X XX 4 4 + + A + + 4- M A A A 4- A 4 4 4 4 4 4 4 4 I 4 4 4 4 + A 4 4 4 4 4- A + 4- 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 + A 4 4 4 + 4- A A 4- 4 4 4 4 4 4 4 4 4 4 4 + + 4 4 A 4 4 4 + 4- A A 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 A 4 4 4 4- A A A 4- A 4 4 A 4 + 4 4 4 4 4 C ardiovascular System Heart AJveolar/bronchiolar carcinoma, metastatic, lung + 4 + 4 4 + 4 4 4- 4- 4- 4- 4- 4 4 4 4 4 4 4 4 4 4 4 X Endocrine System Adrenal gland Spindle cell, adenoma Adrenal gland, cortex Adenoma Adrenal gland, medulla Islets, pancreatic Parathyroid gland Pituitary gland Thyroid gland Follicular cell, adenoma General Body System Tissue NOS Hemangioma Hemangiosarcoma, metastatic, spleen 4 4 4 4 A4 4 4 4 4 A4 4 + 4 4 4 4 4 4 4 4 4 + X 4 4 4 4 A 4 4 4 4 4 A 4 4 -f 4 4 4 4 4 4 4 4 4 + 4 4 I 4 A 4 4 4 MMA 4 4 + 4 4 4 4 4 4 4 4 4 + I I 4 4 A 4 4 4 MA AA 4 A 4 4 4 I I I M4 I I 4 4 4 4 A MM4 I MA 4 4 M4 M4 4 4 I MM4 M 4 4 4 4 A 4 4 4 4 4 A 4 4 M4 4 4 4 4 4 4 4 4 + 4 4 4 4AI 4 4 4 4A4 4 +4 4 4 4 4 4 4 4 4 + + X Lesions in Male Mice 147 T able C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Talc: 18 mg/m3 (continued) Number of Days on Study 7777777777777777777777777 3333333333333333333444444 7777777888889999999000000 Carcass ID Number 2222222223333333333222333 2244555780003333334 1550 11 34280 1294 348 2 3478 9 57 357 13 1 111 11 111 11 11 1111 1111 111 1 Total Tissues/ T um ors Alimentary System Esophagus Gallbladder Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Hemangiosarcoma Hepatocellular carcinoma Hepatocellular adenoma Pancreas Salivary glands Stomach Stomach, forestomach Stomach, glandular +++++++++ +++++++++ +++++++++ +++++++++ +++ +++ +++ MI + + + + MM+ +++++++++ +++++++++ + ++ + ++ + ++ +++ ++ +++ + +++++++++ ++ + + + + + + + + + + + + + + ++ + + + + + + + + + + I + + + ++ + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + ++M + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + ++ + + + + + M+ + + + + + + + ++ + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + XX X +++++++++ +++++++++ + ++ ++++ ++ +++++++++ +++++++++ ++ + ++ + ++ + ++ + ++ + X X ++ + + ++ + + ++ + + + ++ + + ++ + + ++ + + + ++ + + ++ + + ++ + + + ++ + + ++ + + ++ + + + ++ + + ++ + + ++ + + + 46 35 40 37 39 31 36 34 35 36 48 1 11 4 42 47 46 46 43 Cardiovascular System Heart Alveolar/bronchiolar carcinoma, metastatic, lung +++++++++++++++++++++++++ 49 1 Endocrine System Adrenal gland Spindle cell, adenoma Adrenal gland, cortex Adenoma Adrenal gland, medulla Islets, pancreatic Parathyroid gland Pituitary gland Thyroid gland Follicular cell, adenoma +++++++++++++++++++++++++ +++ ++++ ++ + +++ +++ ++ + ++ ++ ++ X + + + + + + + + M+ + + + + + + + + + + + + + + I + I + + + + MMMM+ I + + + + I M+ I + + I I + + + + + M+ MM+ MMM+ M+ + + M+ + + MMM+ + ++ + +++ + ++ + +++ + + ++ + + + + ++ + ++++++++++++++++++++++I ++ XX 47 1 47 1 42 24 26 46 45 2 General Body System Tissue NOS Hemangioma Hemangiosarcoma, metastatic, spleen +2 X1 1 14* Talc, NTP TR 421 Table C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Talc: 18 mg/m1 (continued) Number o f D ays on Study 0 11144445556677777777777 2 115235734557 222 233 33333 845928788 1482 14576666666 Carcass ID Number 11132 122 122222233 11112 22 889 1882 1874848 1048899 111 7350 56 195793 125634 923 134 1 11 1111111 11 11 111 11 11 1 11 Genital System Epididymis Preputial gland Prostate Seminal vesicle Testes + + + + A + + + + + M+ + A + + + + + + M+ + + +++ + + MMMA + + + + + A + + + + + + + + + + + + + + MMMA + + + + A A A MA + + + + + + + + + + + + + + A + + + + + A M+ A + + + + + + + + + + H em atopoietic System Bone marrow Lymph node Lymph node, bronchial Alveolar/bronchiolar carcinoma, metastatic, lung Lymph node, mandibular Hemangiosarcoma, metastatic, spleen Lymph node, mediastinal Lymph node, mesenteric Hemangiosarcoma, metastatic, spleen Spleen Hemangiosarcoma Thymus Alveolar/bronchiolar carcinoma, metastatic, lung + + + + AA + + AAAA + + + + + + + + + + + + + + + + + + + + + + M+ + + + + + + + + + + + + + + MMA + + + + + M+ + + + + + + + + + + + + X M1 1 + A M+ M+ MMMMM+ + + + + + MMMM X MMMMMMMMMMMM+ M+ MM+ MMMMMM M+ + MA + + M+ + A + + A + + M + + + + + + + X + + + .+ A + + + + + A + + + + + + + + + + + + + XX + M+ MM+ + + + + A + M+ + + + + + + M+ + + X Integumentary System Mammary gland Skin 1 I MMMMM+ + MMMMMMMMMMMMMMM +++++++++++++++I ++++++++ M usculoskeletal System Bone Hemangiosarcoma, metastatic, spleen Skeletal muscle Thoracic, alveolar/bronchiolar carcinoma, metastatic, lung + + ++ +++ +++ + ++ + +++ ++ +++ + + X + X Nervous System Brain ++ ++A+++ +++ + ++ + ++ ++ + + ++ + Lesions in Male Mice 149 Table C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Talc: 18 mg/m3 (continued) Number o f D ays on Study 7777777777777777777777777 3333333333333333333444444 7777777888889999999000000 Carcass ID Number 2222222223333333333222333 22445557800033333341550 11 34280 12943482 3478957357 13 1 11111 1111 111 1 111 11 111 1 11 Total T issu es/ Tumors Genital System Epididymis Preputial gland Prostate Seminal vesicle Testes + + + + + + + + + + + +M+ + + + + M+ + + + M+ ++ ++ + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + I + + + + + + + + + + + + + + ++ + + + + + + + + + + + + 42 8 44 39 45 H em atopoietic System Bone marrow Lymph node Lymph node, bronchial Alveolar/bronchiolar carcinoma, metastatic, lung Lymph node, mandibular Hemangiosarcoma, metastatic, spleen Lymph node, mediastinal Lymph node, mesenteric Hemangiosarcoma, metastatic, spleen Spleen Hemangiosarcoma Thymus Alveolar/bronchiolar carcinoma, metastatic, lung +++ ++ ++ ++ ++ ++++ ++ + ++ ++ +++ ++++ ++++ ++++ ++++ ++ ++ ++++ + + + + + + + + + + + + + + + M+ + + + + + + + + + + Mi M+ M+ + MMM+ + M+ MM+ MM+ MMM+ + MMMMMM+ MMMMMMM+ MMMM+ MMMM + + + + + + + + + + + + + + + + + + M+ I + + + + +++++++++++++++++++++++++ + + + + + + M+ + + + + + + M+ + I + + + + + + + 43 48 44 1 19 1 7 40 11 47 2 40 1 Integumentary System Mammary gland Skin +M I M+ + I + M M M M + M M M M M M M M + M M M +++++++++++++++++++++++++ 8 48 M usculoskeletal System Bone Hemangiosarcoma, metastatic, spleen Skeletal muscle Thoracic, alveolar/bronchiolar carcinoma, metastatic, lung +++++++++++++++++++++++++ 49 1 1 1 Nervous System Brain +++++++++++++++++++++++++ 48 150 Talc, NTP TR 421 Table C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Talc: 18 mg/m1 (continued) Num ber of Days on Study 0 11144 4455 5667 7 7777 77 77 7 2 115235734 557 222 233 33 33 3 8 459 28788 1482 1457 66 66 666 C arcass ID N um ber 1113 2 122 122 222 233 1 1 1 12 22 889 188 2 18 7 48 48 104 88 99 11 1 735056 195793 12563492 3 134 111111111111111111111111 R espiratory System Larynx Lung Alveola r/bronchiolar adenoma Alveoiar/bronchiolar adenoma, multiple Alveolar/bronchiolar carcinoma Hemangiosarcoma, metastatic, spleen Hepatocellular carcinoma, metastatic, liver Nose Trachea + +++A++ +++A++A+ ++ ++ ++ +++ ++ ++A++ ++++++ ++ ++ ++ ++ +++ XX X XX X XX + +++A+++++A+++ ++ ++ + + + +++ + + + + A + + A + + A M+ A + + + + + + + + + + Special Senses System Harderian gland Adenoma ++ XX U rinary System Kidney Urinary bladder ++++ + + ++ ++A++ + ++ + + ++ + ++ + + + + +A + + + + + A A + A A + A + + + + + + + Systemic Lesions Multiple organs ++++++++++++++++++++++++ Lesions In Male Mice 151 Table C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Inhalation Study of Talc: 18 mg/m3 (continued) N um ber of Days on Study 7777777777777777777777777 3333333333333333333444444 7 7777 77888 88999 999 90 00 000 Carcass ID N um ber 22 2 22 22 223 333 33 3 333 22 2 3 33 2 24 4555 780 00 333 33 34 155 0 11 34280 1294348 23478957357 13 1111111111111111111111111 Total Tissues/ T um ors R espiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar adenoma, multiple Alveolar/bronchiolar carcinoma Hemangiosarcoma, metastatic, spleen Hepatocellular carcinoma, metastatic, liver Nose Trachea +++++++++++++++++++++++++ +++++++++++++++++++++++++ X XXX X X +++++++++++++++++++++++++ + + + + + I + + M+ + + + + + + + + + + + + + + + M> 48 7 2 2 1 2 47 42 Special Senses System Harderian gland Adenoma ++ XX . 4 4 U rinary System Kidney Urinary bladder +++++++++++++++++++++++++ + + + + + + + + + + + + + + + + + + + + + + + + 4- 48 43 Systemic Lesions Multiple organs +++++++++++++++++++++++++ 49 152 Table C3 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Talc Talc, NTP TR 421 H arderian Gland: Adenoma Overall rales* Adjusted rales*5 Terminal ratesc First incidence (days) Life table tests" Logistic regression testsd Cochran-Armitage testd Fisher exact testd Liven H epatocellular Adenoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Liven H epatocellular Carcinom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Liven H epatocellular Adenoma or Carcinom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Lung: A lveolar/bronchioiar Adenoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 1/46 (2%) 33% 1/30 (3%) 736 (T) P =0.073 P = 0.075 P=0.065 3/45 (7%) 10.0% 3/30 (10%) 736 (T) P=0.489N P=0.493N P=0.515N 6/45 (13%) 16.7% 2 /3 0 (7%) 571 P =0.114 P=0.116 P=0.097 9/45 (20%) 25.6% 5/30 (17%) 571 P = 0.256 P=0.216 P =0.225 6/45 (13%) 20.0% 6/30 (20%) 736 (T) P = 0.224 P=0.251 P=0.210 0/47 (0%) 0.0% 0/28 (0%) _e P=0.514N P=0.514N P=0.495N 9/47 (19%) 29.5% 7/28 (25%) 633 P = 0.050 P=0.061 P=0.070 5/47 (11%) 13.7% 1/28 (4%) 546 P=0.491N P=0.445N P=0.469N 13/47 (28%) 38.1% 8/28 (29%) 546 P = 0.228 P=0.257 P = 0.269 4/47 (9%) 14.3% 4/28 (14%) 736 (T) P=0.411N P=0.411N P=0.342N 4/49 (8%) 12.0% 3/32 (9%) 725 P = 0.204 P=0.216 P =0.201 4/48 (8%) 11.8% 3/32 (9%) 672 P=0.539 P = 0.552 P=0.536 11/48 (23%) 27.3% 5/32 (16%) 438 P=0.187 P = 0.203 P=0.177 14/48 (29%) 34.5% 7/32 (22%) 438 P = 0.230 P=0.223 P=0.217 9/48 (19%) 27.0% 8/32 (25%) 672 P = 0.333 P=0.371 P =0.336 Lesions in Male Mice Table C3 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 Lung; Alveolar/bronchiolar Carcinoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Pituitary Gland (Pars Intermedia): Adenoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Spleen: Hemangiosarcoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test All Organs: Hemangiosarcoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 7/45 (16%) 23.3% 7/30 (23%) 736 (T) P=0.068N P=0.069N P = 0.065 N 12/45 (27%) 40.0% 12/30 (40%) 736 (T) P=0.533N P=0.552N P=0.554N 0/44 (0%) 0.0% 0/29 (0%) - P=0.547N P=0.566N P=0.564N 2/44 (5%) 6.9% 2/29 (7%) 736 (T) P =0.595 P=0.581 P =0.577 4/46 (9%) 12.9% 3/30 (10%) 710 P=0.529N P=0.545N P=0.554N 2/47 (4%) 5.9% 1/28 (4%) 558 P=0.093N P=0.073N P=0.069N 5/47 (11%) 16.4% 4/28 (14%) 558 P=0.063N P=0.043N P=0.043N 2/44 (5%) 6.5% 1/27 (4%) 681 P = 0.238 P = 0.239 P =0.247 0/44 (0%) 0.0% 0/28 (0%) P=0.246N P=0.246N P=0.247N 0/47 (0%) 0.0% 0/28 (0%) - P=0.071N P=0.060N P=0.056N 2/48 (4%) 5.2% 0/32 (0%) 438 P=0.068N P=0.070N P=0.065N 11/48 (23%) 30.8% 8/32 (25%) 438 P=0.426N P = 0.423N P=0.429N 0/46 (0%) 0.0% 0/32 (0%) - 2/47 (4%) 5.5% 0/32 (0%) 672 P=0.650N P=0.668N P=0.666N 3/49 (6%) 8.4% 1/32 (3%) 672 p=0.448N P=0.456N P=0.464N 1S3 154 Talc, NTP TR 421 Table C3 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Talc (continued) 0 mg/m3 6 mg/in3 18 mg/m3 All O rgans: H em angiom a o r H em an g io sarco m a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage lest Fisher exact test All O rgans: M a lig n an t L ym phom a (Lym phocytic, Mixed, Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test All O rg an s: B enign N eoplasm s Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Ail O rg an s: M alig n an t N eoplasm s Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test All O rgans: B enign o r M a lig n a n t N eoplasm s Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 4/46 (9%) 12.9% 3/30 (10%) 710 P=0.515 P =0.505 P = 0.492 0/47 (0%) 0.0% 0/28 (0%) - P=0.071N P=0.060N P=0.056N U ndifferentiated Cell Type) 5/46 (11%) 1/47 (2%) 16.7% 3.6% 5/30 (17%) 1/28 (4%) 736 (T) 736 (T) P=0.019N P=0.116N P=0.019N P=0.116N P=0.020N P=0.097N 11/46 (24%) 35.2% 10/30 (33%) 587 P=0.158 P=0.154 P=0.139 16/47 (34%) 51.1% 13/28 (46%) 633 P=0.135 P=0.188 P=0.199 20/46 (43%) 58.3% 16/30 (53%) 571 P=0.253N P=0.262N P=0.245N 8/47 (17%) 23.3% 4/28 (14%) 546 P=0.012N P=0.005N P=0.005N 26/46 (57%) 76.2% 22/30 (73%) 571 P =0.442 P = 0.344 P =0.399 20/47 (43%) 58.0% 14/28 (50%) 546 P=0.208N P=0.102N P=0.127N 4/49 (8%) 11.4% 2/32 (6%) 672 P=0.590N P=0.598N P=0.607N 0/49 (0%) 0.0% 0/32 (0%) - P=0.027N P=0.027N P = 0.024N 18/49 (37%) 51.4% 15/32 (47%) 672 P=0.127 P=0.138 P=0.128 15/49 (31%) 35.9% 6/32 (19%) 438 P=0.166N P=0.152N P=0.139N 28/49 (57%) 66.5% 18/32 (56%) 438 P=0.554 P =0.503 P=0.558 Lesions in Male Mice 155 Table C3 Statistical Analysis or Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Talc (continued) T)Terminal sacrifice Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, bone marrow, brain, epididymis, gallbladder, heart, itidney, larynx, liver, lung, nose, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, testes, thyroid gland, and urinary bladder, for other tissues, denominator is number of animals necropsied. Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality ' Observed incidence at terminal kill Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. e Not applicable; no neoplasms in animal group 156 Talc, NTP TR 421 Table C4 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Talc* 0 mg/m3 6 m g/m 3 18 mg/m3 D isposition Summary Animals initially in study Early deaths Moribund Natural deaths Survivors Terminal sacrifice Missexed Missing Animals examined microscopically Alim entary System Gallbladder Dilatation Epithelium, hyperplasia, papillary Intestine large, cecum Hyperplasia, lymphoid Intestine large, colon Hyperplasia, lymphoid Intestine large, rectum Serosa, inflammation, suppurative Intestine small, duodenum Hyperplasia, lymphoid Mucosa, atrophy Intestine small, ileum Hyperplasia, lymphoid Mucosa, atrophy Peyer's patch, necrosis Intestine small, jejunum Hyperplasia, lymphoid Mucosa, atrophy Liver Abscess Focal cellular change Hematocyst . Hematopoietic cell proliferation Infarct Inflammation, focal Mineralization, focal Necrosis, focal Pigmentation, hemosiderin, focal Bile duct, hyperplasia, focal Serosa, inflammation, suppurative Pancreas Serosa, inflammation, suppurative Stomach, foreslomach Hyperplasia, squamous, focal Tooth Dysplasia 50 1 16 30 1 2 46 (31) (34) (36) 1 (3%) (32) (32) 3 (9%) (33) 5 (15%) 3 (9%) 1 (3%) (32) 3 (9%) (45) 1 (2%) 4 (9%) 2 (4%) 2 (4%) 4 (9%) (42) (43) 50 2 18 28 1 1 47 (29) (35) 1 (3%) (38) (32) 1 (3%) (30) 7 (23%) (32) 3 (9%) 5 (16%) (31) 3 (10%) (47) 3 (6%) 1 (2%) 2 (4%) 3 (6%) 1 (2%) 5 (11%) (39) (41) 1 (2%) (3) 3 (100%) 50 3 14 32 1 49 (35) 1 (3%) 1 (3%) (37) 3 (8%) (39) (31) (34) 1 (3%) 3 (9%) (35) 5 (14%) 4 (11%) (36) 1 (3%) 2 (6%) (48) 1 (2%) 5 (10%) 1 (2%) 4 (8%) 1 (2%) 1 (2%) 1 (2%) (42) 1 (2%) (46) 1 (2%) Lesions in Male Mice 157 Table C4 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 m g/m 3 Cardiovascular System Heart Thrombosis Coronary artery, mineralization Myocardium, degeneration, focal Myocardium, fibrosis, focal Endocrine System Adrenal gland Spindle cell, hyperplasia Adrenal gland, cortex Atrophy Hyperplasia, focal Vacuolization cytoplasmic, focal Parathyroid gland Cyst Pituitary gland Cyst Pigmentation, lipofuscin Thyroid gland Cyst Follicular cell, hyperplasia General Body System None Genital System Epididymis Inflammation, suppurative Preputial gland Dilatation Inflammation Prostate Inflammation, suppurative Epithelium, hyperplasia Seminal vesicle Inflammation, suppurative Testes Aspermatogenesis, diffuse Atrophy, diffuse Hypospermia Inflammation, suppurative Seminiferous tubule, degeneration, focal H em atopoietic System Bone marrow Hyperplasia Myelofibrosis Myeloid cell, hyperplasia (45) 1 (2%) (43) 38 (88%) (43) 1 (2%) (25) 3 (12%) (44) 1 (2%) 1 (2%) (45) 2 (4%) 4 (9%) (46) 1 (2%) 1 (2%) 1 (2%) (46) 37 (80%) (46) 1 (2%) 3(7% ) (21) 1 (5%) (44) (46) 1 (2%) 8 (17%) (39) 1 (3%) (8) 7 (88%) 3 (38%) (40) 3 (8%) (41) (43) 1 (2%) 3 (7%) {40) 4 (10%) 2 (5%) 4 (10%) (39) 1 (3%) (6) 6 (100%) (43) 7 (16%) 1 (2%) (43) 2 (5%) (44) 2 (5%) 1 (2%) 4 (9%) (42) 1 (2%) 2 (5%) 7 (17%) (49) 1 (2%) (47) 35 (74%) (47) 4 (9%) (26) (46) 1 (2%) (45) 1 (2%) 8 (18%) (42) (8) 8 (100%) 1 (13%) (44) 4 (9%) (39) 1 (3%) (45) 1 (2%) 1 (2%) 1 (2%) (43) 1 (2%) 1 (2%) 158 Taie, NTP TR 421 Table C4 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Talc (continued) 0 0 mg/m 6 mg/m3 18 m g/m3 H em atopoietic System (continued) Lymph node iliac, hyperplasia, lymphoid Iliac, hyperplasia, plasma cell Lumbar, hyperplasia, lymphoid Lumbar, hyperplasia, plasma cell Pancreatic, inflammation, granulomatous Renal, depletion lymphoid Renal, hyperplasia, lymphoid Lymph node, bronchial Abscess Hyperplasia, histiocytic Hyperplasia, histiocytic, lymphoid Hyperplasia, lymphoid Infiltration cellular, mixed cell Inflammation, acute Follicular, necrosis Lymph node, mandibular Hyperplasia, histiocytic Hyperplasia, lymphoid Follicular, necrosis Lymph node, mediastinal Hyperplasia, histiocytic Hyperplasia, lymphoid Lymph node, mesenteric Depletion lymphoid Hyperplasia, lymphoid Infiltration cellular, mixed cell Inflammation, granulomatous Thrombosis Follicular, necrosis Spleen Hematocyst Hematopoietic cell proliferation Hyperplasia, lymphoid Hyperplasia, roast cell Inflammation, granulomatous Lymphoid follicle, depletion lymphoid Lymphoid follicle, necrosis Thvmus Cyst Hyperplasia, lymphoid Inflammation, granulomatous Necrosis Cortex, depletion lymphoid Epithelial cell, hyperplasia, focal Integumentary System Skin Abscess Alopecia Inflammation, acute Ulcer, focal (45) 1 (2%) 1 (2%) 1 (2 7 c) (32) 1 (3 7 c ) 3 (97 c) 3 (97 c ) 1 (3%) 1 (37 c ) (23) (9) 1 (11%) (36) 1 (3%) 4 (11%) 18 (50%) (44) 6 (14%) 3 (7%) 2 (5%) (34) 3 (9%) 1 (3%) 6 (18%) 1 (3%) (44) 1 (2%) (46) 1 (2%) 1 (2%) 1 (2%) (39) 32 (82%) 1 (3%) 10 (26%) 1 (3%) (23) 1 (4%) (10) 1 (10%) 2 (20%) (39) 3 (8%) 20 (51%) 1 (3%) 6 (15%) (44) 1 (2%) 7 (16%) 2 (5%) 1 (2%) 2 (5%) 5 (11%) (33) 2 (6%) 1 (3%) 10 (30%) (45) 1 (2%) 2 (4%) 2 (4%) (48) 1 (2%) 1 (2%) (44) 1 (2%) 42 (95%) 23 (52%) 3 (7%) (19) 1 (5%) 1 (5%) (7) 2 (29%) (40) 2 (5%) 6 (15%) 13 (33%) 1 (3%) 2 (5%) (47) 10 (21%) 3 (6%) 1 (2%) 5 (11%) 1 (2%) (40) 1 (3%) 1 (3%) 2 (5%) 8 (20%) (48) 1 (2%) Lesions in Maie Mice 159 Table C4 Summary or the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Talc (continued) 0 mg/m3 6 m g/m 3 18 mg/m3 M usculoskeletal System Bone Rib, cartilage, fracture healed Nervous System Brain Mineralization, focal (46) 1 (2%) (46) 37 (80%) (47) (47) 39 (83%) (49) (48) 38 (79%) Respiratory System Larynx Inflammation, acute Lung Congestion Hyperplasia, macrophage Inflammation, chronic active Thrombosis Alveolar epithelium, hyperplasia, focal Peribronchiolar, inflammation, chronic active Perivascular, inflammation, suppurative Nose Cytoplasmic alteration, focal Erosion, focal Inflammation, acute (42) 1 (2%) (45) 3 (7%) 3 (7%) 1 (2%) 1 (2%) (45) 5 (11%) 1 (2%) 4 (9%) (41) (47) 1 (2%) 46 (98%) 16 (34%) 1 (2%) (46) 23 (50%) 1 (2%) 4 (9%) Special Senses System Ear Inflammation, granulomatous (1) 1 (100%) Urinary System Kidney Casts protein Cyst Hydronephrosis Inflammation, suppurative, focal Metaplasia, osseous, focal Nephropathy, chronic Capsule, inflammation, suppurative Pelvis, inflammation, suppurative Urinary bladder Dysplasia, focal Inflammation, chronic active Ulcer, focal Transitional epithelium, hyperplasia (45) 1 (2%) 2 (4%) 3 (7%) 3 (7%) 3 (7%) 2 (4%) (43) 1 (2%) 6 (14%) (46) 1 (2%) 5 (11%) 3(7% ) 5 (11%) (38) 5 (13%) 1 (3%) a Number of animals examined microscopically at site and number of animals with lesion (46) 1 (2%) (48) 1 (2%) 48 (100%) 40 (83%) 1 (2%) (47) 40 (85%) 2 (4%) 7 (15%) (48) 3 (6%) 2 (4%) 1 (2%) 1 (2%) (43) 2 (5%) 1 (2%) 160 Talc, NTP TR 421 T 161 APPENDIX D SUMMARY LESIONS IN FEMALE MICE IN THE 2-YEAR INHALATION STUDY OF TALC T a b l e D1 T a b l e D2 T a b l e D3 T a b l e D4 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of T a l c ...................................................................................... Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of T a l c ...................................................................................... Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of T a l c ...................................................................................... Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of T a l c ...................................................................................... 163 166 184 188 162 Talc, NTP TR 421 O Lesions in Female Mice Table Dl summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Talc* 0 mg/m3 6 mg/m3 18 mg/m3 D isposition Summ ary Animals initially in study Early deaths Moribund Natural deaths Survivors Terminal sacrifice Missing Culled Animals examined microscopically Alim entary System Esophagus Gallbladder Intestine large, cecum Leiomyoma Intestine large, colon Leiomyosarcoma Intestine small, ileum Liver Hemangioma Hepatocellular carcinoma Hepatocellular adenoma Mesentery Pancreas Salivary glands Hemangioma Stomach, glandular Cardiovascular System Heart Alveolar/bronchiolar carcinoma, metastatic, lung Endocrine System Adrenal gland Spindle cell, adenoma Adrenal gland, cortex Adenoma Adrenal gland, medulla Pheochromocytoma malignant Pituitary gland Adenoma Carcinoma Thyroid gland Follicular cell, adenoma 50 2 17 30 1 46 (43) (31) (35) (38) (33) (46) 7 (15%) 5 (11%) (2) (42) (46) 1 (2%) (45) (46) (46) 1 (2%) (46) 1 (2%) (41) 1 (2%) (42) 5 (12%) (43) 1 (2%) 50 4 21 23 1 1 48 (47) (28) (29) (33) 1 (3%) (27) (46) 1 (2%) 5 (11%) 1 (2%) (39) (48) (39) (48) 1 (2%) (45) (44) (43) (42) 4 (10%) 2 (5%) (47) 2 (4%) 50 4 21 25 50 (48) (29) (34) 1 (3%) (32) (31) (50) 4 (8%) 4 (8%) (44) (50) (46) (50) (50) (50) (45) (48) 2 (4%) (49) 2 (4%) 163 164 Talc, NTP TR 421 Table D I Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 General Body System Tissue NOS Fibrosarcoma Hemangioma Hemangiosarcoma G enital System Ovary Adenocarcinoma, metastatic, uterus Adenoma Cystadenoma Luteoma Uterus Adenocarcinoma Carcinoma adenosquamous Hematopoietic System Bone marrow Lymph node Lymph node, bronchial Adenocarcinoma, metastatic, kidney Adenocarcinoma, metastatic, uterus Alveolar/bronchiolar carcinoma, metastatic, lung Lymph node, mandibular Lymph node, mediastinal Adenocarcinoma, metastatic, kidney Alveolar/bronchiolar carcinoma, metastatic, lung Lymph node, mesenteric Spleen Hemangiosarcoma Thymus Alveolar/bronchiolar carcinoma, metastatic, lung Integum entary System Mammary gland Fibrosarcoma M usculoskeletal System Bone Vertebra, alveolar/bronchiolar carcinoma, metastatic, lung Nervous System Spinal cord Thoracic, ganglioneuroma (4) 1 (25%) 1 (25%) (38) 1 (3%) 1 (3%) 2 (5%) (44) 1 (2%) (41) (46) (38) 1 (3%) (35) (13) (35) (45) (40) (41) (46) (1) (43) 1 (2%) 1 (2%) (45) (43) (46) (37) 1 (3%) 3 (8%) (38) (17) 1 (6%) 1 (6%) (31) (44) (40) 2 (5%) (45) (48) 1 (2%) (2) 1 (50%) 1 (50%) (46) (49) 1 (2%) (45) (49) (43) (36) (14) (37) (50) 1 (2%) (41) (48) 1 (2%) (50) (1) 1 (100%) Lesions in Female Mice Table D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 Respiratory System Lung Adenocarcinoma, metastatic, kidney Alveolar/bronchiolar adenoma Aiveolar/bronchiolar carcinoma Hemangiosarcoma, metastatic, tissue NOS Hepatocellular carcinoma, metastatic, liver Trachea (46) 3 (7%) 2 (4%) 2 (4%) (40) (48) 1 (2%) 2 (4%) 4 (8%) 2 (4%) (36) Special Senses System Harderian gland Adenocarcinoma Adenoma (2) 2 (100%) (2) 1 (50%) Urinary System Kidney Adenocarcinoma Hepatocellular carcinoma, metastatic, liver Urinary bladder (46) 1 (2%) (44) (46) 1 (2%) (40) System ic Lesions Multiple organs*1 Lymphoma malignant histiocytic Lymphoma malignant lymphocytic Lymphoma malignant mixed Lymphoma malignant undifferentiated cell (46) 2 (4%) 3 (7%) 2 (4%) (48) 3 (6%) 4 (8%) Neoplasm Summ ary Total animals with primary neoplasms0 Total primary neoplasms Total animals with benign neoplasms Total benign neoplasms Total animals with malignant neoplasms Total malignant neoplasms Total animals with metastatic neoplasms Total metastatic neoplasms 31 42 18 23 19 19 3 5 26 33 9 13 19 20 5 13 b Number of animals examined microscopically at site and number of animals with lesion Number of animals with any tissue examined microscopically Primary neoplasms: all neoplasms except metastatic neoplasms (50) 2 (4%) 1 (2%) 1 (2%) (45) (1) 1 (100%) (50) (41) (50) 1 (2%) 3 (6%) 2 (4%) 2 (4%) 21 31 10 13 15 18 1 1 165 166 Talc, NTP TR 421 T able D2 Individual Animal Tumor Pathology of Female Mice in the 2*Year Inhalation Study of Talc: 0 mg/m3 Number o f Days on Study 044455555566666777777 326800045948889222222 0657569428 10362399999 C arcass ID Number 555344344455454433333 3037] 2817702904978888 30 46 70255358 7 766 7 1469 111111111111111111111 Alim entary System Esophagus Gallbladder Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum intestine small, ileum Intestine small, jejunum Liver Hepatocellular carcinoma Hepatocellular adenoma Mesentery Pancreas Salivary glands Hemangioma Stomach Stomach, forestomach Stomach, glandular + + M + + M M + + + + + + + + + + + + + 4- A M A M A M A + A A A A A + A A + + + + 4- A + + A A A + + A + A + + + + + + + + + 4- A + A A A A A A A + A + A + A + + + + + 4- A + A A A A + + A + A + + + + + + M + + 4- A M + MMA + + MA A M+ + + + + + + + M A + A A A A A + A A A A A + + + + + + + 4- A + AAAAAAAAAAA + + A + + + + M A -f A A A A A A A A A A A + + + + + + 4- 4- A + A A A A A + A A A A A + A + + 4- + + 4- + + + + + + -f + + + + + + + + + + + 4- 4- XX XX X X + + + A + A A + + + + I + + + + + + + 4- 4- + + + + + + + + + + + + + + + + + + 4- 4- + + + + + A + + + + + + + + + + + + + 4- 4+ + + + + A + + + + + + + + + + + + + 4- 4+ + + + + A + + + + + + + + + + + + + + 4- Cardiovascular System Heart + + + + + + + + + + + + + + + + + + + + 4- Endocrine System Adrenal gland Spindle cell, adenoma Adrenal gland, cortex Adenoma Adrenal gland, medulla Pheochromocytoma malignant Islets, pancreatic Parathyroid gland Pituitary gland Adenoma Thyroid gland Follicular cell, adenoma + : Tissue examined microscopically A: Autolysis precludes examination 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 44- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4- M 4- 4- 4- i i 4- 4- 4- 4- 4- + 4- 4- 4- 4- 4- M 4- A A M 4- i M i M i M 4- 4- 4- 4- 4- 4- 4- M M 4- M 4- 4- M 4- M I 4- M 4- 4- 4- M M M i M 4- M 4- 4- 4- M M 4- 4- 4- 4- i 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- X XX X A 4- 4- 4- 4- 4- M 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4- 4- 4- M: Missing tissue 1: Insufficient tissue X: Lesion present Blank: Not examined Lesions in Female Mice 167 Table D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 0 mg/m3 (continued) Num ber of Days on Study 77 777777 7 77 77 77 77 77 777 7 77 2333 333333 333 3333 33 33 333 3 90000000 11111122222333333 C arcass ID Number 3444444444444455555445555 9 1144444 57777 700 00 208 33 33 0 180 123500 1489 12499900 189 11] 1111111111111111111111 Total T issu e s/ T um ors Alim entary System Esophagus Gallbladder Intestine large Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Hepatocellular carcinoma Hepatocellular adenoma Mesentery Pancreas Salivary glands Hemangioma Stomach Stomach, forestomach Stomach, glandular + + + + + + + + + + + 4- + + + + + + + + + + + + + ++++++I ++++++++++++++++++ +++++++++++++++++++++++++ + ++ ++ + ++ + +++ ++ + + ++ + ++ ++ + + +++++++++++++++++++++++++ + + + + + + + + + + + M+ + + + + + + + M+ + + + ++ +++++ +++++ ++ +++ +++ ++ ++ + + MM+ + + + + + + + + + + MM+ + + M+ + + + + ++++++++++++ +++ +A++++++++ + + + + + + + + + + + M+ + + + + + + + + + + + + +++++++++++++++++++++++++ XX XX X X + +++++++++++++++++++++++++ ++ ++++ +++ ++++ +++ ++ ++ ++ +++ X +++++++++++++++++++++++++ +++++++++++++++++++++++++ + + ++ ++ + ++ + +++ + + + ++ + ++ + + ++ 43 31 40 35 38 34 35 27 33 33 46 7 5 2 42 46 1 45 45 45 Cardiovascular System Heart +++++++++++++++++++++++++ 46 Endocrine System Adrenal gland Spindle cell, adenoma Adrenal gland, cortex Adenoma Adrenal gland, medulla Pheochromocytoma malignant Islets, pancreatic Parathyroid gland Pituitary gland Adenoma Thyroid gland Follicular cell, adenoma +++++++++++++++++++++++++ X +++++++++++++++++++++++++ X + + + + + + + + + + + + + + + + + + + + + + + +M X + + M+ 1 + I + 1 1 + I I + + + + MI + I + + M+ + + + M+ + M+ I + M+ MM+ + I M+ + + MMM+ + + + + + + + + + + + + + + + + + + + + ++ + + + X + + + + + + + + + + + + + M+ + + + + + + + + + + X 46 1 46 1 41 1 25 23 42 5 43 1 168 Talc, NTP TR 421 Table D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 0 mg/mJ (continued) Number of Days on Study 044455555566666777777 326800045948889222222 065756942810362399999 Carcass ID Number 555344344455454433333 303712817702904978888 3 0 4 6 7 0 2 5 5 3 5 8 7 7 6 6 7 1 469 111111111111111111111 G eneral Body System Tissue NOS Fibrosarcoma Hemangioma Genital System Ovary Adenocarcinoma, metastatic, uterus Adenoma Luteoma Uterus Adenocarcinoma + + + + + + + 1 + + 1 M+ + + + + + + + + Ml + + + + + + + + + + + + + + + + + + + Hematopoietic System Bone marrow Lymph node Lymph node, bronchial Adenocarcinoma, metastatic, uterus Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Thymus + + A + AA + AA + + + + + + + + + + + + + .+ + + + + + + + + + + + + + + + + + + + M+ + + + M+ MM+ + + M+ + + + + + + + + + M+ + + + + + M + + + M + + + + M M + M M + M M M + M + M M M + M + + MM+M + M + + MMM+MM + + + + I + + +M+ + + + ++++A +++++++++++++++ M M + + + M + + + + + + + M + M + + -f + + Integumentary System Mammary gland Skin A + + + +A + I + + +M + + + + + + + + + ++ +++++ ++ ++++ + +++ +++ + Musculoskeletal System Bone ++ +++++ +++++ +++ ++ ++ ++ Nervous System Brain +++++++++++++++++++++ Lesions in Female Mice 169 T a b l e D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 0 mg/m3(continued) N um ber of Days on Study 7777777777777777777777777 2333333333333333333333333 90000000 11111122222333333 C arcass ID N um ber 3444444444444455555445555 9 114444457777700002083333 0 180 12350 0 1489 1249990 0 18 9 1 1 111 11 1111 11 11 11 11 11 111 1 Total Tissues/ T um ors General Body System Tissue NOS Fibrosarcoma Hemangioma +++ X + X 4 1 1 Genital System Ovary Adenocarcinoma, metastatic, uterus Adenoma Luteoma Uterus Adenoca rcinoma + + + M+ + + M+ + + M+ + M+ + + + ] + + + + + X X XX +++ ++++ ++ +++ +++ +++ ++ + ++ ++ X 38 1 1 2 44 1 Hematopoietic System Bone marrow Lymph node Lymph node, bronchial Adenocarcinoma, metastatic, uterus Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Thymus +++++++++++++++++++++++++ +++++++++++++++++++++++++ M+ + + + + + + + I + + + + + + + + + I + + + + + X + + + + MM+ + + + + + + + MM+ + + M+ M+ + + MMMMM+ MMMM+ + MMMMMMM+ MMMM+ M+ + + + + + + + + + + + + + + + + + + + + MM+ +++++++++++++++++++++++++ +++++++++++++++++++++++I + 41 46 38 1 35 13 35 45 40 Integum entary System Mammary gland Skin M usculoskeletal System Bone +++++1 +++++++++++++++++++ +++++++++++++++++++++++++ 41 46 +++++++++++++++++++++++++ 46 Nervous System Brain +++++++++++++++++++++++++ 46 170 Talc, NTP TR 421 Table D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 0 mg/m3 (continued) N um ber of Days on Study 044455555566666777777 326800045948889222222 0657569428 10 362 399999 C arcass ID N um ber 555344344455454433333 3037 128 17702904978888 304670 25535877667 1469 111111111111111111111 Respiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma Hepatocellular carcinoma, metastatic, liver Nose Trachea 4* + + + + A I + 4* + + + + + + + + + + + 4+ + + + + + + + 4* + + + + + + + + + + + 4- X X XX + + 4* + + + + + + + + + + + + + + + + 4- 4A + A + M A A + + + + + + + + + + + + + 4- Special Senses System Harderian gland Adenoma + X U rinary System Kidney Hepatocellular carcinoma, metastatic, liver Urinary bladder + + + + + + + + + + + + + + + + + + + 4- 4- X M + + + + A + + + + + + + + + + + + + 4- 4- Systemic Lesions Multiple organs Lymphoma malignant lymphocytic Lymphoma malignant mixed Lymphoma malignant undifferentiated cell type ++ + + + + + + + + + + + + + + + + 4- 4X X* Lesions in Female Mice 171 Table D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 0 mg/m} (continued) Number o f Days on Study 77 77 77 77777 7777 777 77 777 77 233 333 3333 3333 333 33 33 3333 90000000 11111122222333333 Carcass ID Number 3444444444444455555445555 9 11444445777770000 208 33 33 0 180 12 3500 1489 124 99 900 189 1111111111111111111111111 Total Tissues/ Tumors Respiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma Hepatocellular carcinoma, metastatic, liver Nose Trachea + + + + + + + + + + + + + M+ I + + + + + + + + + ++++ +++++ +++++ ++ +++ ++ +++ + XX X ++++++++++++++++++++ + ++ + + + + + + + + + + + + + + + + + M+ + + + + + + 42 46 3 2 2 46 40 Special Senses System Harderian gland Adenoma +2 X2 Urinary System Kidney Hepatocellular carcinoma, metastatic, liver Urinary bladder +++++++++++++++++++++++++ +++++++++++++++++++++++++ 46 1 44 Systemic Lesions Multiple organs Lymphoma malignant lymphocytic Lymphoma malignant mixed Lymphoma malignant undifferentiated cell type +++++++++++++++++++++++++ X XX X X 46 2 3 2 172 Talc, NTP TR 421 Table D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 6 mg/m* N um ber of Days on Study 004455555566666666666777 2929034556 1224467789900 1 0 22 10 48 4 9 48 18 15 5 6 8 6 2 9 9 9 2 C arcass ID N um ber 10 0 10 1 1 10 10 10 0 1 10 1 10 0 1 10 15 115474 1722583057386 142 7 5 6 5 0 7 6 8 9 5 2O6 16 8 8 19 3 0 8 0 0 111111111111111111111111 Alim entary System Esophagus Gallbladder Intestine large Intestine large, cecum Intestine large, colon Leiomyosarcoma Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Hemangioma Hepatocellular carcinoma Hepatocellular adenoma Pancreas Salivary glands Stomach Stomach, forestomach Stomach, glandular + + + + M + + + + + + + + + + + 4- + + 4- + + + + A MMMA A + + + A A A A A A A A A MA A + A + A + A A A + A + + A + + A A A + A 4- A 4- A + A + AAAAAAA+ + AA + AAA+ AAAAA+ A+ AAAAA + A + + A + + AAA+ A + A + A + A + A + A A A MMI AAAAAAA+ AAAAAAA+ A A AAAA A + AAAAAAA+ + + + A+ + + + MA + MMA A MA + A A A + + AA + AAA + AAAAA + + AA+ AAAAAAAAA + AA+ AAAAAAAAA+ + AA + AAA + AAAAA+ + + + + + + + + A+ + + + + A+ A+ A+ A+ A+ + 4- XX + + + AA + + + + + + + I M+ + A + A + A + + I + + + + + + + + + + + + + + + + + + + + 4- + + + + + + A+ + + + + + + + + + + + A+ + + + + + + + + + A+ + + + + + + + + + + + A+ + + + + + + A + + AA + + + + AA + + A + + A + + + + + A + Cardiovascular System Heart Alveolar/bronchiolar carcinoma, metastatic, lung ++++++++++++++++++++++++ X Endocrine System Adrenal gland Adrenal gland, cortex Adrenal gland, medulla Islets, pancreatic Parathyroid gland Pituitary gland Adenoma Carcinoma Thyroid gland Follicular cell, adenoma + ++A++ ++++ ++++ ++A++ +++ + + + + + A + + + + + + + + M+ + + A + + + + + + + +++A+++++++++ +++A+++ ++++ MI + A A I I + + MI MMM+ + A + MI MMI i I + + A MM+ + MMM+ + M+ + MMI + MI + M M+ + + + + + + + + + + I + + + M+ + MI + + + XX +++A++ ++ + ++ ++ ++ + +++ + ++ + + Lesions in Female Mice 173 Table D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc; 6 mg/m3 (continued) Num ber of Days on Study 777777777777777777777777 1222233333333333333 33333 4999900000 11111222222333 Carcass ID Num ber 000 0 10 000 10 0 11 1111 11 10 11 8 2230 557708 80 1344 66 674 78 5490624687799 18456892780 111111111111111111111111 Total Tissues/ T um ors Alim entary System Esophagus Gallbladder Intestine large Intestine large, cecum Intestine large, colon Leiomyosarcoma Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Hemangioma Hepatocellular carcinoma Hepatocellular adenoma Pancreas Salivary glands Stomach Stomach, forestomach Stomach, glandular ++ 4 ++++ +4 4 ++ + +++ ++ ++ +++ + A+4 +++ 4 4 ++ + 4 4 4 + 4 + ++ +++ ++ A+++ 4 +++ ++ 4 4 + ++4 4 + ++++ + 4 A4 +++++ 4 4 + ++4 4 + 4 4 ++ ++ ++4 A+4+ +++++4 4+4 +4 + 4 ++ ++++4 X A + + M4 + + + 4 4 MI 4 + + + + + + M+ + + M A+++++4 4++4 +++44 4++ ++++4 A + + + + 4 + 4 + 4 4 + + + + 4 I + + + + + MM A+++4++444A4++44 +++++ ++4 A + 4 + + + + + + + 4 + + 4 4 4 + + M+ + + + 4 ++ 4 ++++ 4 4+ +++ 4+ 4 + + + ++ ++4 X XX X X A4+4 ++++++ +++ 4++++++++ +4 ++4++4+44+4+++++4++++++4 A+4 ++++4 ++ +++ ++ 4 +++++++4 A++4 +++4+4 4 +++++ 4++ ++++4 A 4 4 + + 4 4 4 + + 4 4 4 4 + + + + + + + + 4- 4 47 28 34 29 33 1 23 29 25 27 28 46 1 5 1 39 48 45 45 39 Cardiovascular System Heart Alveolar/bronchiolar carcinoma, metastatic, lung +4 444 444 44 4444 44 44 +44 4 44 48 1 Endocrine System Adrenal gland Adrenal gland, cortex Adrenal gland, medulla Islets, pancreatic Parathyroid gland Pituitary gland Adenoma Carcinoma Thyroid gland Follicular cell, adenoma A444 4444 444 4444 44 44+4 44 4 A+4444 444 +4 444 +4 44 4 ++4 44 A4+4 44++4 4 44 +i ++4i 4+4 44+ A i + Mi 4 i 4 Mi + 4 i 4 i i 4 4 4 4 4 4 4 4 MMi I 4 M4 + MMM4 M4 MMi MM4 i 4 M+ 4 4 4 4 4 4 4 4 4 + + 4 + 4 4 + 4 4 M4 4 4 4 + X XX X 4444444444444444444444+4 X .X 45 44 43 20 18 42 4 2 47 2 174 Talc, NTP TR 421 T able D2 Individual Animal Tumor Pathology of Female Mice in the 2-'Year Inhalation Study of Talc: 6 mg/m3 (continued) Number of Days on Study 004455555566 666666666777 2929034556 12 24467789900 1 0 2 2 10 48 49 48 18 15 5686 2 99 92 Carcass ID Number 100 10 1 1 10 10 100 1 10 1 100 1 10 15 1154741722583057386 142 756 50 768 9S20 6 16 88 1930 80 0 11 11 111 111 111 1111 1 111 1 11 General Body System Tissue NOS Genital System Ovary Adenoma Cysladenoma Uterus + + + A + + + + + + + + M + + + A + + 4 + 4 + 4 + + + A + + + + + + + + + + A + A 4- 4- 4- 4- 4 4- 4 Hematopoietic System Bone marrow Lymph node Lymph node, bronchial Adenocarcinoma, metastatic, kidney Alveolar/bronchiolar carcinoma, metastatic, lung Lymph node, mandibular Lymph node, mediastinal Adenocarcinoma, metastatic, kidney Alveolar/bronchiolar carcinoma, metastatic, lung Lymph node, mesenteric Spleen Thymus Alveolar/bronchiolar carcinoma, metastatic, lung 4 + 4 A 4 + A 4- + A + + + A + + + + + + + 4- 4- 4- M 4 A + + + + 4- + + + + + + + 4 4 4 + 4- 4- 4 + 4- M M M + + + M i + + M + + M + + 4 4- M 4- 4- 4- 4- i X X XX M + M M M + + 4- + + + + + M + 4- + 4- + + M + + 4- M M M + M M M M M M M M + + + M M M 4- i M + 4- M X X M M A A A + M + + + + + + A + + A M M + 4- 4 4- 4A + + A + + + + + + + + + + + 4- A 4- 4- 4- 4- 4- 4- 4M M + + + I + 4* + + 1 + + + + 4- M M M + i + + 4- XX Integum entary System Mammary gland Skin Musculoskeletal System Bone Vertebra, alveolar/bronchiolar carcinoma, metastatic, lung Nervous System Brain + + + A + + + + + + + + + + + + M + + + 4- + + 4+ + + + + + + + + + + + + + 4- M + + 4- 4- 4- 4- 4- + + + + + + + + + + + + + + + 4- 4- 4- 4 + 4- + 4- 4X ++++++++++++++++++++++++ Lesions in Female Mice 175 Table D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 6 mg/m3 (continued) N um ber o f D ays on Study 777 7 7 77 7 77 77 7 77 7 77 7 77 7 77 12222333 33 333 333 33 3333 33 4 9 99 9 00 00 0 1 1 11 12 2 22 22 3 3 3 C arcass ID Num ber 0 0 0 0 10 0 0 0 10 0 1 1 1 1 1 11 1 10 1 1 8223055770880 134 4666 7478 54906246 87799 184568927 80 11 1 11 11 11 1 111 1 11 1 11 1 11 1 1 T otal T issues/ T um ors General Body System Tissue NOS + 1 Genital System Ovary Adenoma Cystadenoma Uterus + + + M + + + + + + + + + + + + + + + + + M+ + X X + + + + + 4" + + + + + + + + + + + + + + + + + + 43 1 1 45 H em atopoietic System Bone marrow Lymph node Lymph node, bronchial Adenocarcinoma, metastatic, kidney Alveolar/bronchiolar carcinoma, metastatic, lung Lymph node, mandibular Lymph node, mediastinal Adenocarcinoma, metastatic, kidney Alveolar/bronchiolar carcinoma, metastatic, lung Lymph node, mesenteric Spleen Thymus Alveolar/bronchiolar carcinoma, metastatic, lung A + + + 4 + + + + + + . + + + + + + 4 4 4- 4 4- 4 + + + + +4 + ++ + + + + + + + 4- 4- + 4- 4- 4- 4- + + I + + 4 + + M + + + + + + + + + + + + 4- 4- 4 M M + + + + + + M + + + + + + M + + 4- 4- + 4- 4MM+ MM + M+ + + + + + + MMMMM+ MMMM A + + + + + 1 M + + + M + I + + M 4* 4- M 4- + 4- 4A 4 + + + + + + + + + + + + + + + 4- + + 4- + 4- 4+ + + + 4 + + + + + + + + + + + + 4- + + 4- 4- 4- 4- 43 46 37 1 3 38 17 1 1 31 44 40 2 Integum entary System Mammary gland Skin + + + + + + + I + + + + + + + + + + 4- 4- + + 4 4+ + + + + + + + + + + + + + + + + 4- + + + 4- 4- 4- 45 4b M usculoskeletal System Bone Vertebra, alveolar/bronchiolar carcinoma, metastatic, lung 4*4-4-4*4-4-4-4-4-4-4-4-4-4-4-4-4-4-4-4-4-4"4-4- 48 1 Nervous System Brain +++++++ +++++++++++++++++ 48 176 Talc, NTP TR 421 Table D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 6 mg/m3 (continued) Num ber o f Days on Study 004455555566 66 6666666777 29 2903 4556 12 24 46 77 89 900 1 0 22 104849 48 18 1 5 56 86 299 9 2 C arcass ID N um ber 100 10 1 110 10 100 110 1100 110 15 115474 1722583057 386 142 7565076895206 16 88 1930800 111111111111111111111111 R espiratory System Larynx Lung Adenocarcinoma, metastatic, kidney Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma Hepatocellular carcinoma, metastatic, liver Nose Trachea + + +AAI + + + +A+ + + + + + + + + + + + + ++++++++++++++++++++++++ X X X XX X X + + + + A + + + + + + + + + + + + + + + + + 4- + + + + A A + M+ + A A + A A + + M+ M+ + + + + Special Senses System Eye Harderian gland Adenoma + + U rinary System Kidney Adenocarcinoma Urinary bladder Systemic Lesions Multiple organs Lymphoma malignant lymphocytic Lymphoma malignant mixed + + +A++ ++ ++ ++ + + + +A+ + + + + + + X A A + A + + + + + A + + + A A + A + + + + + + 4- + ++ ++ + +++++ ++++ +++ + ++ +++ XX Lesions in Female Mice 177 T a b l e D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 6 mg/m3 (continued) N um ber of Days on Study 777777777777777777777777 122223333333333333333333 4999900000 11111222222333 C arcass ID N um ber 0 000 10 000 10 0 11 111 111 10 1 1 8223055770880 13446667478 5490624687799 18456892780 1 11 1111 11 11 111 11 111 111 11 Total Tissues/ T um ors Respiratory System Larynx Lung Adenocarcinoma, metastatic, kidney Alveolar/bronchiolar adenoma Alveolar/bronchioiar carcinoma Hepatocellular carcinoma, metastatic, liver Nose Trachea A+++++++++++++++++++++++ ++++++++++++++++++++++++ X X A++++++++++++++ ++++++++ + A + + + + + + + + + + + + + + + + + + + + + MM 43 48 1 2 4 2 46 36 Special Senses System Eye Harderian gland Adenoma 1 +2 X1 U rinary System Kidney Adenocarcinoma Urinary bladder ++++++++++++++++++++++++ A + + + + + + + + + + + 4- + + + + + + + + + + + 46 1 40 Systemic Lesions Multiple organs Lymphoma malignant lymphocytic Lymphoma malignant mixed ++++++++++++++++++++++++ X X XXX 48 3 4 17 Talc, NTP TR 421 Table I)2 Individual Animai Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 18 mg/m1 Number o f Days on Study 0 0 0 0 0 0 0 4 5 5 5 5 5 5 5 6 6 66 666 7 7 7 222222270 145 56844566890 11 0 8 8 8 8 8 8 3 8 6 8 4 8 9 12 6 5 15 626 6 8 Carcass ID Number 2 12222 23 32 2333 23 232 2 2222 2 9900000440315672352236965 26 13 46 79629 63 00 8 22 786 66 79 1111111111111111111111111 A lim entary System Esophagus Gallbladder Intestine large Intestine large, cecum Leiomyoma Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Hepatocellular carcinoma Hepatocellular adenoma Pancreas Salivary glands Stomach Stomach, forestomach Stomach, glandular + + + + + M+ + M+ + + + + + + + + + + + + + + + A MMA A A MM+ MA A A A + A A + A A A + + A + AAA+ +AA+ +A +A+A+ A +AAA + +AA+ AAA+ AAA+ + A+AAA+ A+AAA+ + AA+ AAA+ AAAI + A +AAA+ AAAAA + +AA+ A A A + + A A MI M MA + A + A + MA M+ + MA + AAA +AAA + +A + A+ A + A + AAAA+AA+ A A A + A A A + + A MA A A + A A A A A A + A A + AAAAAAA + AA+ AAA+ A+ AAAA +AA + AAA + AAA+ AAAA+ A + AAAAAA + AA + ++ + ++++ + ++ +++ +++ + ++ + ++ + ++ X XX A + + + MA + + + A + + + A + + + + + + + + + A + +++++++++++++++++++++++++ + ++++++ ++++ + +++ +++ ++ ++++ + +++++++++++++++++++++++++ + + ++ +++ ++A++AI ++ + +A+ ++ +++ Cardiovascular System Heart +++++++++++++++++++++++++ Endocrine System Adrenal gland Adrenal gland, cortex Adrenal gland, medulla Islets, pancreatic Parathyroid gland Pituitary gland Adenoma Thyroid gland Follicular cell, adenoma +++++++++++++++++++++++++ + + ++ + +++ ++ ++++ ++ ++++ ++ +++ + I i M+ + I + + + + + + + + + + + + + + + + + + A + + I MA M+ + MM+ + I I M+ + + Mi I + A + + MM+ + MMMMM + + + + MM+ + I + MM+ + + + + + + MI + + + + + + + + + + + + + + + + + + + X + + M+ + + + + + + + + + + + + + + + + + + + + + General Body System Tissue NOS Hemangioma Hemangiosarcoma + X L esio n s in F em ale M ice 179 T a b l e D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 18 mg/m3 (continued) N um ber of Days on Study 7 777 77 77 77 777 77 77 7 77 7777 7 2222233333333333333333333 999990000 1111122222233333 C arcass ID N um ber 22 222 2 2222 22 33 3 33 33 3 222 33 123346688999 1122255558925 094800 16848979 46945869029 111 11 11111 11 11 11 1 111 11 1 11 Total T issu e s/ T um ors Alim entary System Esophagus Gallbladder Intestine large Intestine large, cecum Leiomyoma Intestine large, colon Intestine large, rectum Intestine small Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Hepatocellular carcinoma Hepatocellular adenoma Pancreas Salivary glands Stomach Stomach, forestomach Stomach, glandular 4 4 + 4 + + 4 4 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4 + 4 + + M 4 4 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4- 4- 4- 4- 4 + + + + 4 + 4 + 4- 4- 4- 4- 4- 4- 4- 4- 4 4- 4 4" 4- 4- 4- 4- + + 4 4 + + 4 + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- X 4 4 4 4 + 4 4 4 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + 4 4 + M 4 M + + 4- M 4- 4- M M 4- 4- i 4- 4- 4- 4- 4- 4- 4- 4 4 + 4 4 + + 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4 M 4 + 4 M + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- M 4- 4- 4 + 4 + + 4 4 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4 4 + + + 4 4 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4 4 + + + + 4 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- X XX XX + 4 + + + + + 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4 4 + + 4 4 + + 4- 4- 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4 + + + + 4 + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 44 + 4 + + + + 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- + + + + + 4 + + 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 48 29 36 34 1 32 27 34 27 31 31 50 4 4 44 50 50 50 46 C ardiovascular System Heart 4 4 + + 4 4 + 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 50 Endocrine System Adrenal gland Adrenal gland, cortex Adrenal gland, medulla Islets, pancreatic Parathyroid gland Pituitary gland Adenoma Thyroid gland Follicular cell, adenoma 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4 4 4- 4- 4- 4- 4- 4 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4 4 4 4 4 4- 4- i 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4 4- i M i 4- i 4- l 4- 4- 4- M i 4- i 4- 4- i i 4- i 4- 4 M i 4- 4- 4- i M M M 4- i 4- 4- M M 4- 4- M i M 4- 4- 4 + 4- 4- 4- 4- 4- 4- 4- 4- 4 4- 4- 4- 4- 4- 4- 4 4- 4- 4- 4- 4- 4 44 Mi M4 44 X 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4- 4 4 4 XX 50 50 45 23 25 48 2 49 2 General Body System Tissue NOS Hemangioma Hemangiosarcoma + X - 2 1 1 180 Talc, NTP TR 421 Table D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study or Talc: 18 mg/m3 (continued) Num ber o f D ays on Study 0000000455555556666666777 222222270145568445668901 1 0888888386848912651562668 C arcass ID N um ber 2122222332233323232222222 9900000440315672352236965 2613467962963008227866679 1111111111111111111111111 Genital System Ovary Uterus Carcinoma adenosquamous + + + + + + + + + + + + + + + 1 + M+ + M+ + + + ++M+ + + + + + + + + + + + + + + + + + + + + + H em atopoietic System Bone marrow Lymph node Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Hemangiosarcoma Thymus Integum entary System Mammary gland Fibrosarcoma Skin + + + + + + +A+AA+ +AA+ + + + + + + + + + + M+ + + + + + + + + + + + + + + + + + + + + + + MMM+ MMM+ + 1 + + + + + + + + + + + + + + + + MMM+ + + + + + + + MM + + + + + + + + + + + MMMMMMMMMM+ MMMMMM + + + M + M+ + MMA + MMM+ + A + A + + + M+ + + + M + M + + + ++++ + ++ + +++ ++ + ++ + + +++ + ++ MM+ M+ + MM + + + + + + + + + + + + M+ + MI + + M + + + + + + + 4-I + + + + + + + + + + + + + + +++ + ++ +++++ +++ ++ ++ ++ + +++ M usculoskeletal System Bone +++++++++++++++++++++++++ Nervous System Brain Spinal cord Thoracic, ganglioneuroma +++++++++++++++++++++++++ R espiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma Hemangiosarcoma, metastatic, tissue NOS Nose Trachea + +M + + + ++ + + + ++ + + + ++ + + ++ + + + + + + ++ ++ + + + + + + + + ++ + + + + + + ++ + ++ + + + + + + + X X X ++ + + + + + + ++ + ++ + + + + + + + + + + + + + + + + +A + M+ + + + + + + Lesions in Female Mice 181 Table D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 18 mg/m3 (continued) N um ber of Days on Study 7777777777777777777777777 2222233333333333333333333 999 9900 00 11 1 11222 22 2 3 33 33 C arcass ID N um ber 2222222222223333333322233 123346688999 1122255558925 094800 16848979 4694 5869029 1 111 1 111 1111 11 11 111 1 111 11 Total T issu e s/ T um ors Genital System Ovary Uterus Carcinoma adenosquamous + + + + + + + + + + + + + + + + M+ + + + + + + + ++++ +++ +++ ++++ ++ + ++ +++ ++ + X 46 49 1 Hematopoietic System Bone marrow Lymph node Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Lymph node, mesenteric Spleen Hemangiosarcoma Thymus ++ ++++ ++ ++++ ++ ++ +++ + ++ +++ +++++++++++++++++++++++++ ++ ++++ +++ +++ ++ ++ +++ + ++ +++ M+ + MI M+ + M+ + + + + MM+ + + + + M+ M+ MMMMMMMM + M + + M + MMMMM + M + + MM M+ + + + + + + + + + + + + + + + + + + + + + + M ++ ++++ ++ ++++ +++ + ++ ++ ++ ++ + X ++ ++++ +++ +++ ++ +++ ++ ++ + ++1 45 49 43 36 14 37 50 1 41 Integum entary System Mammary gland Fibrosarcoma Skin +++++++++++++++++++++++++ X +++++++++++++++++++++++++ 48 1 50 M usculoskeletal System Bone +++++++++++++++++++++++++ 50 Nervous System Brain Spinal cord Thoracic, ganglioneuroma +++++++++++++++++++++++++ + X 50 1 1 R espiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma Hemangiosarcoma, metastatic, tissue NOS Nose Trachea I ++++++++++++++++++++++++ +++++++++++++++++++++++++ X + + + + + + + + + + + + + + + + + + + + + + + + .+ M + .+ + + + + + + + + + + M I + + + + + + + + + + 48 50 2 1 1 50 45 182 Talc, NTP TR 421 Table D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 18 mg/mJ (continued) Number of Days on Study 0000000455555556666666777 2222222701455684456689011 0888888386848912651562668 Carcass ID Number 2122222332233323232222222 9900000440315672352236965 2613467962963008227866679 1111111111111111111111111 Special Senses System Harderian gland Adenocarcinoma + X Urinary System Kidney Urinary bladder +++ +++ + +++ ++ ++ ++ ++ ++ ++ + ++ AA + + + +A + + + +A + + + A + A + +A + AA + Systemic Lesions Multiple organs Lymphoma malignant histiocytic Lymphoma malignant lymphocytic Lymphoma malignant mixed Lymphoma malignant undifferentiated cell type +++++++++++++++++++++++++ X X X Lesions in Female Mice 1 X3 T a b l e D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Inhalation Study of Talc: 18 mg/m3 (continued) N um ber of Days on Study 7777777777777777777777777 2222233333333333333333333 9999900001 1 11 122222233333 C arcass ID N um ber 2222222222223333333322233 1233466889991 122255558925 0948001684897946945869029 1111111111111111111111111 Tot al T issu e s/ T um ors Special Senses System Harderian gland Adenocarcinoma 1 1 U rinary System Kidney Urinary bladder +++++++++++++++++++++++++ +++++++++++++++++++++++++ 50 41 Systemic Lesions Multiple organs Lymphoma malignant histiocytic Lymphoma malignant lymphocytic Lymphoma malignant mixed Lymphoma malignant undifferentiated cell type +++++++++++++++++++++++++ XXX X X 50 1 3 2 2 184 Talc, N TP TR 421 T a b l e D3 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Talc 0 mg/m3 6 mg/m3 18 mg/m3 L iver H epatocellular Adenoma Overall rales* Adjusted ratesb Terminal ratesc First incidence (days) Life table tests" Logistic regression tests** Cochran-Armitage test** Fisher exact test3 L iver H epatocellular Carcinom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test L iver H epatocellular Adenoma or Carcinom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Lung: Aiveolar/bronchiolar Adenoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test Lung: A lveolar/broncbiolar Carcinom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 5/46 (11%) 16.7% 5/30 (17%) 729 (T) P =0.565 P=0.603N P=0.523N 7/46 (15%) 19.1% 3/30 (10%) 426 P=0.308N P=0.243N P=0.197N 11/46 (24%) 31.1% 7/30 (23%) 426 P=0.329N P=0.253N P=0.184N 3/46 (7%) 10.0% 3/30 (10%) 729 (T) P=0.505N P=0.467N P=0.425N 2/46 (4%) 6.7% 2/30 (7%) 729 (T) P=0.383N P=0.325N P=0.309N 1/47 (2%) 4.3% 1/23 (4%) 729 (T) P=0.169N P-0.169N P=0.097N 5/47 (11%) 18.4% 3/23 (13%) 645 P=0.487N P=0.372N P=0.364N 6/47 (13%) 22.5% 4/23 (17%) 645 P=0.262N P=0.147N P=0.131N 2/49 (4%) 6.7% 1/23 (4%) 559 P=0.589N P=0.499N P=0.470N 4/49 (8%) 11.6% 0/23 (0%) 491 P =0.286 P =0.356 P=0369 4/50 (8%) 14.0% 2/25 (8%) 581 P=0.602N P-0.539N P=0.447N 4/50 (8%) 15.4% 3/25 (12%) 718 P=0.344N P=0.255N P=0.216N 7/50 (14%) 25.2% 5/25 (20%) 581 P=0.330N P=0.227N P=0.163N 2/50 (4%) 6.4% 1/25 (4%) 548 P=0.562N P=0.515N P=0.460N 1/50 (2%) 2.6% 0/25 (0%) 558 P=0.539N P=0.500N P=0.468N Lesions in Fem ale Mice T a b l e D3 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rales Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 5/46 (11%) 16.7% 5/30 (17%) 729 (T) P=0.337N P=0.269N P=0.235N Ovary; Luteoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 2/38 (5%) 8.0% 2/25 (8%) 729 (T) P=0.177N P=0.177N P=0.146N Pituitary Gland (Unspecified Site): Adenoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 5/42 (12%) 15.1% 2/30 (7%) 683 P=0.239N P=0.189N P=0.133N Pituitary Gland (Unspecified Site): Carcinoma Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 0/42 (0%) 0.0% 0/30 (0%) - P = 0.591 N P=0.515N P=0.542N Pituitary Gland (Unspecified Site): Adenoma or Carcinoma Overall rates 5/42 (12%) Adjusted rates 15.1% Terminal rates 2/30 (7%) First incidence (days) 683 Life table tests P=0.216N Logistic regression tests P=0.150N Cochran-Armitage test P=0.111N Fisher exact test 6/49 (12%) 173% 1/23 (4%) 491 P =0.394 P =0.519 P = 0.545 0/43 (0%) 0.0% 0/21 (0%) e P=0.277N P=0.277N P=0.217N 4/43 (9%) 18.2% 4/22 (18%) 729 (T) P=0.610 P=0.604N P=0.485N 2/43 (5%) 5.5% 0/22 (0%) 534 P =0.237 P=0.274 P =0.253 6/43 (14%) 22.7% 4/22 (18%) 534 P = 0.352 P=0.451 P=0.517 3/50 (6%) 8.9% 1/25 (4%) 548 P=0.428N P = 0.367N P =0.31 IN 0/46 (0%) 0.0% 0/24 (0%) - P=0.246N P=0.246N P=0.202N 2/48 (4%) 7.1% 1/25 (4%) 665 P=0.290N P=0.220N P=0.166N 0/48 (0%) 0.0% 0/25 (0%) - - - 2/48 (4%) 7.1% 1/25 (4%) 665 P=0.290N P=0.220N P=0.166N 185 186 Talc, NTP TR 421 T able D3 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Talc (continued) i 0 mg/m 1 6 mg/m 7 18 mg/m All Organs: H em angiom a or H em angiosarcom a Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 2/46 (4%) 6.7% 2/30 (7%) 729 (T) P =0.323 P =0.356 P=0.399 1/49 (2%) 4.3% 1/23 (4%) 729 (T) P=0.593N P=0.593N P=0.476N All Organs: M alignant Lym phom a (H istiocytic, Lym phocytic, Mixed, or U ndifferentiated Cell Type) Overall rates 7/46 (15%) 7/49 (14%) Adjusted rates 21.3% 26.7% Terminal rates 5/30 (17%) 5/23 (22%) First incidence (days) 509 628 Life table tests P=0.358 P=0.454 Logistic regression tests P = 0.406 P = 0.607 Cochran-Armitage test P=0.514 Fisher exact test P=0.563N All Organs: Benign N eoplasm s Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 18/46 (39%) 54.5% 15/30 (50%) 683 P=0.148N P=0.094N P=0.050N 9/49 (18%) 36.4% 8/23 (35%) 559 P=0.125N P=0.044N P=0.022N All Organs: M alignant N eop lasm s Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 19/46 (41%) 51.9% 13/30 (43%) 426 P=0.372N P=0.241N P=0.143N 19/49 (39%) 55.4% 9/23 (39%) 491 P = 0.340 P=0.546N P=0.483N All Organs: Benign or M alignant N eoplasm s Overall rates Adjusted rates Terminal rates First incidence (days) Life table tests Logistic regression tests Cochran-Armitage test Fisher exact test 31/46 (67%) 81.4% 23/30 (77%) 426 P=0.141N P=0.036N P=0.011N 26/49 (53%) 75.1% 15/23 (65%) 491 P =0.537 P=0.162N P=0.112N 3/50 (6%) 10.1% 2/25 (8%) 473 P =0.434 P = 0.495 P = 0.540 8/50 (16%) 27.4% 5/25 (20%) 642 P = 0.387 P =0.463 P=0.571 10/50 (20%) 33.0% 6/25 (24%) 548 P=0.145N P=0.071N P=0.033N 15/50 (30%) 45.6% 8/25 (32%) 473 P=0.441N P=0.279N P=0.173N 21/50 (42%) 58.9% 11/25 (44%) 473 P=0.168N P=0.035N P=0.011N Lesions in Female Mice 187 Table 1)3 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Talc (continued) (T)Terminal sacrifice Number o f neoplasm-bearing animals/number o f animals examined. Denominator is number of animals examined microscopically (or adrenal gland, bone marrow, brain, clitoral gland, gallbladder, heart, kidney, larynx, liver, lung, nose, ovary, pancreas, parathyroid gland, pituitary gland, salivary gland, spleen, thyroid gland, and urinary bladder lor other tissues, denominator is number of animals necropsied. Kaplan-Meier estimated neoplasm incidence at the end o f the study after adjustment for intercurrenl mortality ^ Observed incidence at terminal kill Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that exposed group. The life table test regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armiiage and Fisher exact tests compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N. e Not applicable; no neoplasms in animal group m Talc, NTP TR 421 Table D4 Summary of the Incidence of Nonneoplastic Lesions in Female Mice In the 2-Year Inhalation Study of Talc* 0 mg/m3 6 mg/m3 18 mg/m3 Disposition Summary Animals initially in study Early deaths Moribund Natural deaths Survivors Terminal sacrifice Missing Culled Animals examined microscopically A lim entary System Intestine large, cecum Hyperplasia, lymphoid Serosa, inflammation, suppurative Intestine large, colon Serosa, inflammation, suppurative Intestine small, duodenum Ulcer, focal Mucosa, atrophy Serosa, inflammation, suppurative Intestine small, ileum Hyperplasia, lymphoid Mucosa, atrophy Peyer's patch, necrosis Serosa, inflammation, suppurative Intestine small, jejunum Mucosa, atrophy Serosa, inflammation, suppurative Liver Eosinophilic focus Fibrosis, focal Focal cellular change Hematopoietic cell proliferation Inflammation, focal Necrosis, focal Pigmentation, hemosiderin, focal Centrilobular, degeneration Centrilobular, necrosis, coagulative Serosa, inflammation, suppurative Sinusoid, inflammation Pancreas Inflammation, focal Acinus, hyperplasia, focal Serosa, inflammation, suppurative Salivary glands Inflammation, acute Stomach Serosa, inflammation, granulomatous Serosa, inflammation, suppurative Stomach, forestomach Hyperplasia, mast cell, focal Hyperplasia, squamous, focal Ulcer, focal 50 2 17 30 1 46 (35) (38) (27) 1 (4%) 2 (7%) (33) 1 (3%) 4 (12%) (33) 2 (6%) (46) 2 (4%) 1 (2%) 1 (2%) 1 (2%) 4 (9%) 2 (4%) (42) 1 (2%) 1 (2%) (46) (45) 1 (2%) (45) 2 (4%) 1 (2%) 50 4 21 23 1 1 48 (29) 1 (3%) (33) 2 (6%) (25) 6 (24%) 2 (8%) (27) 1 (4%) 6 (22%) 2 (7%) (28) 7 (25%) 2 (7%) (46) 1 (2%) 1 (2%) 3 (7%) 2 (4%) 2 (4%) 2 (4%) 1 (2%) 1 (2%) 1 (2%) 7 (15%) (39) 5 (13%) (48) 1 (2%) (45) 2 (4%) (45) 4 (9%) 3 (7%) 50 4 21 25 50 (34) 1 (3%) (32) (27) 4 (15%) (31) 6 (19%) 1 (3%) 1 (3%) (31) 3 (10%) 1 (3%) (50) 1 (2%) 2 (4%) 1 (2%) 2 (4%) 5 (10%) (44) 2 (5%) 4 (9%) (50) 1 (2%) (50) 1 (2%) 1 (2%) (50) 1 (2%) 2 (4%) Lesions in Female Mice 189 Table D4 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 mg/m3 Alimentary System (continued) Stomach, glandular Inflammation, suppurative Ulcer, focal ' Forestomach, inflammation, focal Cardiovascular System Heart Myocardium, degeneration, focal Myocardium, inflammation, focal Myocardium, mineralization, focal Pericardium, inflammation, suppurative Endocrine System Adrenal gland Capsule, inflammation, suppurative Corticomedullary junction, hemorrhage Spindle cell, hyperplasia Adrenal gland, cortex Cyst Inflammation, suppurative, focal Vacuolization cytoplasmic, focal Adrenal gland, medulla Hyperplasia, focal Parathyroid gland Hyperplasia Pituitary gland Cyst Hemorrhage, focal Hyperplasia, focal Pigmentation, lipofuscin Thyroid gland Cyst Inflammation, acute, focal C-cell, hyperplasia Follicular cell, hyperplasia General Body System Tissue NOS Thrombosis, chronic Genital System Ovary Abscess Cyst Thrombosis Uterus Angiectasis Hyperplasia, histiocytic, focal Metaplasia, squamous (45) 1 (2%) (A 6 ) 1 (2%) 1 (2%) 1 (2%) (46) 4 (9%) 2 (4%) 46 (100%) (46) 2 (4%) 3(7% ) (41) 2 (5%) (23) 1 (4%) (42) 2 (5%) 2 (5%) 2 (5%) 1 (2%) (43) 2 (5%) 1 (2%) 9 (21%) (^) 1 (25%) (38) 4 (11%) 6 (16%) 1 (3%) (44) (39) 1 (3%) 1 (3%) (48) 1 (2%) 2 (4%) (45) 7 (16%) 3 (7%) 45 (100%) (44) 3(7% ) (43) (18) (42) (47) 12 (26%) (1) (43) 10 (23%) 11 (26%) 2(5% ) (45) 1 (2%) (46) 1 (2%) 2 (4%) (50) 4 (8%) (50) 5 (10%) 1 (2%) 47 (94%) (50) 1 (2%) (45) (25) (48) (49) 2 (4%) 1 (2%) 10 (20%) (2) (46) 7 (15%) 10 (22%) (49) 1 (2%) 1 (2%) 190 Talc, NTP TR 421 Taule D 4 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Talc (continued) 0 m g/m 3 6 m g/m 3 18 m g/m 3 G enital System (continued) Uterus (continued) Thrombosis Endometrium, hyperplasia, cystic Mucosa, inflammation, suppurative Serosa, inflammation, suppurative H em atopoietic System Bone marrow Hyperplasia Myelofibrosis Myeloid cell, hyperplasia Lymph node Iliac, hyperplasia, lymphoid Iliac, inflammation Pancreatic, hyperplasia, lymphoid Pancreatic, infiltration cellular, mixed cell Pancreatic, follicular, necrosis Renal, hyperplasia, lymphoid Renal, infiltration cellular, mixed cell Renal, inflammation Renal, follicular, necrosis Lymph node, bronchial Hyperplasia, histiocytic Hyperplasia, lymphoid Infiltration cellular, mixed cell Inflammation, acute Lymph node, mandibular Cyst Depletion lymphoid Hyperplasia, histiocytic Hyperplasia, lymphoid Hyperplasia, plasma cell Infiltration cellular, mixed cell Inflammation Follicular, necrosis Lymph node, mediastinal Hyperplasia, histiocytic Hyperplasia, lymphoid Infiltration cellular, mixed cell Lymph node, mesenteric Depletion lymphoid Hematocyst Hyperplasia, histiocytic Hyperplasia, lymphoid Hyperplasia, plasma cell Infiltration cellular, mixed cell Inflammation Follicular, necrosis Spleen Congestion Hematopoietic cell proliferation Hyperplasia, lymphoid 1 (2%) 34 (77%) 3 (7%) 1 (2%) (41) 1 (2%) 28 (68%) 1 (2%) (46) 1 (2%) 1 (2%) 1 (2%) (38) 1 (3%) 1 (3%) (35) 1 (3%) 1 (3%) 1 (3%) (13) 1 (8%) 1 (8%) (35) 5 (14%) 3 (9%) (45) 2 (4%) 8 (18%) 5 (11%) 30 (67%) 7 (16%) 4 (9%) (43) 4 (9%) 23 (53%) 6 (14%) (46) 2 (4%) 1 (2%) 2 (4%) (37) 25 (68%) 16 (43%) 1 (3%) (38) 1 (3%) 1 (3%) 1 (3%) 1 (3%) (17) 3 (18%) 1 (6%) (31) 1 (3%) 1 (3%) 2 (6%) 5 (16%) 2 (6%) 12 (39%) (44) 12 (27%) 8 (18%) 35 (71%) 4 (8%) 2 (4%) (45) 5 (11%) 27 (60%) 3 (7%) (49) 1 (2%) 1 (2%) 1 (2%) 1 (2%) 1 (2%) 2 (4%) 1 (2%) 1 (2%) 1 (2%) (43) 39 (91%) 20 (47%) 1 (2%) (36) 1 (3%) 3 (8%) 1 (3%) (14) 2 (14%) 2 (14%) (37) 2(5% ) 1 (3%) 1 (3%) 2 (5%) 1 (3%) 5 (14%) 1 (3%) 7 (19%) (50) 10 (20%) 6 (12%) lesions in Female Mice 191 t ABLE D 4 ummary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Talc (continued) 0 mg/m 6 mg/m3 18 m g/m3 H em atopoietic System (continued) Spleen (continued) Inflammation, suppurative Capsule, inflammation, suppurative Lymphoid follicle, depletion lymphoid Lymphoid follicle, necrosis Thymus Cyst Hyperplasia, plasma cell Inflammation, suppurative Necrosis Cortex, depletion lymphoid Integumentary System Mammary gland Abscess Edema Skin Alopecia M usculoskeletal System Bone Periosteum, femur, proliferation connective tissue Nervous System Brain Hydrocephalus Mineralization, focal 2 (4%) 2 (4%) 2 (4%) 2 (4%) (40) 2 (5%) 3 (8%) 8 (20%) (41) 1 (2%) (46) 2 (4%) (46) 1 (2%) (46) 36 (78%) 3 (7%) 3 (7%) 4 (9%) (40) 2 (5%) 1 (3%) 1 (3%) 5 (13%) 12 (30%) (45) (46) 2 (4%) (48) (48) 2 (4%) 33 (69%) 1 (2%) 3 (6%) 5 (10%) 2 (4%) (41) 1 (2%) 15 (37%) (48) 1 (2%) (50) (50) (50) 29 (58%) Respiratory System Larynx Inflammation, acute Lung Congestion Hyperplasia, histiocytic Hyperplasia, macrophage Inflammation, chronic active Metaplasia, osseous, focal Alveolar epithelium, hyperplasia, focal Perivascular, inflammation, suppurative Pleura, inflammation, suppurative Nose Cytoplasmic alteration, focal Developmental malformation Erosion, focal Inflammation, acute Ulcer, focal (42) 1 (2%) (46) 1 (2%) 2 (4%) 1 (2%) 1 (2%) (46) 29 (63%) 1 (2%) 3 (7%) 6 (13%) 1 (2%) (43) (48) 3 (6%) 45 (94%) 25 (52%) 3 (6%) 2 (4%) (46) 37 (80%) 4 (9%) (48) (50) 1 (2%) 43 (86%) 38 (76%) 1 (2%) 1 (2%) 5 (10%) (50) 40 (80%) 1 (2%) 5 (10%) ' 192 Talc NTP TR 421 T a b l e D4 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Talc (continued) 0 mg/m3 6 mg/m3 18 m g/m3 Special Senses System Eye Inflammation, suppurative Harderian gland Inflammation, suppurative (1) 1 (100%) (2) (2) 1 (50%) Urinary System Kidney Casts protein Infarct Inflammation, focal Metaplasia, osseous, focal Nephropathy, chronic Capsule, inflammation, suppurative Renal tubule, hyperplasia, focal Urinary bladder Serosa, inflammation, suppurative Submucosa, hyperplasia, lymphoid Submucosa, inflammation, suppurative (46) 1 (2%) 1 (2%) 1 (2%) 1 (27c) 3 (7%) (44) 1 (2%) (46) 2 (4%) 1 (2%) 1 (2%) 1 (2%) 6 (13%) 1 (2%) (40) 3 (8%) Number of animals examined microscopically at site and number of animals with lesion (1) (50) 1 (2%) 2 (4%) 5 (10%) (41) 3 (7%) 1 (2%) 193 APPENDIX E ORGAN WEIGHTS AND ORGAN-WEIGHT-TO-BODY-WEIGHT RATIOS Table El T a b l e E2 T a b l e E3 T a b l e E4 T a b l e E5 T a b l e E6 T a b l e E7 T a b l e E8 T a b l e E9 6-Month Interim Evaluation in the Lifetime Inhalation Study of Talc in Rats ............. lL-Month Interim Evaluation in the Lifetime Inhalation Study of Talc in R a t s ............... 18-Month Interim Evaluation in the Lifetime Inhalation Study of Talc in R a t s ............... 24-Month Interim Evaluation in the Lifetime Inhalation Study of Talc in Rats .............. End of the Lifetime Inhalation Study of Talc in R a t s .............................................................. 6-Month Interim Evaluation in the 2-Year Inhalation Study of Talc in Mice ................ 12-Month Interim Evaluation in the 2-Year Inhalation Study of Talc in M ic e .................. 18-Month Interim Evaluation in the 2-Year Inhalation Study of Talc in M ic e ................. End of the 2-Year Inhalation Study of Talc in M ic e ................................................................ 194 195 196 L97 198 199 200 201 202 194 Talc, NTP TR 421 T a b l e El Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats at the 6-Month Interim Evaluation in the Lifetime Inhalation Study of Talc3 0 mg/m3 6 mg/m3 IB m g/m 3 M ale n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative F em ale n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 3 379 2 2.061 0.073 5.44 0.22 1.087 0.024 2.87 0.07 1.203 0.055 3.17 0.16 12.969 0.336 34.20 0.79 1.196 0.049 3.15 0.11 3 216 + 10 1.801 0.020 8.39 0.33 0.679 0.023 3.16 0.11 0.700 0.043 3.25 0.17 7.579 0.502 35.13 1.09 1.006 0.112 4.71 0.65 3 365 9 1.962 0.035 5.38 0.22 0.984 0.047 2.69 0.07 1.155 0.028 3.16 0.01 11.658 0.483 31.89 0.65 1.201 0.060 3.29 0.19 3 210 5 1.800 0.030 8.57 0.28 0.691 -4- 0.031 3.29 0.13 0.775 0.025 3.69 0.10 7.253 0.172 34.51 0.33 0.986 0.064 4.69 0.29 3 351 + 4** 1.964 0.041 5.59 0.10 1.008 0.018 2.87 0.03 1.143 0.025 3.25 -4- 0.04 11.644 0.613 33.11 1.43 1.600 0.073' 4.55 0.19*' 3 212 7 1.860 0.031 8.82 0.39 0.716 0.055 3.38 0.20 0.751 0.030 3.55 0.07 6.875 0.409 32.47 1.21 1.090 0.010 5.17 0.21 Significantly different (P<0.05) from the control group by Williams' or Dunnett's test ** PiO.Ol . ` Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight (mean standard error) Organ Weight Analyses 195 Table E2 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats at the 11-Month Interim Evaluation in the Lifetime Inhalation Study of Talc2 0 mg/m1 6 mg/m3 18 mg/m3 Male n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 2 425 10 2.018 0.010 4.75 0.13 1.161 0.080 2.73 0.12 1.313 0.008 3.09 0.09 12.824 0.065 30.20 0.86 1.228 0.143 2.90 0.40 3 406 15 1.616 0.306 3.97 0.74 1.051 0.063 2.58 0.06 1.242 0.062 3.07 0.26 12.454 0.424 30.72 1.47 1.152 0.043 2.85 0.18 3 395 14 2.020 0.012 5.13 0.16 1.079 0.048 2.73 0.09 1.216 0.069 3.07 0.07 12.223 0.618 30.92 0.50 1.979 0.077** 5.02 0.16** Female n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 3 254 1 1.863 0.003 7.36 0.22 0.858 0.032 3.38 0.06 0.830 0.007 3.28 0.11 7.878 0.275 31.13 1.53 0.959 0.037 3.79 0.20 3 249 5 1.867 0.036 7.52 0.18 0.796 0.020 3.20 0.06 0.839 0.002 3.38 0.07 7.774 0.130 31.30 0.47 1.039 0.034 4.18 0.09 3 247 10 1.845 0.030 7.50 0.19 0.753 0.063 3.05 0.19 0.735 0.034* 2.99 0.13 7.537 0.354 30.57 0.50 1.551 0.163** 6.27 0.48* * * Significantly different (PS0.05) from the control group by Williams' or Dunnett's test * PsO .O l * Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight (mean standard error) - 196 Talc, NTP TR 421 Table E3 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats at the 18-Month Interim Evaluation in the lifetime Inhalation Study of Talc* 0 mg/m3 6 mg/m3 18 mg/m3 Male n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 3 446 + 14 2.019 0.043 433 0.10 1.077 0.065 2.41 0.09 1.913 0.599 4.27 + 1.31 14.329 1.434 32.10 3.01 1.691 + 0.100 3.78 0 .1 3 3 428 10 1.965 0.035 4.60 + 0.17 1.027 0.030 2.40 0.07 1328 0.063 3.10 0.12 13.866 0.882 3238 1.68 1.852 0.058 4.34 0.21 2 430 + 2 2.092 0.004 4.86 0.01 1.131 + 0.103 2.63 0.23 1317 0.023 3.06 0.06 12.520 + 0.189 29.10 0.56 3.169 0.121' 7.36 + 0.25 ' Female n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 3 305 5 1.840 0.028 6.04 + 0.17 0.772 0.015 Z53 + 0.08 0.929 0.023 3.05 0.12 8.750 0.223 28.71 + 035 1.130 0.026 3.71 0.12 3 275 + 4** 1.827 + 0.045 6.63 + 0.11* 0.706 0.010* 2.56 0.03 0.902 0.038 3.28 0.17 8340 + 0.648 31.03 2.47 1.395 0.046* 5.07 0.11** 3 280 + 4* 1.847 0.013 6.61 0.13* 0.765 0.011 2.74 0.01* 0.955 + 0.047 3.41 0.13 8.904 0396 3134 + 1.94 2.600 + 0.030*' 931 + 0.18** * Significantly different (P<0.05) from the control group by Williams' or Dunnett's test PiO.Ol * Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight (mean + standard error) ' Organ Weight Analyses 197 T able E4 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats at the 24-Month Interim Evaluation in the Lifetime Inhalation Study of Talc* 0 m g/m 3 6 m g/m 3 18 m g/m 3 Male n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 3 406 29 2.068 0.015 5.15 0.42 1.065 0.022 2.66 0.25 1.720 0.138 4.25 0.32 15.298 0.187 38.11 3.23 1.766 0.177 4.40 0.55 6 422 12 2.023 0.025 4.81 0.11 1.126 0.044 2.69 0.18 1.577 0.048 3.76 0.19 14.924 0.480 35.55 1.80 2.150 0.230 5.18 0.69 2 392 30 1.989 0.008 5.10 0.37 0.993 0.026 2.54 0.13 1.649 0.068 4.24 0.50 14.344 1.253 37.05 6.03 2.473 0.674 6.48 2.21 F em ale n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 5 296 17 1.821 0.023 6.24 0.42 0.826 0.014 283 0.19 1.118 0.055 3.82 0.26 11.218 0.527 38.38 2.74 1.014 0.104 3.40 0.23 9 296 10 1.826 0.011 6.24 0.21 0.826 0.032 2.81 0.10 ' 1.137 0.044 3.85 0.10 12.127 0.672 41.16 2.12 1.447 0.219 4.88 0.67 3 262 25 1.865 0.012 7.23 0.63 0.824 0.045 3.16 0.13 1.021 0.022 3.97 0.44 9.966 0.246 38.84 4.59 3.261 12.73 1.62** * H * Significantly different (PsO.Ol) from the control group by Williams' or Dunnett's test * Organ weights and body weights are given in grams; organ-weight-to-body-weighl ratios are given as mg organ weight/g body weight (mean standard error) 198 Talc, NTP TR 421 Table E5 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats at the End of the Lifetime Inhalation Study of Talc* 0 mg/m3 6 mg/m3 18 mg/m3 M ale n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 8 379 17 2.030 0.016 5.45 0.28 1.385 0.104 3.68 0.26 1.899 0.151 5.09 0.49 15.501 0.861 41.03 1.67 2.154 0.124 5.76 0.38 12 397 6 2.041 0.015 5.16 0.09 1.288 0.041 3.26 0.13 1.847 0.125 4.69 0.37 16.562 0.540 41.92 1.73 2.509 0.068 6.34 0.21 13 326 12** 2.014 0.019 6.29 0.25* 1.302 0.064 4.05 0.22 1.737 0.101 5.39 0.35 14.055 0.936 42.85 1.76 4.026 0.196** 12.65 0.85 * F em ale n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 12 260 14 1.975 0.122 8.03 0.95 1.020 0.039 4.03 0.24 1.313 0.047 5.21 0.34 12005 0.660 46.35 1.68 1.575 0.109 6.11 0.35 13 247 14 1.860 0.020 7.89 0.51 1.006 0.026 4.33 0.39 1.235 0.049 5.22 0.36 12.567 0.903 51.25 2.90 2.673 0.362* * 11.77 2.10* 9 231 9 1.847 0.028 8.06 0.27 1.047 0.027 4.58 0.20 1.281 0.079 5.66 0.55 12.247 0.678 53.65 3.82 4.050 0.228* * 17.83 1.43* * Significantly different (PS0.05) from the control group by Williams' or Dunnett's test P<0.01 * Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight (mean standard error) Organ Weight Analyses 199 Table E6 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice at the 6-Month Interim Evaluation In the 2-Year Inhalation Study of Talc* 0 mg/mJ 6 m g /m 3 18 mg/m3 Male n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 4 31.3 0.9 0.431 0.028 13.81 0.90 0.159 0.003 5.10 0.07 0.303 0.022 9.66 0.40 1.737 0.079 55.51 1.06 0.165 0.008 5.29 0.35 4 31.1 0.9 0.458 0.006 14.74 0.23 0.165 0.008 5.31 0.33 0.297 0.018 9.58 -f; 0.70 1.792 0.066 57.75 2.77 0.149 -j- 0.010 4.78 0.27 4 32.1 0.6 0.469 0.008 14.60 0.38 0.157 0.011 4.88 0.25 0.292 0.011 9.10 0.33 1.731 0.060 53.84 1.19 0.173 0.017 5.35 0.44 Female n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 4 27.1 0.9 0.474 Hk 0.007 17.52 0.36 0.142 0.004 5.27 0.30 0.201 0.011 7.40 0.20 1.541 0.099 56.86 2.92 0.190 0.019 7.11 0.96 4 27.2 1.7 0.482 0.008 17.85 0.81 0.133 0.005 4.92 0.19 0.203 0.004 7.53 0.34 1.640 0.138 60.01 1.74 0.164 0.011 6.03 0.28 4 29.5 1.4 0.474 0.019 16.10 0.67 0.145 0.006 4.92 0.15 0.217 0.008 737 0.13 1.628 0.033 55.38 1.91 0.178 0.011 6.04 0.26 a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight (mean standard error) 200 Talc, NI P TR 421 Table E7 Organ Weights and Organ-Welght-to-Body-Welght Ratios for Mice at the 12-Month Interim Evaluation in the 2-Year Inhalation Study of Talc* 0 mg/m3 6 mg/m3 18 mg/m3 Male n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 4 34.6 1.7 0.478 0.020 13.87 0.31 0.196 0.023 5.62 0.37 0.334 0.007 9.71 0.28 1.612 0.052 46.77 0.79 0.157 0.009 4.54 0.17 4 37.2 0 3 0.475 0.009 1Z76 0.16 0.195 0.005 5.23 0.10 0.339 0.020 9.12 0.52 1.886 0.124 50.72 3.25 0.216 0.018 5.80 0.46 4 33.1 13 0.475 0.009 14.39 038 0.205 0.023 6.21 0.69 0.311 _ 0.027 9.41 036 1.928 0.240 58.55 8.01 0.243 0.032' 7.30 0.72*' Female n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 3 32.1 2.4 0.478 0.006 15.04 1.16 0.151 0.004 4.72 0.23 0.225 0.010 7.03 0.22 1.470 0.105 46.04 3.71 0.151 0.019 4.68 0.23 4 333 1.3 0.488 0.005 14.74 0.70 0.162 0.008 4.91 0.42 0.231 0.008 6.97 0.40 1.796 0.036* 54.20 235 0.191 0.014 5.78 0.61 4 28.7 12 0.491 0.008 17.16 035 0.190 0.010* 6.64 0.47* 0.230 0.011 8.01 0.10 1.477 0.093 51.40 Z48 0.207 0.015* 7.19 0.24 * * Significantly different (P<0.05) from the control group by Williams' or Dunnett's test PsO.Ol * Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight (mean standard error) Organ Weight Analyses 201 Table E8 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice at the 18-Month Interim Evaluation in the 2-Year Inhalation Study of Talc1 0 mg/nr' 6 mg/m1 18 mg/m3 M ale n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 4 33.1 3.0 0.467 0.007 14.51 1.44 0.193 0.017 6.18 1.29 0.342 0.007 10.66 1.23 1.844 0.228 57.08 7.95 0.229 0.034 7.45 2.01 4 37.5 2.1 0.470 0.009 12.63 0.58 0.186 0.011 5.00 0.35 0.361 0.021 9.66 0.47 1.796 0.080 48.07 1.26 0.238 0.013 6.42 0.57 4 35.4 1.7 0.496 0.014 14.10 0.76 0.203 0.006 5.77 0.22 0.350 0.009 9.91 0.22 1.748 0.113 49.28 1.45 0.345 0.032' 9.79 0.91 Fem ale n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 4 32.1 + 1.2 0.483 0.007 15.10 0.59 0.155 0.008 4.85 0.28 0.238 0.009 7.41 0.28 1.446 0.056 45.10 1.35 0.223 0.008 6.96 0.07 4 31.9 1.6 0.467 0.019 14.73 0.90 0.154 0.011 4.87 0.47 0.233 0.011 7.35 0.45 1.592 0.034 50.17 2.02 0.242 0.018 7.65 0.73 4 27.6 1.0** 0.501 0.038 18.33 1.91 0.164 0.010 5.96 0.48 0.228 0.007 8.32 0.55 1.318 0.055b 48.69 0.30b 0.299 0.018*' 10.90 0.87* * Significantly different (P<0.05) from the control group by Williams' or Dunnett's test * PS0.01 1 Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body b wn=ei3~ght (mean standard error) 202 Talc, NTP TR 421 Table E9 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice at the End of 2-Year Inhalation Study of Talc* 0 mg/m3 6 mg/m3 18 mg/m3 M ale n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 30 33.4 0.5 0.461 0.004 13.90 0.22 0.183 0.003 5.52 0.12 0.361 0.010 10.85 0.27 1.845 0.064 55.64 2.21 0.252 0.008c 7.47 0.25c 28 32.1 0.8 0.458 0.004 14.50 0.34 0.181 0.004 5.68 0.10 0.362 0.010 11.28 0.16 1.733 0.073b 53.14 1.72b 0.258 0.009b 8.01 0.24b 32 31.2 0.4* 0.460 0.005 14.78 0.19* 0.183 0.005 5.88 0.15 0.354 0.006 11.34 0.18 1.535 0.033* *c 49.27 1.03*c 0.408 0.011** 13.08 0.33* * Female n Necropsy body wt Brain Absolute Relative Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 30 31.4 0.6 0.484 0.004 15.53 0.26 0.164 0.005 5.24 0.15 0.251 0.007^ 8.03 0.17d 1.816 0.089 57.41 2.25 0.276 0.014 8.80 0.42 23 31.7 0.7 0.469 0.006 14.93 0.28 0.190 0.009* 6.02 0.28* * 0.265 0.010 8.38 0.27 1.770 0.107f 55.45 3.13f 0.293 0.012 9.28 0.34 25 30.7 0.5 0.477 0.003 15.59 0.20 0.163 0.003 5.32 0.09 0.257 0.007e 837 0.14e 1.761 0.083e 56.94 1.93e 0.410 0.010** 13.39 0.28** Significantly different (P<0.05) from the control group by Williams' or Dunnett's test P<0.01 Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body b weight (mean standard error) n=27 c n=28 d e n=29 n=24 f n=22 203 APPENDIX F 4-WEEK INHALATION STUDIES IN RATS AND MICE Materials and M e t h o d s ................................................................................................................................................. 204 T a b l e FI Experimental Design and Materials and Methods in the 4-Week Inhalation Studies of T a l c ....................................................................................... 206 Results ........................................................................................................................................................................... 208 T a b l e F2 Survival and Mean Body Weights of Rats in the 4-Week Inhalation Study of Talc .......... 209 T a b l e F3 Organ Weights and Organ-Weight-to-Body-Weight Ratios of Rats in the 4-Week Inhalation Study of Talc ......................................................................................... 210 T a b l e F4 Lung Talc Burden of Rats in the 4-Week Inhalation Study of T a lc ........................................ 211 T able F5 Normalized Lung Talc Burden of Rats in the 4-Week Inhalation Study of Talc ................... 211 T a b l e F6 Survival and Mean Body Weights of Mice in the 4-Week Inhalation Study of T a l c ........... 212 T able F7 Organ Weights and Organ-Weight-to-Body-Weight Ratios of Mice in the 4-Week Inhalation Study of Talc ............................................................................................ 213 T able F8 Lung Talc Burden of Mice in the 4-Week Inhalation Study of T a lc ......................................... 214 T a b l e F9 Normalized Lung Talc Burden of Mice in the 4-Week Inhalation Study of Talc ................ 214 204 Talc, NTP TR 421 M a t e r ia l s a n d M e t h o d s Procurement and Characterization of Talc Talc was obtained from Walsh and Associates (North Kansas City, MO) in one lot (W101882). Identity and purity analyses were performed by the analytical chemistry laboratory, Midwest Research Institute (Kansas City, MO). The study chemical, a finely powdered white solid, was identified as talc by infrared spectroscopy, elemental analysis, and microscopic analyses. The moisture content of the bulk chemical was analyzed and was determined to be stable throughout the studies. Bulk chemical studies were not conducted due to the physical and chemical properties of talc. The compound was stored in sealed Nalgene containers. Generation and Monitoring of Chamber Concentrations Talc aerosols were generated in a fluidized bed generator by injecting filtered air into the bed. Samples were collected continuously during the 6-hour exposure day on glass fiber filters. Only one sampling port position was used each day to collect the samples from each chamber. Once a week, samples were collected on Zefiuor filters so that the magnesium content of aerosolized talc could be determined and be compared to the magnesium content of bulk talc. Cascade impactor samples were collected 3 to 6 times a week to determine aerosol particle size. The amount of talc collected on the filters and impactor stages was quantitated gravimetrically. The extent of carry-over of the stainless steel material from the fluidized bed generator was quantitated by measuring the amount of acid-soluble nickel and chromium in filter samples taken from the exposure atmosphere twice during the studies. Study Design Groups of 10 male and 10 female F344/N rats and B6C3F, mice were exposed by inhalation to talc at target concentrations of 0, 2, 6, and 18 mg/m3. Rats and mice were exposed for 6 hours daily, 5 days a week, for 4 weeks. Source and Specification of Animals Male and female F344/N rats were obtained from Lovelace Inhalation Toxicology Research Institute (Albuquerque, NM). Male and female B6C3F, mice were obtained from Simonsen Laboratory (Gilroy, CA). Rats and mice were held 3 weeks before the studies began, and were 6 to 7 weeks old when the studies began. Animal health was monitored by serologic analyses during the studies under the protocols of the NTP Sentinel Animal Program. Animal Maintenance Rats and mice were housed individually throughout the studies. Drinking water was available ad libitum. Further details of animal maintenance are given in Table Fl. Clinical Examinations and Pathology All rats and mice were observed twice daily. Clinical observations and body weights were recorded at the beginning of the studies, each week, and at the end of the studies. Organ weights were recorded for the heart, right kidney, liver, and lung at the end of the studies. A necropsy was performed on all animals. During necropsy, all organs and tissues were examined for grossly visible lesions. A complete histopathologic examination was performed on all 18 mg/m3and control animals. Tissues for microscopic examination were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned to a thickness of 5 /im, and stained with hematoxylin and eosin. Lung Burden Analysis Groups of five male and five female rats and mice were analyzed for lung talc burden. Lungs were homogenized using water and the proteins were precipitated with 70% perchloric acid. The individual 4-Week Inhalation Studies 205 samples were filtered and washed with 5% trichloroacetic acid (TCA) to remove perchlorates. Washing continued until magnesium levels in the wash were within 10% of levels in the TCA solution (< 0.03 ppm magnesium). Filters and tissue residues were placed in 15-mL porcelain crucibles, dried slowly (200 C), and then ashed at 600 C for 1 hour. Ashed samples were transferred to Teflon beakers using 2 mL HC1 and evaporated to dryness. Samples were then digested in hydrofluoric acid (HF), and the HF evaporated. Additional HF was added and reevaporated. Sulfuric acid was added to remove trace HF, and samples were then diluted with distilled water and analyzed for magnesium by atomic absorbance (Perkin Elmer, Model 307, Atomic Absorption Spectrophotometer) with a magnesium hollow cathode lamp and an air acetylene flame (Hanson et al., 1985). 206 Talc, NTP TR 421 Table FI Experimental Design and Materials and Methods in the 4-Week Inhalation Studies of Talc Study Laboratory Lovelace Inhalation Toxicology Research Institute (Albuquerque, NM) Strain and Species Rats: F344/N rats Mice: B6C3Fj mice Anim al Source Rats: Lovelace Inhalation Toxicology Research Institute (Albuquerque, NM) Mice: Simonsen Laboratory (Gilroy, CA) Tim e Held Before Studies 3 weeks Average Age W hen Placed on Studies 6-7 weeks D ate o f First Exposure Rats: 20 April 1983 Mice: 16 June 1983 Duration o f Exposure 6 hours/day, 5 days/week for 4 weeks Date o f Last Exposure Rats: 18 May 1983 Mice: 13 July 1983 Average Age W hen Killed 10 to 11 weeks M ethod o f Sacrifice lntraperitoneal injection of T-61 solution Necropsy Dates Rats: 19-20 May 1983 Mice: 14-15 July 1983 Size o f Study Groups 10 males and 10 females * M ethod o f Animal D istribution Randomized by weight A nim als per Cage 1 M ethod o f Anim al Identification Ear tag and toe clip D iet NIH-07 Rat and Mouse Ration (Zeigler, Bros., Gardner, PA) available a d lib itu m during non-exposure periods M axim um Storage Tim e for Feed 90 days * 4-Week Inhalation Studies 207 Table FI Experimental Design and Materials and Methods in the 4-Week Inhalation Studies of Talc (continued) Water Automatic Watering System (Edstrom Industries, Waterford, Wl), available a d lib itu m Cages Stainless steel mesh cages (Hazleton, Aberdeen, MD), changed weekly Cham bers Stainless steel multitiered whole-body exposure chambers (H2000 and H1000, Hazleton Systems, Aberdeen, MD) washed weekly Excreta Pan Techboard untreated paper (Shepherd Specialties Paper, Inc., Kalamazoo, Ml), changed twice a day Filters Room Air and Chamber Air High Efficiency Particulate Air (HEPA) Filter, MIL Spec M1L-F-51068C (Flanders, Washington, DC), changed as required Animal Room Environm ent Rats Average temperature: 23 C Relative humidity: 40.3% Fluorescent light: 12 hours/day Room air changes: minimum of 10 changes/hour Mice Average temperature: 24 C Relative humidity. 42% Fluorescent light: 12 hours/day Room air changes: minimum of 10 changes/hour Exposure C oncentrations 0, 2, 6, and 18 mg/m3 by inhalation Type and Frequency o f Observation Observed twice daily body weights and clinical findings recorded at study initiation and weekly thereafter N ecropsy Necropsy was performed on all animals. Organ weights recorded for heart, right kidney, liver, and lung H istopathology Complete histopathologic examinations performed on all 18 mg/m3 and control animals. In addition to tissue masses, gross lesions, and associated lymph nodes, tissues examined included: larynx, lung, nasal turbinates, trachea, and tracheobronchial lymph nodes. Lung Talc Burden Groups of 5 male and 5 female rats and mice were evaluated for lung talc burden. 208 Talc, NTP TR 421 R esults Rats All rats survived to the end of the study and there were no clinical findings related to talc exposure. The final mean body weights of exposed male and female rats were similar to the controls (Table F2). There were no significant increases in any organ-weight-to-body-weight ratios in male or female rats (Table F3). The talc lung burdens increased with talc exposure level (Table F4); however, the ratio of lung burden to exposure concentration was somewhat higher at the 6 and 18 mg/m3exposure levels (Table F5). The increase in talc lung burden with exposure concentration may have been because the maximum ability of the respiratory tract to clear particles was exceeded at the 6 and 18 mg/m3exposure levels. There was a minimal increase in the number of intra-alveolar macrophages in the lung of male and female rats exposed to 18 mg/m3. The lesion was diffuse in nature and in no instance were clusters of greater than three alveolar macrophages observed. The individual macrophages were slightly larger than normal and had cytoplasm which contained fine eosinophilic granules. Mice One male mouse exposed to 2 mg/m3and one male mouse exposed to 6 mg/m3died before the end of the study. The final mean body weights of exposed male and female mice were similar to those of the controls (Table F6). There were no clinical findings associated with exposure to talc aerosols. There were no significant changes in any organ-weight-to-body-weight ratios in exposed male or female mice (Table F7). Talc lung burdens increased with talc exposure level (Table F8). However, the ratio of lung burden to exposure concentration was constant at all exposure levels (Table F9). In contrast to rats, the maximum ability of the respiratory tract to clear particles was apparently not exceeded at the 18 mg/m3 level. The only lesions related to inhalation of talc aerosols were observed in the lung of male and female mice exposed to 18 mg/m3. The changes were minimal and consisted of a diffuse increase in the number of intra-alveolar macrophages. In most cases, pulmonary macrophages did not exceed two per alveolus, but occasional clusters of up to 10 alveolar macrophages were observed. The individual macrophages were two to three times normal size with foamy granular cytoplasm. 4-Week Inhalation Studies 209 Table F2 Survival and Mean Body Weights of Rats in the 4-Week Inhalation Study of Talc1 D ose (mg/rn*) Survival* Initial M ean Bodv W eieh tb (s) Final Change Final W eight R elative to Controls (%) M ale 0 2 6 18 5/5 144 5 221 5 5/5 144 5 233 9 5/5 150 8 223 9 5/5 145 4 213 4 78 3 89 12 73 + 2 6+11 105 101 % Fem ale 0 2 6 18 5/5 110 2 151 2 5/5 109 2 151 + 5 5/5 110 2 150 6 5/5 110 2 150 2 41+2 42 6 40 6 40 + 2 100 100 100 1 Number of animals surviving/number initially in group b Weights and weight changes are given as mean standard error. Subsequent calculations are based on animals surviving to the end of the studies. 210 Talc, NTP TR 421 Table F3 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 4-Week Inhalation Study of Talc* 0 mg/m1 2 mg/m3 6 mg/m3 18 mg/m3 M ale n Necropsy body wt Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 5 219 5 0.796 0.020 3.64 0.07 0.882 0.047 4.02 0 .1 4 8.640 0.383 39.42 1.10 0.990 0.025 4.53 0.13 5 226 9 0.828 0.020 3.67 0.09 0.856 0.040 3.82 0.30 8.952 0.614 39.90 3.59 1.058 0.039 4.68 0.06 5 218 9 0.828 0.020 3.82 0.19 0.856 0.040 3.93 0.09 8.952 0.614 40.96 1.71 1.050 0.040 4.83 0 .1 8 5 212 10 0.826 0.070 3.89 0.21 0.898 0.035 4.25 0.09 9.076 0.520 42.82 0.59 0.994 0.042 4.70 0.07 Female n Necropsy body wt Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 5 148 1 0.600 0.019 4.05 0.11 0.628 0.014 4.24 0.06 5.950 0.286 40.17 1.63 0.846 0.032 5.72 0.20 5 144 5 0.632 0.023 4.40 0.15 0.638 0.025 4.43 0 .1 0 5.766 0.262 40.26 2.46 0.820 0.035 5.69 0.06 5 146 5 0.630 0.023 4.31 0.06 0.638 0.025 437 0.15 5.766 0.262 39.43 1.21 0.822 0.040 5.62 0.17 5 150 1 0.632 0.022 4.23 0.17 0.630 0.025 4.21 0.17 6.156 0.269 41.20 1.94 0.866 0.035 5.79 0.23 * Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight (mean standard error) 4-Week Inhalation Studies Table F4 Lung Talc Burden of Rats in the 4-Week Inhalation Study of Talc* 0 mg/m3 2 m g/m 3 6 mg/m3 M ale n Mg laic Mg talc/g lung Fem ale n Mg talc Mg talc/g lung 5 4.28 1.63 4.50 1.86 5 0.58 0.24 0.66 0.27 5 81.60 2.06** 78.80 2.75 * 4 56.50 1.56* 76.00 3.24* 5 186.00 9.27** 190.00 7.75 ** 5 127.20 9.27* 185.00 10.41 **b 211 18 mg/m3 5 846.00 45.45** 842.00 69.96* * 5 546.00 35.16** 770.00 51.28** Significantly different (P<0.05) from the control group by Dunn's or Shirley's test P<0.01 * Mean standard error b n=4 T a b l e F5 Lung Talc Burden (Normalized to Exposure Concentration) of Rats in the 4-Week Inhalation Study of Talc* 0 m g/m 3 2 mg/m3 6 m g/m 3 M ale n Fem ale n 5 5 5 34.25 1.21 4 33.05 1.40** 5 44.22 1.80** 4 43.03 2.41 18 mg/m3 5 49.52 4.12* 5 45.30 3.01 * Significantly different (P<0.01) from the control group by Dunn's or Shirley's test * Mean standard error; units are presented as Mg talc/g control lung per mg/m3. b Values of magnesium in sample pools of 2 to 3 control lungs were less than the limit of detectability (0.1 ppm). Therefore no equivalent of measurement of talc was calculated to be present in control lungs. 212 Talc, NTP TR 421 Table F6 Survival and Mean Body Weights of Mice in the 4-Week Inhalation Study of Talc D ose (m g /m 1) Survival* Initial M ean Body W eishtb fa) Final Change Final W eight Relative to Controls (%) M ale 0 2 6 18 Female 0 2 6 18 5/5 25.8 0.1 28.1 0.2 2.3 0.2 4/5 25.8 0.2 27.5 0.4 1.9 0.4 4/5 25.8 0.2 27.3 0.3 1.5 0.4 5/5 25.8 0.2 27.0 0.7 1.2 0.6 5/5 20.6 0.2 22.7 1.1 2.1 1.2 5/5 20.6 0.2 22.6 0.9 2.0 0.9 5/5 20.7 0.2 23.6 0.8 2.9 0.8 5/5 20.6 0.2 22.7 0.7 2.1 0.8 98 97 % 99 104 100 * Number of animals surviving/number initially in group b Weights and weight changes are given as mean standard error. Subsequent calculations are based on animals surviving to the end of the studies. . 4-Week Inhalation Studies 213 Table F7 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 4-Week Inhalation Study of Talc* 0 mg/ro3 2 mg/m3 6 mgZm3 18 mg/m3 M ale n Necropsy body wt Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 5 28.8 0.3 0.194 0.015 6.73 0.47 0.278 0.007 9.66 0.26 1.868 0.051 64.89 1.53 0.254 0.007 8.83 0.29 4 24.8 0.6* 0.198 0.021 7.97 0.87 0.245 0.012 9.86 0.33 1.383 0.079* * 55.59 2.23** 0.230 0.007 9.26 0.12 4 26.8 0.5 ** 0.235 0.018 8.78 0.62 0.263 0.009 9.81 0.28 1.673 0.045 62.49 0.70 0.288 0.038 10.72 1.31 5 25.3 0.1** 0.218 0.010 8.62 0.36 0.242 0.009* 9.57 0.34 1.678 0.050 66.36 1.77 0.228 0.007 9.02 0.28 F em ale n Necropsy body wt Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lungs Absolute Relative 5 23.1 0.3 0.168 0.014 7.26 0.54 0.192 0.004 8.31 0.23 1.458 0.057 63.02 2.02 0.218 0.005 9.43 0.16 5 20.9 0.7* 0.162 0.016 7.78 0.82 0.174 0.005** 8.33 0.14 1.208 0.025** 57.88 1.14* 0.215 0.009b 10.47 0.12* *b 5 22.3 0.4 0.180 0.011 8.05 0.44 0.188 0.002 8.44 0.20 1.374 0.029 61.56 0.62 0.234 0.005 10.49 0.23** 5 22.7 0.3 0.152 0.015 6.67 0.61 0.198 0.002 8.71 0.10 1.458 0.029 64.12 1.07 0.220 0.005 9.67 0.17 * Significantly different (P50.05) from the control group by Williams' or Dunnett's test ** PiO.Ol * Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios are given as mg organ weight/g body weight (mean standard error) b n=4 214 Table F8 Lung Talc Burden or Mice in the 4-Week Inhalation Study of Talc* 0 m g/m 3 2 mg/m3 6 mg/m3 M ale n jig talc g talc/g lung Fem ale n 1g talc #tg talc/g lung 5 _b - 5 _ - 5 19.60 1.29 128.0 9.7 5 15.40 1.21 101.6 8.4 5 50.20 2.84 322.0 19.6 5 49.80 1.66 328.0 13.6 Talc, NTP TR 421 18 mg/m3 5 197.00 5.75 1,138.0 10.7 5 180.60 6.61 1,162.0 66.4 * Mean standard error b Values of magnesium in sample pools of 2 to 3 control lungs were less than the limit of detectability (0.1 ppm). Therefore no equivalent of measurement of talc was calculated to be present in control lungs. Table F9 Lung Talc Burden (Normalized to Exposure Concentration) of Mice in the 4-Week Inhalation Study of Talc* 0 m g/m3 2 mg/m3 6 mg/m3 M ale n Fem ale n 5 -b 5 - 5 58.170 4.405 5 46.180 3.820 5 56.480 3.443 5 57.540 2.372 18 mg/m3 5 55.240 0.512 5 56.400 3.223 * Mean standard error, units are presented as /ig talc/g control lung per mg/m3. b Values of magnesium in sample pools of 2 to 3 control lungs were less than the limit of detectability (0.1 ppm). Therefore no equivalent of measurement of talc was calculated to be present in control lungs. 215 APPENDIX G LUNG BURDEN, PULMONARY FUNCTION, AND LUNG BIOCHEMISTRY IN RATS Me t h o d s ....................................................................................................................................................................... Table G l Number of Rats Evaluated for Lung Talc Burden, Pulmonary Function, and Lung Biochemistry ................................................................................................................. Table G2 Lung Talc Burden (Normalized to Control Lung Weight) of Rats .................................... Table G3 Lung Talc Burden (Normalized to Exposure Concentration) of Rats .............................. Table G4 Bronchoalveolar Lavage Fluid Enzymes of Rats at the 24-Month Interim Evaluation ........................................................................................... Table G5 Bronchoalveolar Lavage Fluid Cell Populations of Rats at the 24-Month Interim Evaluation .......................................................................................... Table G6 Viability and Phagocytic Activity of Macrophages in Bronchoalveolar Fluid of Rats at the 24-Month Interim Evaluation ........................................................................... Table G7 Lung Collagen Metabolism and Protein Synthesis in Rats at the 24-Month Interim Evaluation .......................................................................................... Table G8 Proteinase Activity in Lavage Fluid and Lung Homogenate Supernatant Fluid of Rats at the 24-Month Interim Evaluation ........................................................................... Table G9 Respiratory Frequency of R a t s ..................................................................................................... Table G10 Total Lung Capacity of Rats ....................................................................................................... Table G il Total Lung Capacity/Kilogram Body Weight of R a t s ............................................................ Table G12 Tidal Volume of Rats ..................................................................................................................... Table G13 Minute Volume of R a t s .................................................................................................................. Table G14 Minute Volume/Kilogram Body Weight of R a ts ....................................................................... Table G15 Residual Volume of R a t s ............................................................................................................... Table G16 Residual Volume/Total Lung Capacity of Rats ....................................................................... Table G17 Vital Capacity of R a t s ..................................................................................................................... Table G18 Vital Capacity/Total Lung Capacity of Rats ............................................................................ Table G19 Forced Vital Capacity of R a t s ....................................................................................................... Table G20 Forced Vital Capacity/Kilogram Body Weight of Rats .......................................................... Table G21 Functional Residual Capacity of Rats ......................................... Table G22 Functional Residual Capacity/Total Lung Capacity of Rats ............................................... Table G23 Total Pulmonary Resistance of R a t s .......................................................................................... Table G24 Maximum Quasistatic Compliance of R a t s ............................................................................... Table G25 Quasistatic Chord Compliance of Rats ..................................................................................... Table G26 Dynamic Compliance of R a t s ....................................................................................................... Table G27 Peak Expiratory Flow of R a t s ....................................................................................................... Table G28 Peak Expiratory Flow/ForcedVital Capacity of R a t s ............................................................... Table G29 Expiratory Flow 10% Forced Vital Capacity of Rats .............................................................. Table G30 Expiratory Flow 10% Forced Vital Capacity/Forced VitalCapacity of R a t s ....................... Table G31 Expiratory Flow 25% Forced Vital Capacity of Rats .............................................................. Table G32 Expiratory Flow 25% Forced Vital Capacity/Forced VitalCapacity of R a t s ....................... Table G33 Expiratory Flow 50% Forced Vital Capacity of Rats .............................................................. Table G34 Expiratory Flow 50% Forced Vital Capacity/Forced VitalCapacity of R a t s ....................... Table G35 Mean Midexplratory Flow of R a t s ............................................................................................... Table G36 Mean Midexplratory Flow/Forced Vital Capacity of R a t s .................................................... Table G37 Carbon Monoxide Diffusing Capacity of R a t s ......................................................... Table G38 Carbon Monoxide Diffusing Capacity/Lung Volume of R a t s .................................... Table G39 Carbon Monoxide Diffusing Capacity/Kilogram Body Weight of Rats ................. Table G40 Percent Forced Vital Capacity Expired in 0.1 Second of R a t s ......................................... Table G41 Slope III of N2 Washout of R a t s .................................................................................................. 216 220 221 221 222 222 223 223 224 225 225 226 226 227 227 228 228 229 229 230 230 231 231 232 232 233 233 234 234 235 235 236 236 237 237 238 238 239 239 240 240 241 216 Talc, NTP TR 421 M ethods Lung Burden Lung talc burden was measured to determine the relationship between the exposure concentration and the amount of talc deposited and retained within the pulmonary region of the respiratory tract. The method used for analyzing for talc in lungs has been published (Hanson et al., 1985). Lung burdens were determined on three male and three female rats from each exposure group sacrificed at 6, 10, 18, and 24 months after the start of exposure. The analysis was based on determination of acid-insoluble magnesium in the lung. Midwest Research Institute reported that the value for the magnesium was 19.33% for batch 02 and 19.47% for batch 03. These values and the results of the analysis at Lovelace Inhalation Toxicology Research Institute were close to the theoretical value of magnesium for talc (19.22%). Since rats sacrificed at 27, 47, and 79 weeks had been exposed to only batch 02 of talc, 19.33% magnesium was used to calculate the quantity of talc for these rats. Because batch 03 was used for the last 4 months of exposure and lung burdens of rats after 105 weeks of exposure to talc would be expected to contain substantial amounts of batch 03 talc, 19.47% magnesium was used to calculate the quantity of talc deposited in the lungs of these rats. All operations in conjunction with tissue analysis for talc were done while wearing talc-free gloves. Left lung lobes were weighed at necropsy and stored frozen (-20 C) until used. Lungs were homogenized using water and the proteins were precipitated with 70% perchloric acid. The individual samples were filtered and washed with 5% trichloroacetic acid (TCA) to remove perchlorates. Washing continued until magnesium levels in the wash were within 10% of levels in the TCA solution (< 0.03 ppm magnesium). Filters and tissue residues were placed in 15 mL porcelain crucibles, dried slowly (200 C), and then ashed at 600 C for 1 hour. Ashed samples were transferred to Teflon beakers using 2 mL HC1 and evaporated to dryness. Samples were then digested in hydrofluoric acid (HF), and the HF evaporated. Additional HF was added and reevaporated. Sulfuric acid was added to remove trace HF, and samples were then diluted with distilled water and analyzed for magnesium by atomic absorbance (Perkin Elmer, Model 306, Atomic Absorption Spectrophotometer) with a magnesium hollow cathode lamp and an air acetylene flame (Hanson et al., 1985). Pulmonary Function Groups of 10 male and 10 female rats from each exposure group were assigned for respiratory function analyses. Respiratory function was measured at 6 months, 10 months, and 18 months. At 24 months of exposure, respiratory function was performed on all surviving rats not assigned to the lifetime study. Respiratory function was measured by noninvasive techniques, using methods previously published (Harkema et al., 1982). Tests were conducted using a 1.4 L combination flow and pressure plethysmograph. Flows were measured by measuring differential pressures across a wire screen pneumotachograph in the plethysmograph wall. Volumes were obtained by integration (Model 6, Pulmonary Mechanics Analyzer, Buxco Electronics, Sharon, CT). In the pressure mode, used only for measuring functional residual capacity, the pneumotachograph hole was sealed and volume changes were measured as pressure changes. The plethysmograph was maintained at approximately 37 C by a resistance element. Transpulmonary pressure was measured using transducers connected to the external airway and a liquid-filled, 2.2 mm O.D. esophageal catheter. A positive-negative pressure respirator system was used to induce quasistatic and forced respiratory movements. Reservoirs maintained at +40 and -50 cm H20 were connected to the airway by solenoid valves. Inspiratory and quasistatic expiratory flow rates were limited by calibrated needle valves to 5 and 3 mL/sec, respectively. Inspirations were stopped automatically at a transpulmonary pressure of 30 cm H20 , defining the lung volume at that distending pressure as total lung capacity. Forced inhalations were induced from total lung capacity by opening the airway to the negative pressure Lung Burden, Pulmonary Function, and Lung Biochemistry in Rats 217 reservoir via a rapidly opening valve having a 9.5 mm I.D., with no intentional flow restriction between the valve and the reservoir. The rats were anesthetized with halothane and intubated orally with a tracheal catheter 5.5 cm long x 1.8 mm I.D., fabricated from a 14-gauge intravenous catheter as previously described (Mauderly, 1977). The breathing port in the plethysmograph wall was a Luer fitting drilled to 2.5 mm I.D. The frequency response of the plethysmograph-respirator-tracheal catheter system has been tested and found adequate for forced expiratory events in rats. No phase lag among flow, pressure, and volume signals has been found in the frequency range of spontaneous breathing. Rats were anesthetized, intubated and placed prone in the plethysmograph. The esophageal catheter was adjusted to maximize the recorded transpulmonary pressure signal. Anesthetic depth was adjusted to yield a respiratory frequency of 50 to 60 per minute. Respiratory frequency, tidal volume, minute volume, dynamic lung compliance, and total pulmonary resistance were recorded during spontaneous respiration, time-averaged by a data logger and displayed on a teletype terminal. Prior to each subsequent measurement procedure, the rat's lung was manually inflated with a syringe to induce apnea. A quasistatic deflation from total lung capacity to residual volume allowed measurement of vital capacity and the quasistatic expiratory pressure-volume curve. Quasistatic lung chord compliance was measured as the slope of the curve over the chord between the apneic lung volume and the volume at +10 cm H20 pressure. Maximum quasistatic compliance was measured as the steepest slope of the pressure-volume curve over any 2 cm H ,0 pressure interval. Functional residual capacity was measured by the barometric method (Dubois et al., 1956) from recordings of lung volume and airway pressure changes as the rat resumed breathing against a blocked airway. From these measurements, all subdivisions of lung volume, including residual volume, were calculated. Alveolar-capillary gas exchange was evaluated by a single-breath, CO diffusing capacity test (Ogilvie et al., 1957). The lungs were inflated with a gas mixture containing CO and Ne in air to 20 cm H20 transpulmonary pressure. After 6 seconds, one-half of the gas was withdrawn and the remaining gas collected for analysis by gas chromatography. The lung volume when inflated with the mixture was measured by neon dilution. A forced inhalation was performed as described above, and the maneuver analyzed by a microprocessor in the data logger (Model D-12, Buxco). Data included forced vital capacity (FVC), the percentage of FVC exhaled in 0.1 second, flow rates at peak flow, and at 50%, 25%, and 10% of FVC. A single-breath nitrogen washout was performed by recording volume and nitrogen concentration of expirate during a slow deflation after an inflation to total lung capacity with oxygen. The slope of phase III ("alveolar plateau") of the washout curve was calculated to assess the uniformity of intrapulmonary gas distribution. Lung Biochem ist^ All surviving rats from each exposure group (the 3 males and 3 females originally assigned for lung burden/histology and the 10 males and 10 females from physiology/biochemistry) were sacrificed after 105 weeks of exposure. The rats were anesthetized with halothane and sacrificed by exsanguination from the abdominal aorta or renal artery. The heart and lung block tvas removed, the right apical, right cardiac, and right intermediate portions of each rat lung were given endobronchial saline lavage (6 mL total volume in three, 2.0 mL washes of saline), and the bronchoalveolar lavage (BAL) fluid was centrifuged at 300 x G to separate the cells from the supernatant fluid. 218 Talc, NTP TR 421 Airway Fluid Enzymes and Cytology Measurements In this study, BAL fluid was analyzed to determine the degree of: 1) Cell injury as indicated by concentration of lactate dehydrogenase (LDH). 2) Chronic inflammatory response as indicated by presence of increased numbers of polymorphonuclear leukocytes (PMN) and pulmonary alveolar macrophages (AM) as well as increased protein and alkaline phosphatase activity. 3) Lysosomal activation as indicated by B-glucuronidase and acid proteinase activity. Elevated enzyme activities have been observed in BAL fluid from rodents exposed to particles. These enzymes may be associated with the breakdown of necrotic tissues. 4) Response to oxidant injury as indicated by increased glutathione reductase activity. The supernatant fluid was analyzed by spectrophotometric, kinetic, and enzymatic analyses for the activities of B-glucuronidase, LDH, glucose-6-phosphate dehydrogenase, alkaline phosphatase, glutathione reductase, and glutathione peroxidase. Acid proteinase was measured by the release of radiolabeled globin peptides from the trichloroacetic acid-precipitable protein substrate, and total protein was analyzed colorimetrically (Henderson et at., 1985). Numbers of total nucleated cells recovered in lavage fluid were determined using a cell counter (Coulter Electronic, Hileah, FL) or a hemocytometer. Cytocentrifuge preparations of resuspended cells were made, stained with Wright's stain (Diff-Quick, Curtin Matheson Scientific, Denver, CO) and the differential cell count determined. Alveolar macrophages (AM) were recovered from BAL fluid of the same rats as described above. The cells (1 x 106) were suspended in Roswell Park Memorial Institute (RPMI) 1640 culture medium and pelleted by centrifugation and the supernatant removed. Cells were resuspended in 1 mL of a 1% suspension of IgG antibody-sensitized sheep red blood cells (SRBC) in RPMI 1640. The antibody-sensitized SRBC were made as previously described (Harmsen and Jeska, 1980). The subagglutinating titer of heat-inactivated rabbit anti-SRBC serum was used to sensitize the SRBC. The AM and SRBC suspensions were incubated at 37 C for 1 hour in a humidified atmosphere of 5% C 0 2 in air. The AM and SRBC were sedimented by centrifugation and the supernatant discarded. Unphagocytized SRBC were removed by lysing the red blood cells with water for 30 seconds. Lysing of unphagocytized SRBC was stopped by the addition of an equal volume of saline and cytocentrifuge preparations were made. The slides were stained with Wright's stain (Diff-Quik, American Scientific Products, McGaw Park, IL) and the percent of AM phagocytizing SRBC was determined by light microscopy. Three fields of 100 cells per preparation were counted. Viability was determined by trypan blue exclusion. Lung Tissue Collagen and Proteinase In this study, rats sacrificed at 105 weeks of talc exposure were used for collagen metabolism, protein synthesis, and proteinase activity measurements. Tissue and BAL fluid from single rats were used for analyses. To estimate collagen and protein synthesis, `"C-proline (0.1 /Ci/g body weight) was injected intraperitoneally approximately 2 to 3 hours prior to sacrifice. Lung lobes to be analyzed for collagen were frozen in liquid nitrogen and pulverized. The pulverized lungs were extracted overnight in 0.5 M acetic acid at 4 C, and centrifuged to separate the insoluble material from the supernatant fluid. The supernatant fluid was separated into high and low molecular weight fractions using Amicon Cones with a size cutoff of approximately 50 kDa. All samples for collagen analyses from lung and lavage supernatant fluid were hydrolyzed for approximately 18 hours in 6N HC1 at 110 C to convert proteins to their individual amino acids, were evaporated to dryness to remove the HC1, and were resuspended in 0.001 N HC1 prior to analysis. Lung Burden, Pulmonary Function, and Lung Biochemistry in Rats 219 Collagen quantity was measured and multiplied by 7.46 to convert BAL or lung tissue hydroxyproline content to BAL or lung tissue collagen content, taking into account that collagen is approximately 13% hydroxyproline by weight (Neuman and Logan, 1950). Radioactive proline and hydroxyproline were quantitated in the low molecular weight supernatant fluid fraction and in a sample containing both the high molecular weight supernatant fluid fraction and the acetic acid insoluble fraction. Following this, the radioactive proline and hydroxyproline quantities were used to calculate the noncollagenous protein synthesis, the collagen production, and the intracellular collagen degradation. Noncollagenous protein synthesis was measured as the total radioactive proline incorporation into lung tissue minus the incorporation into lung tissue which was related to collagen synthesis. The radioactive proline in collagen was assumed to be equal to the radioactive hydroxyproline, thus, incorporation into collagen was calculated as twice the radioactive hydroxyproline. Collagen production (% of newly synthesized protein that was collagen) was calculated as the percentage of the total incorporation of proline into all proteins constituted by collagen, and adjusted for the 5.4-fold difference in the content of total amino acids (proline and hydroxyproline) between collagen and noncollagenous protein (Pickrell et ai, 1987). Intracellular collagen degradation (as a percent of newly synthesized collagen) was calculated as the percentage of total radioactive hydroxyproline in collagen constituted by low molecular weight radioactive hydroxyproline-containing peptides. Lung tissue proteinase activity was measured as the release of 14C-leucine from prelabeled globin at pH 4.2 and 7.5 (Gregory and Pickrell, 1982; Harkema et al., 1984; Pickrell et al., 1987). Acid proteinase activity was inhibited by leupeptin to indicate either neutrophil and macrophage cathepsin B (inhibited) or macrophage cathepsin D (not inhibited)-like activity. Neutral proteinase activity was inhibited by 1,10-phenanthroline to indicate either macrophage elastase (inhibited) or neutrophil elastase-cathepsin G (not inhibited)-like activity. 220 Talc, NTP TR 421 Table G1 Number of Rats Evaluated for Lung Talc Burden, Pulmonary Function, and Lung Biochemistry 0 mg/m3 M ale 6 mg/mJ 18 mg/m3 0 mg/m3 Fem ale 6 mg/m3 Lung Burden 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim Pulm onary Function 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim Lung Biochem istry 24-Month Interim - - 9 9 9 3 3 3 3 3 6 10 10 10 6 6 a Lung burden not measured in 0 mg/m3 rats. 3 3 3 9 10 10 10 3 2 - 33 - 33 - 23 - 23 10 10 10 10 10 10 9 99 6 93 5 93 Lung Burden, Pulmonary Function, and Lung Biochemistry in Rats Table G2 Lung Talc Burden (Normalized to Control Lung Weight) of Rats* 6 m onths 12 m onths 18 m onths M ale 0 mg/m3 6 mg/m3 18 mg/m3 _b 2.63 0.24 10.83 0.23 4.38 0.59* 20.96 204* 7.31 0.71 27.57 0.91* Fem ale 0 mg/m3 6 mg/m3 18 mg/m3 2.43 0.19 8.34 0.12 4.71 0.26* 14.16 3.36 7.66 0.34** 24.33 0.63* * Significantly different (PiO.05) from the 6 month group by Dunn's or Shirley's test P<0.01 * Mean standard error, units are presented as mg lalc/g control lung. No measurements taken 221 24 months 10.45 1.26** 24.15 3.41* 9.10 0.88** 29.40 2.40** Table G3 Lung Talc Burden (Normalized to Exposure Concentration) of Rats* ______________ M ale________________ 6 mg/m3 18 mg/m3 _______________ F em ale______________ 6 mg/m3 18 mg/m3 6-Month Interim 12-Month Interim 18-Month Interim 24-Month Interim 0.439 0.040 0.731 0.098 1.22 0.12 1.74 0.21 0.602 0.013* 1.165 0.113* 1.53 0.05 1.34 0.19 0.406 0.032 0.785 0.043 1.28 0.06 1.52 0.15 0.464 0.007* 0.787 0.187 1.35 0.04 1.63 0.13 Significantly different (P<0.05) from the 6 mg/m3 group by Dunn's or Shirley's test Mean standard error, units are presented as mg talc/g control lung per mg talc/m3. 222 Talc, NTP TR 421 Table G4 Bronchoalveolar Lavage Fluid Enzymes or Rats at the 24-Month Interim Evaluation' 0 mg/m3 6 mg/m3 18 mg/m3 M ale B-Glucuronidase Lactate Dehydrogenase Alkaline Phosphatase Glutathione Reductase Total Proteinb 1.09 0.40 1,634 545 364.7 147 103.03 16.43 1.78 0.40 18.86 3.20* 3,193 606 572.8 86.8 99.35 19.79 3.12 0.64 Fem ale -Glucuronidase Lactate Dehydrogenase Alkaline Phosphatase Glutathione Reductase Total Protein 3.33 0.97 1,655 266 427.8 30.9 100.6 1.7 1.20 0.22 41.05 4.39** 3,906 444* 853.6 79.7* 135.2 22.4 4.30 0.36** * Significantly different (P<0.05) from the control group by Dunn's or Shirley's test PiO.Ol 4 Mean standard error, units presented as mlU/g control lung. Mean standard error; units presented as mg/g control lung. 89.24 14.24** 8,262 380* 1,604.7 143* 110.99 51.27 5.79 0.55* 154.16 17.21** 14,436 1,218** 2,504.7 221 ** 460.0 44.8* 12.% 0.28* * Table G5 Bronchoalveolar Lavage Fluid Cell Populations of Rats at the 24-Month Interim Evaluation' 0 mg/m3 6 mg/m3 18 m g/m 3 M ale Polymorphonuclear Cells Lymphocytes Macrophages Epithelial Cells 0.333 0.167 0.000 0.000 93.67 3.72 6.00 3.61 24.417 2.557* 0.500 0.258 70.25 2.53* 4.83 1.41 Fem ale Polymophonuclear Cells Lymphocytes Macrophages Epithelial Cells 0.625 0.315 0.000 0.000 . 91.38 1.75 8.00 2.01 25.778 2.673* * 0.722 0.188* 71.22 2.95** 2.28 0.50* Significantly different (Pi0.05) from the control group by Dunn's or Shirley's test PiO.Ol Mean standard error, units presented as percent of total cells. 32.500 3.000' 0.500 0.500 62.75 1.75* 4.25 1.75 37.000 1.528* * 1.333 0.667* 57.33 4.67* * 4.33 2.60 Lung Burden, Pulmonary Function, and Lung Biochemistry in Rats 223 Table G6 Viability and Phagocytic Activity or Macrophages in Bronchoalveolar Fluid of Rats at the 24-Month Interim Evaluation 0 m g/nr1 6 m g/ra1 18 m g/m J M ale Viability" Phagocytic Activity11 63.67 5.91 83.13 4.54 66.73 1.59 63.12 8.14 Fem ale Viability Phagocytic Activity 82.65 9.65 75.60 5.14 74.64 3.24 66.51 8.09 a Mean standard e rro r units are presented as percent viable cells. Mean standard e rro r units are presented as percent cells phagocytizing sheep erythrocytes. c n= l; no standard error calculated 57.70 5.00 65.30c 61.00 4.42 70.15 2.85 Table G7 Lung Collagen Metabolism and Protein Synthesis in Rats at the 24-Month Interim Evaluation 0 mg/m1 6 m g/m J 18 mg/mJ M ale Lavage Fluid Collagenous Peptides8 Total Lung Collagenb Collagen Production1 Collagen Degradation"1 Non-Collagenous Protein Synthesis' 39.79 13.87 1.58 31.67 142.1 5.07 0.60 0.17 1.72 14.5 46.99 15.98 1.60 27.74 199.8 6.51 0.39* 0.17 1.42 2 2.1 " Fem ale Lavage Fluid Collagenous Peptides Total Lung Collagen Collagen Production Collagen Degradation Non-Collagenous Protein Synthesis 78.27 1 1 .6 4 14.32 0.66 0.982 0.185 14.41 2.44 173.9 34.5 115.36 8.61* 19.95 1.58* 1.804 0.144* 21.59 4.99 325.8 90.9 * Significantly different (P<0.05) from the control group by Dunn's or Shirley's test * P<0.01 8 Mean standard e rro r units are presented as gg/g control lung. b Mean standard e rro r units are presented as mg/g control lung. c Mean standard e rro r units are presented as percent new protein. d Mean standard e rro r units are presented as percent new collagen. e Mean standard e rro r units are presented as disintegrations per minute x 10"3/g control lung. 79.21 13.73 18.88 3.35* 1.63 0.22 9.18 2.38* 312.2 10.6* 174.71 13.56** 36.47 3.39** 2.264 0.347** 9.38 1.63 554.3 107* 224 Talc, NTP TR 421 Table G8 Proteinase Activity in Lavage Fluid and Lung Homogenate Supernatant Fluid of Rats at the 24-Month Interim Evaluation* 0 m g/w 3 6 m g/nr 18 m g/m3 M ale Lavage Fluid Acid Proteinase Cathepsin D Cathepsin B Homogenate Supernatant Fluid Acid Proteinase Cathepsin D Cathepsin B Neutral Proteinase PMN Elastase Cathepsin G Macrophage Elastase Collagenase 0.994 0.147 0.924 0.329 0.147 0.415 10.92 0.64 8.53 0.91 2.39 0.41 0.715 -f- 0.168 0.490 0.218 0.225 0.099 1.866 0.599 1.267 0.174 0.150 0.094 17.51 14.04 3.48 2.417 1.936 0.482 0.90* 0.62* 0.37 0.304* 0.242* 0.077 Fem ale Lavage Fluid Acid Proteinase Cathepsin D Cathepsin B Homogenate Supernatant Fluid Acid Proteinase Cathepsin D Cathepsin B Neutral Proteinase PMN Elastase Cathepsin G Macrophage Elastase Collagenase 1.52 0.015 1.61 0.12 0.015 0.26 14.04 0.95 10.05 0.68 3.99 0.58 0.648 0.087 0.785 0.142 0.054 0.037 3.46 0.33* 1.310 0.292* 2.15 0.22 29.43 1.18** 22.97 1.07** 6.46 0.60* 5.040 0.418** 4351 0.261 ** 0.683 0.175* * Significantly different (P<0.05) from the control group by Dunn's o r Shirley's test PiO.Ol * Mean standard error, units are presented as mg/hour per gram control lung. n = l; no standard error calculated 4.307 0.218* 2.420 0.147** 1.887 0.365 25.13 1.50** 21.03 1.56** 4.10 0.06* 4.505b 4.457 0.377** 0.000b 6.05 4.043 2.01 0.73* * 0.578* * 0.17 38.61 131** 30.25 1.60** 8.37 0.42* * 12.293 1.598** 10.313 2.694 * 2.012 1.126* Lung Burden, Pulmonary Function, and Lung Biochemistry in Rats Table G9 Respiratory Frequency of RatsJ 0 mg/m3 6 m g/m 3 M ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 57.11 0.86 55.33 1.11 56.50 1.34 57.67 1.20 55.00 Hh 1.13 56.10 0.92 55.40 1.08 56.50 1.80 Fem ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 52.10 0.55 53.60 0.73 55.44 1.12 57.67 1.23 54.50 1.19 53.70 1.10 54.56 0.93 54.44 0.93 Significantly different (P<0.05) from the control by Dunn's or Shirley's test * Mean standard e rro r units are presented as breaths/min. 225 18 mg/m3 54.00 0.75* 53.50 0.99 54.60 1.13 56.67 1.86 54.30 0.90 55.20 0.94 55.22 1.41 59.00 0.58 Table G10 Total Lung Capacity of Rats1 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 19.86 0.54 20.06 0.32 20.30 0.45 20.50 0.83 19.48 0.46 18.44 0.39** 18.87 0.41* 20.20 0.28 F em ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 14.20 0.25 13.29 0.21 13.94 0.26 14.85 0.31 14.56 0.27 12.91 0.17 12.68 0.28* * 13.73 0.34* * Significantly different (P<0.05) from the control by Dunn's o r Shirley's test * P =<.01 * Mean standard error, units are presented as mL. 18 mg/m3 19.25 0.39 17.67 0.45* * 16.34 0.52* 16.47 1.53 13.80 0.27 12.06 0.26* 11.43 0.31** 11.50 1.07** 226 Table G il Total Lung Capacity/Kiiogram Body Weight of Rats' 0 mg/m' 6 mg/m1 M ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 51.63 1.05 47.71 0.99 45.92 1.58 51.05 4.36 51.45 1.03 44.11 0.87* 42.98 1.15 48.49 1.40 F em ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 67.73 1.26 55.21 + 1.91 45.78 1.26 49.03 1.31 67.06 1.65 52.37 1.05 43.40 1.18 48.93 2.49 * Significantly different (P<0.05) from the control by Dunn's or Shirley's test PiO.Ol ' Mean standard error, units are presented as mlVkg. Talc, NTP TR 421 18 mg/m1 52.32 0.78 43.42 0.74" 38.74 1.50" 44.16 1.29 65.41 1.50 50.24 1.19 43.26 2.42 44.54 0.51 Table G12 Tidal Volume of Rats' 0 m g/m1 6 mg/m1 M ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 1.83 0.09 1.94 0.06 1.66 0.08 1.50 + 0.00 1.90 0.08 1.91 0.06 1.63 0.08 1.85 0.16 Fem ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 1.65 0.07 1.66 0.07 1.54 0.04 1.43 0.08 1.53 0.11 1.68 0.06 1.34 0.06* 1.39 0.09 Significantly different (PS0.05) from the control by Dunn's or Shirley's test Mean standard error, units are presented as mL. 18 mg/m1 2.01 0.10 1.93 0.06 1.74 0.08 2.13 0.19' 1.40 0.071 1.43 0.09 1.40 0.03' 1.37 0.15 Lung Burden, Pulmonary Function, and Lung Biochemistry in Rats Table G13 Minute Volume of Rats4 0 mg/m3 6 m g/m 3 M ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 102.5 3.9 104.5 3.4 97.34 2.79 92.53 2.64 104.8 4.2 106.2 2.3 90.83 3.45 107.25 6.34 F em ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 85.43 4.22 87.89 3.95 87.14 2.71 83.87 5.04 83.05 4.44 88.18 3.26 73.54 3.02* 79.64 5.29 ** Significantly different (P =0.01) from the control by Dunn's or Shirley's lest 4 Mean standard error, units are presented as mL/min. 227 IS m g/m 3 104.8 3.4 100.5 2.8 95.87 4.61 117.77 11.70 76.36 78.78 76.83 82.07 3.45 3.81 2.29** 5.95 Table G14 Minute Volume/Kilogram Body Weight of Rats4 0 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 266.0 7.0 247.9 5 .9 219.4 4.6 229.5 12.7 F em ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim . 408.7 23.3 365.0 18.9 286.2 11.0 276.9 17.4 4 Mean standard error, units are presented as mLVmin per kg. 6 mg/m3 277.7 12.8 254.5 7.2 206.8 8.2 256.9 14.8 381.7 19.5 359.3 18.1 250.6 7.6 282.5 21.4 18 mg/m3 285.1 9.5 247.6 8.2 226.8 10.5 319,9 38.1 362.7 19.2 330.1 20.7 291.6 17.7 328.8 57.7 228 Table G15 Residual Volume of Rats* 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 2 9 0 0.21 206 0.17 1.96 0.15 3.23 0.48 2.99 0.17 1.63 0.10 1.74 0.13 283 0.19 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 218 0.14 1.22 0.15 1.28 0.11 1.68 0.11 239 0.22 1.25 0.17 1.52 0.13 1.72 0.23 * Significantly different (PiO.Ol) from the control by Dunn's o r Shirley's test * Mean standard error, units are presented as m L Talc, NTP TR 421 18 mg/m3 2.64 0.11 1.70 0.16 1.98 0.16 2 2 0 0.32 2.47 0.15 1.65 0.14 1.83 0.13' 1.73 0.19 Table G16 Residual Volume/Total Lung Capacity of Rats* 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.146 0.009 0.102 + 0.008 0.097 * 0.007 0.157 0.019 0.154 0.009 0.088 0.005 0.092 0.007 0.140 0.010 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.153 0.009 0.091 0.010 0.091 0.007 0.113 0.007 0.163 0.013 0.096 0.013 0.120 0.010* 0.125 0.016 * Significantly different (Pi0.05) from the control by Dunn's or Shirley's test PiO.Ol * Mean standard error, units are presented as mL/mL. 18 mg/m3 0.137 0.004 0.096 0.008 0.121 0.010 0.133 0.011 0.179 + 0.011 0.137 0.01 r 0.160 0.009' 0.151 0.005 Lung Burden, Pulmonary Function, and Lung Biochemistry in Rats Table G17 Vital Capacity of Rats*1 0 fflg/m1 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 16.% 0.49 18.01 0.27 18.35 0.45 17.27 0.48 16.49 0.44 16.82 037* 17.15 0.38 1735 0.34 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 12.02 0.22 12.06 0.20 12.66 0.21 13.15 0.27 12.17 0.20 11.68 0.18 11.14 0.31 ** 11.99 0.32* Significantly different (P<0.05) from the control by Dunn's or Shirley's test PiO.Ol 1 Mean standard error, units are presented as mL. 229 18 mg/m3 16.61 032 15.97 0.42* * 14.36 0.51 ** 14.27 1.26 11.33 0.28 10.40 035* * 9.61 0.26* * 9.77 0.90** Table G18 Vital Capadty/Total Lung Capacity of Rats1 0 mg/m3 6 mg/m3 Male 6-Month Interim 11 -Month Interim 18-Month Interim 24-Month Interim 0.854 0.009 0.898 0.008 0.904 0.007 0.843 .0.19 0.846 0.009 0.912 0.005 0.909 0.006 0.859 0.010 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.847 0.009 0.908 0.010 0.908 0.007 0.886 0.007 0.837 0.013 0.905 0.012 0.879 0.010* 0.874 0.016 * Significantly different (P<0.05) from the control by Dunn's or Shirley's test * PsO.Ol 1 Mean standard e rro r units are presented as mL/mL. 18 mg/m3 0.863 0.004 0.904 0.008 0.878 0.010 0.867 0.011 0.821 0.011 0.862 0.010* 0.841 0.009* * 0.849 0.005 230 Table G19 Forced Vital Capacity of Rats* 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 17.88 0.40 19.03 0.38 19.45 0.45 17.27 0.61 12.53 0.33 12.86 0.25 13.39 0.24 13.08 0.30 17.15 0.45 18.07 0.43* 17.92 0.34* 17.53 0.46 12.38 0.26 1244 0.26 11.91 0.28* * 12.33 0.33 Significantly different (P<0.05) from the control by Dunn's or Shirley's test P<0.01 * Mean standard error; units are presented as mL. Talc, NTP TR 421 18 mg/m3 17.38 0.41 17.25 0.45' 15.28 0.56 14.90 1.08 11.27 0.33* 11.22 0.25 ** 10.24 0.27 * 10.03 0.93* Table G20 Forced Vital Capacity/Kilogram Body Weight of Rats* 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 46.48 0.61 45.26 0.95 44.00 1.56 42.85 2.67 45.32 1.18 43.22 0.95 40.82 1.01 42.00 0.93 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 59.78 1.75 53.35 1.68 43.95 1 .1 8 43.23 1 .5 1 57.01 1.49 50.43 1.16 40.76 1.08 43.87 2.08 * Significantly different (PiO.05) from the control by Dunn's or Shirley's test PS0.01 a Mean standard error; units are presented as mL/kg 18 mg/m3 47.32 1.25 42.42 0.89 36.23 1-57" 40.18 2.32 53.37 1.48* 46.69 0.90* 38.75 Z17** 38.85 0.48 Lung Burden, Pulmonary Function, and Lung Biochemistry in Rats T a b l e G21 Functional Residual Capacity of Rats' 0 mg/m ' 6 mg/m3 M ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 4.48 0.25 3.34 0.24 3.24 0.16 4.53 0.52 4.48 0.22 3.16 0.09 3.07 0.11 3.98 0.24 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 3.51 0.12 2.78 0.12 2.47 0.08 3.07 0.13 3.72 0.16 2.74 0.10 2.82 0.12** 3.31 0.26 Significantly different (PS0.05) from the control by Dunn's or Shirley's test PiO.Ol 1 Mean standard error, units are presented as mL. 231 18 mg/rn3 4.17 0.10 3.19 0.12 3.53 0.14 4.37 0.59 3.57 0.15 2.87 0.14 3.17 0.141 3.27 0.18 T a b l e G22 Functional Residual Capacity/Tota! Lung Capacity of Rats* 6 mg/rn3 M ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.226 0.012 0.166 0.011 0.159 0.006 0.220 0.020 0.230 0.009 0.172 0.006 0.163 0.005 0.197 0.012 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.248 0.008 0.209 0.008 0.177 0.007 0.207 0.009 0.255 0.009 0.212 0.006 0.223 0.010* 0.240 0.016 * Significantly different (P<0.05) from the control by Dunn's or Shirley's test ** P<0.01 * Mean standard error, units are presented as mL/mL 18 mg/m3 0.217 0.006 0.181 0.007 0.217 0.008* * 0.268 0.042 0.258 0.008 0.238 0.010* 0.277 0.010* * 0.287 0.021* 232 Table G23 Total Pulmonary Resistance of Rats* 0 mg/m3 6 mg/m3 M ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.117 0.097 0.075 0.110 0.018 0.007 0.014 0.025 0.105 0.010 0.107 0.010 0.096 0.009 0.087 0.028 F em ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.144 0.008 0.131 0.008 0.130 0.012 0.138 0.020 0.143 0.016 0.150 0.009 0.131 0.016 0.131 0.014 * Significantly different (PsO.05) from the control by Dunn's or Shirley's test * Mean standard error; units are presented as cm H20/m L per second. Talc, NTP TR 421 18 mg/m3 0.115 0.009 0.098 0.008 0.120 0.009* 0.067 0.020 0.152 0.014 0.146 0.010 0.180 0.010* 0.150 0.035 Table G24 Maximum Quasistatic Compliance of Rats* 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 1.97 0.15 2.32 0 .1 0 2.35 0.07 2.00 0.30 1.84 0.12 1.92 0.12* 2.09 0.16 2.01 0.11 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 1.37 0.11 1.273 0.062 1.704 0.108 1338 0.055 1.47 0.11 1.276 0.033 1.123 0.050** 1.263 0.062* * Significantly different (PsO.05) from the control by Dunn's or Shirley's test P50.01 * Mean standard error; units are presented as mL/cm H20 . 18 mg/m3 2.01 0.13 1.91 0.09* 1.57 0.07* 1.48 0.20 137 0.08 0.968 0.057** 0.908 0.068* 0.883 0.093* * Lung Burden, Pulmonary Function, and Lung Biochemistry in Rats Table G25 Quasistatic Chord Compliance of Rats1 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 1.18 0.05 1.34 0.02 1.343 0.037 1.167 0.104 1.16 0.04 1.20 0.04* 1.205 0.040* 1.220 0.035 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.824 0.030 0.841 0.020 0.879 0.019 0.883 0.035 0.895 0.091 0.809 0.016 0.749 0.027* * 0.764 0.024* * Significantly different (P<0.05) from the control by Dunn's or Shirley's lest ** P<0.01 4 Mean standard error, units are presented as mL/cm H20 . 233 18 mg/m3 1.17 0.03 1.15 0.04** 0.982 0.037* 0.890 0.124 0.802 0.024 0.684 0.025 ** 0.607 0.030** 0.573 0.084* Table G26 Dynamic Compliance of Rats4 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.546 0.053 0.748 0.041 0.990 0.080 0.930 0.173 0.575 0.043 0.647 0.048 0.741 0.043* 0.987 0.130 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.399 0.029 0.492 0.024 0.618 0.053 0.650 0.065 0.445 0.032 0.426 0.027* 0.527 0.027 0.618 0.045 * Significantly different (PS0.05) from the control by Dunn's o r Shirley's test ** PiO.Ol 4 Mean standard error, units are presented as mLVcm H ,0 . 18 mg/m3 0.536 0.058 0.687 0.046 0.685 0.050* * 1.173 0.186 0380 0.034 0.393 0.020** 0372 0.025** 0377 0.077* 234 Table G27 Peak Expiratory Flow of Rats2 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 139.9 1.9 136.6 1.4 132.2 1.2 126.1 2.7 138.7 2.8 133.5 4.5 132.3 0.7 124.5 1.9 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 120.1 8.7 125.3 4.3 120.6 3.0 117.1 2.5 122.3 6.6 123.9 4.9 113.2 2.3 116.7 3.4 Significantly different (PsO.Ol) from the control by Dunn's or Shirley's test 2 Mean standard error; units are presented as mL/second. Talc, NTP TR 421 18 mg/m3 132.5 4.0 132.9 2,0 129.5 0.6* 124.0 1.0 113.5 5.7 123.2 2.1 1143 2.5 110.1 4.7 Table G28 Peak Expiratory Flow/Forced Vital Capacity of Rats* 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 7.85 0.18 7.21 0 .1 8 6.82 0.15 7.31 0.14 8.12 0.22 7.44 0.33 7.40 0.14* 7.13 0.21 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 9 3 6 0.62 9.73 0.22 9.02 0.20 8.% 0.22 9.82 0.35 9.95 0.31 9.57 0.37 9.47 0.19 * Significantly different (P<0.05) from the control by Dunn's or Shirley's test PsO.Ol 2 Mean standard error; units are presented as mL/second per mL. 18 mg/m3 7.63 0.16 7.74 0.20 837 0.29' 8.40 0.52 10.08 0.47 11.01 0.23* 11.21 0.32* 11.16 1.13" Lung Burden, Pulmonary Function, and Lung Biochemistry in Rats Table G29 Expiratory Flow 10% Forced Vital Capacity of Rats* 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 28.22 2.04 26.33 1.82 19.00 1.87 11.33 1.20 24.20 1.77 20.80 1.14* 18.00 1.61 18.67 1.50 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month interim 17.40 2.88 19.20 2.36 19.67 1.62 12.67 1.65 18.10 3.10 19.50 1.97 19.00 1.45 18.44 1.51 Significantly different (PsO.05) from the control by Dunn's or Shirley's test * Mean standard error; units are presented as mLVsecond. 235 18 mg/m3 19.60 2.67' 21.60 1.50 20.70 1.17 18.33 1.76 16.60 2.68 23.30 2.29 21.78 0.66 17.00 2.52 Table G30 Expiratory Flow 10% Forced Vital Capacity/Forced Vital Capacity of Rats* 0 mg/m3 6 mg/m3 M ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 1.58 0.11 1.39 0.10 0.986 0.106 0.661 0.085 1.41 0.09 1.16 0.08 1.002 0.085 1.057 0.065* F em ale 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 1.37 0.21 1.47 0.17 1.47 0.13 0.959 0.109 1.43 1.55 1.62 1.488 0.23 0.14 0.15 0.102* Significantly different (P<0.05) from the control by Dunn's or Shirley's test PiO.Ol * Mean standard error, units are presented as mLVsecond per raL. 18 mg/m3 1.13 1.27 1.372 1.256 0.16* 0.11 0.085* 0.188* 1.45 _ 0.22 2.07 0.19** 2.14 - 0.09* * 1.693 0.170* 236 Table G31 Expiratory Flow 25% Forced Vital Capacity of Rats* 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 6336 3.30 62.00 289 50.50 2.57 47.00 2.89 55.00 5.57 60.40 3.46 54.20 245 5133 3.97 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 44.30 7.73 50.40 5.68 52.33 4.57 40.67 3.80 41.20 7.14 43.00 5.69 42.56 4.76 49.33 6 .1 7 Significantly different (P<0.05) from the control by Dunn's o r Shirley's test PiO.Ol * Mean standard error; units are presented as mL/second. Talc, NTP TR 421 18 mg/m3 44.30 6.59** 5930 3.36 62.20 280* * 60.00 3.79 35.60 5.59 54.60 4.01 49.00 3.67 46.00 1249 Table G32 Expiratory Flow 25% Forced Vital Capacity/Forced Vital Capacity of Rats* 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 3.56 0.19 3.26 0.14 2.61 0.15 2.72 0.07 3.21 0.31 3.35 0.20 3.03 0.14* 2.92 0.20 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 3.50 0.59 3.88 0.42 3.91 0.34 3.10 0.24 3.25 0.53 3.43 0.44 3.60 0.44 3.95 0.44 Significantly different (P<0.05) from the control by Dunn's or Shirley's test * PsO.Ol * Mean standard error, units are presented as mL/second per m L 18 mg/m3 2.54 0.37 3.47 0.24 4.14 0.27' 4.06 0.35 3.10 0.43 4.88 0.37 4.75 0.27 4.66 1.28 Lung Burden, Pulmonary Function, and Lung Biochemistry in Rats Table G33 Expiratory Flow 50% Forced Vital Capacity of Rats* 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 111.33 7.11 111.7 4.4 98.75 6.00 99.33 10.17 94.00 7.61 100.1 7.1 97.10 3.59 92.33 4.47 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 75.30 1 1 .9 8 85.50 8.87 93.00 8.40 86.50 7.12 73.90 10.54 78.00 10.09 76.11 9.60 85.89 10.40 * Significantly different (P<0.05) from the control by Dunn's or Shirley's test * Mean standard error, units are presented as mLVsecond. 237 18 mg/m3 78.70 10.05* 102.1 6.2 107.70 5.25 94.67 9.02 66.00 8.52 94.10 537 87.67 6.91 83.67 23.90 Table G34 Expiratory Flow 50% Forced Vital Capacity/Forced Vital Capacity of Rats** 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 6.23 0.39 5.86 0.18 5.08 030 5.73 0.44 5.50 0.47 5.55 0.40 5.43 0.21 5.30 0.36 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 5.95 0.90 6.58 0.62 6.92 0.59 6.63 0.55 5.85 0.77 6.21 0.77 6.48 0.90 6.88 0.77 Significantly different (P<0.05) from the control by Dunn's or Shirley's test * PsO.Ol * Mean standard error; units are presented as mL/second per m L 18 mg/m3 4.49 0.55* 5.95 0.40 7.18 0.50** 638 0.62 5.79 0.67 8.39 0.48' 8.49 0.54 830 2.50 238 Table G35 Mean Midexpiratory Flow of Rats' 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 101.90 5.20 102.52 3.54 93.12 3.99 87.13 6.27 89.30 7.34 94.92 6.02 91.41 2.81 87.78 3.74 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 71.07 12.01 81.38 7.94 85.98 6.80 78.28 5.27 70.72 10.66 73.24 9.19 69.51 7.53 79.94 9.44 * Significantly different (P<0.05) from the control by Dunn's or Shirley's test ' Mean standard error; units are presented as mLVsecond. Talc, NTP TR 421 18 mg/m3 74.27 9.38' 94.11 4.51 98.44 3.67 90.33 7.07 60.65 7.99 87.91 5 .0 4 81.79 5.58 75.13 19.66 Table G36 Mean Midexpiratory Flow/Forced Vital Capacity of Rats' 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 5.71 0.30 5.39 0.16 4.78 0.13 5.04 0.24 5.23 0.44 5.27 0.35 5.11 0.18 5.03 0.29 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 5.62 0.91 6.27 0.56 6.41 0.48 5.99 039 5.59 0.78 5.83 0.70 5.90 0.72 6.40 0.69 Significantly different (P<0.05) from the control by Dunn's or Shirley's test PS0.01 Mean standard error; units are presented as mlVsecond per mL. 18 mg/m3 4.24 0.51 5.49 032 6.55 039' 6.10 0.56 531 0.62 7.85 0.451 7.94 0.41 7.65 213 Lung Burden, Pulmonary Function, and Lung Biochemistry in Rats Table G37 Carbon Monoxide Diffusing Capacity of Rats* 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.364 0.014 0.400 0.010 0.338 0.022 0.303 0.027 0.347 0.008 0.373 0.010 0.301 0.015 0.288 0.011 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.238 0.012 0.233 0.008 0.233 0.010 0.198 0.007 0.241 0.008 0.231 0.005 0.207 0.009 0.183 0.006 * Significantly different (P<0.05) from the control by Dunn's or Shirley's test ** P< 0.01 * Mean standard error; units are presented as mL/minute per mm Hg. 239 18 mg/m3 0.336 0.010 0.331 0.020* * 0.235 0.009* * 0.177 0.035* 0.213 0.010 0.190 0.003* 0.137 0.011** 0.113 0.017* Table G38 Carbon Monoxide Diffusing Capacity/Lung Volume of Rats3 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.020 0.001 0.021 0.000 0.017 0.001 0.015 0.002 0.020 0.000 0.021 0.001 0.025 0.008 0.015 0.001 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.019 0.001 0.018 0.001 0.017 0.001 0.013 0.001 0.019 0.001 0.019 0.000 0.016 0.001 0.013 0.001 * Significantly different (PS0.05) from the control by Dunn's or Shirley's test * PsO.Ol 3 Mean standard error, units are presented as mL/minute per mm Hg per m L 18 mg/m3 0.019 0.000 0.019 0.001* 0.014 0.001* 0.010 0.002* 0.017 0.001 0.017 0.000* 0.012 0.001 * * 0.009 0.001 240 Table G39 Carbon Monoxide Diffusing Capacity/Kilogram Body Weight of Rats* 0 mg/m1 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.949 0.039 0.951 0.021 0.759 0.043 0.749 0.056 0.917 0.017 0.892 0.021 0.683 0.029 0.691 0.025 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 1.13 0.05 0.968 0.045 0.766 0.034 0.656 0.031 1.11 0.04 0.939 0.033 0.705 0.028 0.650 0.027 Significantly different (P<0.05) from the control by Dunn's or Shirley's test PsO.Ol * Mean standard error, units are presented as mL/minute per mm Hg per kg. Talc, NTP TR 421 18 mg/m3 0.914 0.026 0.812 0.043 * 0.554 0.016** 0.465 0.062* 1.01 0.04 0.792 0.019* 0.502 0.028** 0.435 0.036* Table G40 Percent Forced Vital Capacity Expired in 0.1 Second of Rats* 0 mg/m3 6 mg/m3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 61.11 1.52 58.22 0.98 55.00 0.63 58.67 1.20 59.80 215 57.40 2.74 58.30 0.90* 57.00 1.71 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 62.80 5.17 67.00 2.82 66.44 2.60 65.83 2.09 64.20 3.82 65.20 3.61 64.56 3.57 66.78 3.31 Significantly different (P<0.05) from the control by Dunn's or Shirley's test ** PsO.Ol * Mean standard error; units are presented as percent forced vital capacity. 18 mg/m3 53.90 2.64 60.30 1.63 66.50 2.13** 64.00 2.89 63.40 3.86 75.50 1.78* 75.78 1.56* 73.00 9.17 Lung Burden, Pulmonary Function, and Lung Biochemistry in Rats Table G41 Slope III of N2 Washout of Rats* 0 mg/m3 6 mg/on 3 Male 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.400 0.023 0.449 0.019 0.393 0.037 0.627 0.077 0.431 0.037 0.446 0.037 0361 0.035 0.438 0.045 Female 6-Month Interim 11-Month Interim 18-Month Interim 24-Month Interim 0.587 0.059 0.704 0.027 0.601 0.053 0.535 0.040 0.528 0.049 0.735 0.029 0.699 0.074 0.580 0.071 * Significantly different (P<0.05) from the control by Dunn's or Shirley's test ** PiO.Ol * Mean standard error, units are presented as percent N2/mL. 241 18 mg/m3 0.481 0.049 0.437 0.040 0.555 0.041* 0.597 0.083 0.596 0.042 0.813 0.076 1.008 0.087** 1.520 0.409* 242 Talc, NTP TR 421 243 APPENDIX H LUNG BURDEN AND LUNG BIOCHEMISTRY IN MICE Me t h o d s ........................................................................................................................... 244 Table HI Number of Mice Evaluated for Lung Talc Burden and Lung Biochemistry ........................... 247 Table H2 Lung Talc Burden (Normalized to Control Lung Weight) of Mice .......................................... 248 Table H3 Lung Talc Burden (Normalized to Exposure Concentration) of Mice .....................................248 Table H4 Bronchoalveolar Lavage Fluid Enzymes of Mice at the 6-Month Interim E valu ation ................................................................................................. 249 Table H5 Bronchoalveolar Lavage Fluid Enzymes of Mice at the 12-Month Interim Evaluation .............................................................................................. 249 Table H6 Bronchoalveolar Lavage Fluid Enzymes of Mice at the 18-Month Interim Evaluation .............................................................................................. 250 Table H7 Bronchoalveolar Lavage Fluid Enzymes of Mice at the 24-Month Interim E v a lu a tio n ................................... 250 Table H8 Bronchoalveolar Lavage Fluid Cell Populations of Mice at the 6-Month Interim E valu ation ................................................................................................. 251 Table H9 Bronchoalveolar Lavage Fluid Cell Populations of Mice at the 12-Month Interim E v a lu a tio n ..............................................................................................251 Table H10 Bronchoalveolar Lavage Fluid Cell Populations of Mice at the 18-Month Interim Evaluation .............................................................................................. 252 Table H ll Bronchoalveolar Lavage Fluid Cell Populations of Mice at the 24-Month Interim E v a lu a tio n .............................................................................................. 252 Table H12 Phagocytic Activity of Macrophages in Bronchoalveolar Fluid of Mice at the 12-Month Interim E v a lu a tio n .................................................................................................. 253 Table H13 Phagocytic Activity of Macrophages in Bronchoalveolar Fluid of Mice at the 18-Month Interim Evaluation .............................................................................................. 253 Table H14 Viability and Phagocytic Activity of Macrophages in Bronchoalveolar Fluid of Miceat the 24-Month Interim E va lu a tio n ............................... 254 Table H15 Measurements of Lung Collagen in Miceat the 6-Month Interim E v a lu a tio n 255 ... Table H16 Measurements of Lung Collagen in Miceat the 12-Month Interim Evaluation 255 Table H17 Measurements of Lung Collagen in Miceat the 18-Month Interim Evaluation 255 Table H18 Lung Collagen Metabolism and Protein Synthesis In Mice at the 24-Month Interim Evaluation .............................................................................................. 256 Table H19 Proteinase Activity in Lavage Fluid and Lung Homogenate Supernatant Fluid of Mice at the 6-Month Interim E va lu a tio n ................................................................................. 257 Table H20 Proteinase Activity in Lavage Fluid and Lung Homogenate Supernatant Fluid of Mice at the 12-Month Interim Evaluation .............................................................................. 258 Table H21 Proteinase Activity in Lavage Fluid and Lung Homogenate Supernatant Fluid of Mice at the 18-Month Interim Evaluation .............................................................................. 259 Table H22 Proteinase Activity in Lavage Fluid and Lung Homogenate Supernatant Fluid of Mice at the 24-Month Interim Evaluation .............................................................................. 260 244 Talc, NTP TR 421 M ethods Lung Burden Lung talc burden was measured to determine the relationship between the exposure concentration and the amount of talc deposited and retained within the pulmonary region of the respiratory tract. The method used for determination of talc in the lungs of rats and mice has been published (Hanson et al., 1985). Lung talc burdens were determined on the left lung of four male and four female mice from each exposure group sacrificed at 6, 12, and 18 months after the start of exposure. At 24 months, lung burdens were determined on the left lungs of two mice from the biochemistry group. The analysis was based on determination of acid insoluble magnesium in the lung. Midwest Research Institute reported that the value for the magnesium was 19.33% for batch 02, and 19.47% for batch 03. The values reported by Midwest Research Institute and the results of the analysis at Lovelace Inhalation Toxicology Research Institute were close to the theoretical value of magnesium for talc (19.22%). Since mice sacrificed at 6, 12, and 18 months had been exposed to only batch 02 of talc, 19.33% magnesium was used to calculate quantity of talc for these mice. Since batch 03 was used for the last 4 months of exposure, and lung burdens of mice after 24 months of exposure talc would be expected to contain substantial amounts of batch 03 talc, 19.47% magnesium was used to calculate quantity of talc in lungs for these mice. All operations in conjunction with the tissue analysis for talc were done with talc-free gloves. Left lung lobes were weighed at necropsy and stored frozen (-20 C) until used. Lungs were homogenized using water and the proteins precipitated with 70% perchloric acid. The individual samples were filtered and washed with 5% trichloroacetic acid (TCA) to remove perchlorates. Washing continued until magnesium levels in the wash were within 10% of levels in the TCA solution (< 0.03 ppm magnesium). Filters and tissue residues were placed in 15-mL porcelain crucibles, dried slowly (200 C), and then ashed at 600 C for 1 hour. Ashed samples were transferred to Teflon beakers using 2 mL HC1 and evaporated to dryness. Samples were digested in hydrofluoric acid (HF), and the HF evaporated. Additional HF was added and reevaporated. Sulfuric acid was added to remove trace HF, and samples were diluted with distilled water and analyzed for magnesium by atomic absorbance (Perkin Elmer, Model 306, Atomic Absorption Spectrophotometer) with a magnesium hollow cathode lamp and an air acetylene flame (Hanson et al., 1985). Lung Biochemistry In this study, bronchoalveolar lavage (BAL) fluid enzyme activity and cell numbers were measured as biochemical and cytological indicators of pulmonary injury from inhalation of talc. Four male and four female mice from each exposure group were sacrificed at 27, 52, and 79 weeks, and all remaining lung toxicology mice were sacrificed at 24 months. Numbers of animals sacrificed at each interim evaluation are shown in Table HI. Mice were anesthetized with halothane and sacrificed by exsanguination from the abdominal aorta or renal artery. The heart and lung block were removed. Mice were administered endobronchial saline lavage (3 to 4 mL total volume in four, 0.75 to 1.0 mL washes) and the BAL fluid centrifuged at 300 x G to separate the cells from the supernatant fluid. At all sacrifices, biochemical analyses were done on lavage fluid from single mice. At the 24-month terminal sacrifice where lung burden measurements were also performed on the left lung lobes, mouse lavage fluids were paired (from two mice) to obtain sufficient cells for the analyses and paired mouse lung tissue samples (from two mice) were analyzed to obtain sufficient lung tissue for collagen analyses. Lung Burden and Lung Biochemistry of Mice 245 Airway Fluid Enzymes and Cytology In this study, BAL fluid was analyzed to determine degree of: 1) Cell injury as indicated by quantities of BAL fluid lactate dehydrogenase (LDH). 2) Chronic inflammatory response as indicated by presence of increased numbers of polymorphonuclear leukocytes (PMN) and pulmonary alveolar macrophages (AM) as well as increased BAL fluid protein and alkaline phosphatase activity. 3) Lysosomal activation as indicated by quantities of BAL fluid B-glucuronidase and acid proteinase. Elevated quantities of these enzymes have been observed in BAL fluid from rodents exposed to particulates. These enzymes may be associated with the breakdown of necrotic tissues. 4) Response to oxidant injury as indicated by increased quantities of glutathione reductase and peroxidase activity. The supernatant fluid was analyzed for the activities of B-glucuronidase, LDH, glucose-6-phosphate dehydrogenase, alkaline phosphatase, glutathione reductase, and glutathione peroxidase by spectrophotometric, kinetic, and enzymatic techniques. Acid proteinase was measured by release of radiolabeled globin from the trichloroacetic acid precipitatible protein substrate, and total protein was analyzed colorimetrically (Henderson et al., 1985). B-Glucuronidase was not performed at the 6-month interim evaluation, but was performed at all other sacrifice times. Numbers of total nucleated cells recovered in lavage fluid were determined on each sample using a cell counter (Coulter Electronics, Hileah, FL) or a hemocytometer. Cytocentrifuge preparations of resuspended cells were made, stained with Wrights stain (Diff-Quik, Curtin Matheson Scientific, Denver, CO) and differential cell counts were determined. At the 6-, 12-, and 18-month interim sacrifices, analyses were done on individual mice. Alveolar macrophages (AM) were recovered from BAL fluid of the same mice as described above. Cells (0.5 x 106) in Roswell Park Memorial Institute (RPMI) culture medium were pelleted by centrifugation and the supernatant removed. Cells were resuspended in 1 mL of a 1% suspension of IgG antibodysensitized sheep red blood cells (SRBC) in RPMI 1640. The antibody sensitized SRBC were made as previously described (Harmsen and Jeska, 1980). The subagglutinating titer of heat-inactivated rabbit anti-SRBC serum was used to sensitize the SRBC. The AM and SRBC suspensions were incubated at 37 C for 1 hour in a humidified atmosphere of 5% C 0 2 in air. The AM and SRBC were sedimented by centrifugation and the supernatant discarded. Unphagocytized SRBC were removed by lysing the red blood cells with water for 30 seconds. The lysing of unphagocytized SRBC was stopped by the addition of an equal volume of saline and cytocentrifuge preparations were made. The slides were stained with a rapid Wright's stain (Diff-Quik, American Scientific Products, McGaw Park, IL) and the number of AM phagocytizing 0, 1, 2, 3 to 4, and > 4 SRBC was determined by light microscopy. Three fields of 100 cells per preparation were counted. Viability of macrophages was not determined at the 6-, 12-, and 18-month week sacrifices because the small number of cells recovered from these mice lungs precluded the measurement of cell viability. Viability determination of macrophages was made on macrophages obtained at the final sacrifice because sufficient numbers of cells were generally available at this time. Lung Tissue Collagen and Proteinase At 6-, 12-, and 18-month sacrifices, collagen content of lungs and lavage fluid was measured. At the 24-month sacrifice, additional collagen metabolism and protein synthesis measurements were made on survivors from each group. Proteinase activities were measured at all sacrifice times. 246 Talc, NTP TR 421 The supernatant BAL fluid was analyzed for hydroxyproline and acid proteinase. Lung tissue and bronchoalveolar lavage (BAL) fluid samples were hydrolyzed with 6N HC1 at 110 C for approximately 18 hours to convert proteins to their individual amino acids. Collagen quantity was measured and multiplied by 7.46 to convert BAL or lung tissue hydroxyproline content to BAL or lung tissue collagen content, taking into account that collagen is approximately 13% hydroxyproline by weight (Neuman and Logan, 1950). Additional collagen metabolism measurements were made on the mice sacrificed after 24 months of talc exposure to further define collagen metabolism. Approximately 2 to 3 hours prior to sacrifice, HC-proline (0.1 /LtCi/g body weight) was injected intraperitoneally to estimate collagen and protein synthesis. Radioactive proline and hydroxyproline were quantitated in lung hydrolysate. Following this, the radioactive proline and hydroxyproline quantities were used to calculate the noncollagenous protein synthesis, and the collagen production. Noncollagenous protein synthesis was indicated as total '"C-proline incorporation into lung tissue minus the incorporation into lung tissue which was related to collagen synthesis. The radioactive proline in collagen was assumed to be equal to the radioactive hydroxyproline, thus, incorporation into collagen was calculated as twice the radioactive hydroxyproline. Collagen production (% of newly synthesized protein that was collagen) was calculated as the percent of the total incorporation of proline into all proteins constituted by collagen, and adjusted for the 5.4-fold difference in the content of total amino acids (proline and hydroxyproline) between collagen and noncollagenous protein (Pickrell et al., 1987). At each sacrifice time, lung tissue proteinase activity was measured as the release of I4C-leucine from prelabeled globin at pH 4.2 and 7.5 (Gregory and Pickrell, 1982; Harkema et al., 1984; Pickrell et al., 1987). Acid proteinase activity was inhibited by leupeptin to indicate either cathepsin B (inhibited) or cathepsin D (not inhibited)-like activity. Neutral proteinase activity was inhibited by 1,10-phenanthroline to indicate either macrophage elastase (inhibited) or neutrophil elastase-cathepsin G (not inhibited)-like activity. Lung Burden and Lung Biochemistry of Mice Table HI Number of Mice Evaluated for Lung Talc Burden and Lung Biochemistry ________________ M ale________________ 0 mg/m3 6 mg/m3 18 mg/m3 _______________ F em ale 0 mg/m3 6 mg/m3 Lung Burden 6-Month Interim 12-Month Interim 18-Month Interim 24-Month Interim Lung Biochem istry 6-Month Interim 12-Month Interim 18-Month Interim 24-Month Interim - - 4 4 4 9 2 4 2 8 4 4 4 8 a Lung burden nol measured in 0 mg/m3 mice 4 4 1 6 4 4 4 6 -4 -4 -4 6 44 44 44 76 4 4 3 5 4 4 4 5 247 248 T a b l e H2 Lung Talc Burden (Normalized to Control Lung Weight) of Mice3 6 m onths 12 months 18 m onths M ale 0 mg/m3 6 mg/m3 18 mg/m3 _b 0.415 0.114 1.41 0.29 1.084 0.130 9.00 1.45* 0.426 0.040 8.36c Fem ale 0 mg/m3 6 mg/m3 18 mg/m3 0.524 0.056 1.35 0.24 0.707 0.170 6.17 1.39* 1.387 0.178** 7.83 1.36* Significantly different (P<0.05) from the 6 month group by Dunn's or Shirley's test P<0.01 3 Mean standard error; units are presented as mg talc/g control lung. Not examined c n = l; no standard error calculated Talc, NTP TR 421 24 months 2.973 0.762* 19.73 4.03** 2.667 0.720* 20.05 0.98* * T a b l e H3 Lung Talc Burden (Normalized to Exposure Concentration) of Mice3 6 mg/m3 Male 18 mg/m3 Female 6 mg/m3 18 mg/m3 6-Month Interim 12-Month Interim 18-Month Interim 24-Month Interim 0.069 0.019 0.181 0.022 0.071 0.007 0.496 0.127 0.078 0.016 0.500 0.081* 0.464b 1.096 0.224* 0.087 0.009 0.118 0.028 0.231 0.030 0.445 0.120 0.075 0.013 0.343 0.077* 0.435 0.075 1.114 0.055* Significantly different (PsO.05) from the 6 mg/m3 group by Dunn's or Shirley's test 3 Mean standard error, units are presented as mg talc/g control lung per mg talc/m3 b n = l; no standard error calculated Lung Burden and Lung Biochemistry or Mice 249 Table H4 Bronchoalveolar Lavage Fluid Enzymes of Mice at the 6-Month Interim Evaluation1 0 mg/m3 6 mg/m3 18 mg/m3 M ale Lactate Dehydrogenase Glutathione Reductase Total Proteinb 1,408 658 148.4 33.8 3.57 0.89 Fem ale Lactate Dehydrogenase Glutathione Reductase Total Proteinb 1,988 157 206.8 14.7 2.55 0.53 a Mean standard error; units are presented as mlU/g control lung. b Mean standard error; units are presented as mg/g control lung. 1,317 106 123.3 28.3 1.92 0.70 2/351 180 166.0 21.3 4.43 0.34 2,107 336 227.2 65.6 6.24 1.23 1,400 197 148.5 29.4 6.89 4.29 Table H5 Bronchoalveolar Lavage Fluid Enzymes of Mice at the 12-Month Interim Evaluation3 0 m g/m 3 6 mg/m3 18 mg/m3 M ale -Glucuronidase Lactate Dehydrogenase Glutathione Reductase Total Proteinb 0.188 0.114 1,107.6 545 89.50 11.65 2.21 0.74 0.486 0.346 540.2 59.0 91.67 6.60 1.56 0.33 F em ale* -Glucuronidase Lactate Dehyrogenase Glutathione Reductase Total Proleinb 0.073 0.073 1,209.7 305 113.57 19.78 3.54 1.27 0.413 0.251 447.5 76.1 97.93 14.93 3.61 1.38 * Significantly different (P<0.05) from the control group by Dunn's or Shirley's test * PiO.Ol 3 Mean standard error; units are presented as mlU/g control lung. Mean standard error, units are presented as mg/g control lung. 12.787 3.604 1,487.1 456 302.40 65.15 6.19 2.63 9.786 2.271 1,805.3 285 198.65 23.44 4.82 2.88 250 T a lc , N T P T R 421 T able H6 Bronchoalveolar Lavage Fluid Enzymes of Mice at the 18-Month Interim Evaluation9 0 mg/m3 6 mg/m3 18 mg/m3 M ale -Glucuronidase Lactate Dehydrogenase Glutathione Reductase Total Proteinb 0.000 0.000 434.0 45.7 63.93 14.16 3.43 0.62 1.344 1.267 642.4 119 106.38 12.15 6.23 0.97* Fem ale B-Glucuronidase Lactate Dehydrogenase Glutathione Reductase Total Protein1* 4.243 4.203 501.4 46.9 73.19 14.94 2.96 0.40 0.334 0.334 404.2 97.6 71.27 12.11 3.41 0.92 Significantly different (P<0.05) from the control group by Dunn's or Shirley's test P<0.01 9 Mean standard error; units are presented as mlU/g control lung. Mean standard error; units are presented as mg/g control lung. 9.937 4.196" 1,039.9 168" 217.18 45.29* 9.45 1.95** 19.064 9.200 1,217.6 255* 240.55 44.06* 9.59 1.23* Table H7 Bronchoalveolar Lavage Fluid Enzymes of Mice at the 24-Month Interim Evaluation9 0 mg/m3 6 m g/m 3 18 mg/m3 M ale -Glucuronidase Lactate Dehydrogenase Glutathione Reductase Total Proteinb 0.000 0.000 1,769 259 73.66 9.75 1.69 0.20 1.811 0.878" 1,439 295 87.55 25.16 2.34 0.22 F em ale -Glucuronidase Lactate Dehydrogenase Glutathione Reductase Total Protein1* 0.000 0.000 1,082 155 68.66 7.42 1.111 0.310 2.624 1.176** 1,596 197* 73.37 13.91 0.872 0.261 Significantly different (P<0.05) from the control group by Dunn's or Shirley's test P<0.01 9 Mean standard error; units are presented as m|U/g control lung. b Mean standard error; units are presented as mg/g control lung. 16.571 3.932* * 2,965 1 3 1 * 229.53 58.46* 4.68 0.70" 13.778 2.640" 2,026 279* 163.46 33.43* 2.228 0.501 Lung Burden and Lung Biochemistry of Mice 251 Table H8 Bronchoalveolar Lavage Fluid Cell Populations of Mice at the 6-Month Interim Evaluation3 0 mg/inJ 6 mg/m3 18 mg/m3 M ale Polymorphonudeated Cells Lymphocytes Macrophages Epithelial Cells 0.250 0.250 0.750 0.750 92.50 3.23 6.500 3.775 3.250 1.250 0.750 0.479 95.75 1.44 0.250 0.250 Fem ale Polymorphonuclear Cells Lymphocytes Macrophages Epithelial Cells 0.000 0.000 0.000 0.000 95.00 2.16 5.00 2.16 1.250 0.629* 1.000 1.000 94.75 1.44 3.00 1.73 Significantly different (P<0.05) from the control group by Dunn's or Shirley's test P<0.01 3 Mean standard error; units are presented as percent of total cells. 12.000 3.764 0.000 0.000 84.75 2.95 3.250 1.250 1.750 0.854' 0.000 0.000 96.00 1.22 2.25 1.31 T a b l e H9 Bronchoalveolar Lavage Fluid Cell Populations of Mice at the 12-Month Interim Evaluation*3 0 mg/m3 6 m g/m 3 18 m g/m 3 M ale Polymorphonuclear Cells Lymphocytes Macrophages Epithelial Cells 26.75 15.12 0.750 0.250 70.50 14.56 2.00 1.41 7.50 5.85 2.250 1.436 83.25 6.91 7.00 2.12 Fem ale Polymorphonuclear Cells Lymphocytes Macrophages Epithelial Cells 1.33 1.33 1.000 0.577 92.67 0.33 5.00 1.53 34.50 10.27* 3.500 1.500 58.25 11.65 3.75 1.75 * Significantly different (P<0.05) from the control group by Dunn's or Shirley's test 3 Mean standard error, units are presented as percent of total cells. 15.00 14.01 0.333 0.333 73.33 12.14 11.33 7.36 2.25 0.85 0.000 0.000 91.00 2.04 6.75 2.84 252 Talc, NTP TR 421 Table H10 Bronchoalveolar Lavage Fluid Cell Populations of Mice at the 18-Month Interim Evaluation' 6 m g/m 3 18 m g/m 3 M ale Polymorphonuclear Cells Lymphocytes Macrophages Epithelial Cells 0.250 0.250 0.000 0.000 89.00 * 1.22 10.75 1.44 8.750 4.404 0.500 0.500 82.75 5.81 8.00 4.74 F em ale Polymorphonuclear Cells Lymphocytes Macrophages Epithelial Cells 0.250 0.250 0.000 0.000 84.50 5.52 15.25 5.54 1.000 0.577 0.000 0.000 92.67 0.88 6.33 0.88 * Significantly different (P<0.05) from the control group by Dunn's or Shirley's test a Mean standard error, units are presented as percent of total cells. 19.000 6.258* 1.000 0.577 75.75 4.73 4.25 2.39 16.000 * 3.606' 1.333 0.882' 79.00 3.06 3.67 2.33 Table HII Bronchoalveolar Lavage Fluid Cell Populations of Mice at the 24-Month Interim Evaluation3 0 mg/m3 6 m g/m 3 18 mg/m3 M ale Polymorphonuclear Cells Lymphocytes Macrophages Epithelial Cells 0.200 0.200 0.000 0.000 89.10 2.50 10.70 2.61 13.000 2.345* 0.375 0.239 78.25 1.61 8.38 1.01 F em ale Polymorphonuclear Cells Lymphocytes Macrophages Epithelial Cells 0.000 0.000 0.000 0.000 86.38 3.57 13.63 3.57 7.500 1.607* 0.500 0.500 87.00 2.08 5.00 1.00 * Significantly different (P<0.05) from the control group by Dunn's or Shirley's test * * P<0.01 3 Mean standard error; units are presented as percent of total cells. 16.500 1.803* * 0.500 0.289 80.33 0.60* 2.67 1.59 20.667 5.918** 0.500 0.500 73.67 8.46 5.17 3.03 Lung Burden and Lung Biochemistry of Mice Table H12 Phagocytic Activity of Macrophages in Bronchoalveolar Fluid of Mice at the 12-Month Interim Evaluation* 0 m g/m ] 6 m g/m 3 M ale Phagocytic Activity 85.50 1.44 56.10 2.23* Fem ale Phagocytic Activity 77.07 9.88 52.10 9.22 Significantly different (P<0.05) from the control by Dunn's or Shirley's test P<0.01 * Mean standard error, units are presented as percent cells phagocytizing sheep erythrocytes. 253 IS m g/m 3 16.77 2.98** 17.37 6.17* Table H13 Phagocytic Activity of Macrophages in Bronchoalveolar Fluid of Mice at the 18-Month Interim Evaluation* 0 m g/m 3 6 m g/m 3 M ale . Phagocytic Activity 37.43 8.55 14.10 4.54 Fem ale Phagocytic Activity 46.85 11.08 20.03 7.45 Significantly different (PsO.05) from the control by Dunn's or Shirley's test Mean standard error, units are presented as percent cells phagocytizing sheep erythrocytes. 18 m g/m 3 11.98 2.22* 6.65 0.35* 254 Talc, NTP TR 421 Table H14 Viability and Phagocytic Activity of Macrophages in Bronchoalveolar Fluid of Mice at the 24-Month Interim Evaluation 0 mg/m' 6 tng/m ' 18 mg/niJ M ale Viability* Phagocytic Activity15 79.20 3.44 37.14 9.80 64.60 4.15 11.90 4.64 Fem ale Viability Phagocytic Activity 60.50 8.80 21.57 6.77 47.17 2.74 13.60 4.71 Significantly different (P<0.05) from the control by Dunn's or Shirley's test PsO.Ol * Mean standard error, units are presented as percent viable cells. Units are presented as percent cells phagocytizing sheep erythrocytes. 83.23 0.87 3.56 2.25 59.77 3.21 4.35 2.65* Lung Burden and Lung Biochemistry or Mice Table H15 Measurements of Lung Collagen in Mice at the 6-Month Interim Evaluation 0 mg/m3 6 mg/m3 M ale Lavage Fluid Collagenous Peptides*4 Total Lung Collagen1* Fem ale Lavage Fluid Collagenous Peptides Total Lung Collagen 67.13 9.76 7.42 0.48 42.92 8.49 4.69 0.35 2 Mean standard error, units are presented as (tg/g control lung. b Mean standard error, units are presented as mg/g control lung. 24.83 8.18 7.51 1.38 70.83 9.09 5.85 0.89 Table H16 Measurements of Lung Collagen in Mice at the 12-Month Interim Evaluation 0 m g/m 3 6 m g/m 3 M ale Lavage Fluid Collagenous Peptides4 Total Lung Collagen1* Fem ale Lavage Fluid Collagenous Peptides Total Lung Collagen 74.23 9.42 11.94 0 47 89.88 12.99 11.64 0.48 68.73 4.11 12.44 0.82 73.66 11.58 11.84 0.45 * Significantly different (P<0.05) from the control by Dunn's or Shirley's test 4 Mean standard error, units are presented as jtg/g control lung. Mean standard error, units are presented as mg/g control lung. Table H17 Measurements of Lung Collagen in Mice at the 18-Month Interim Evaluation 0 m g/m 3 6 m g/m 3 M ale Lavage Fluid Collagenous Peptides4 Total Lung Collagen1* Fem ale Lavage Fluid Collagenous Peptides Total Lung Collagen 42.54 2 .1 5 6.60 0.49 54.09 11.27 6.16 0.25 51.18 5.40 7.13 0.30 37.68 6.01 6.96 0 .3 1 * Significantly different (P<0.01) from the control by Dunn's or Shirley's lest 4 Mean standard error; units are presented as gg/g control lung. Mean standard e rro r units are presented as mg/g control lung. 255 18 mg/m3 79.64 18.03 12.27 4.53 51.17 5.14 11.00 3.88 18 m g/m 3 117.62 11.07* 13.30 1 .1 1 108.55 7.56 13.78 1.09 18 m g/m 3 70.67 8.41 ** 9.70 0.70* * 64.88 6.56 7 .3 4 0 .4 3 256 Talc, NTP TR 421 T a b l e H18 Lung Collagen Metabolism and Protein Synthesis in Mice at the 24-Month Interim Evaluation 0 m g/m 3 6 m g/m3 18 m g/m3 M ale Lavage Fluid Collagenous Peptides* Total Lung Collagenb Collagen Production0 Non-Collagenous Protein Synthesis"1 54.39 4.42 8.53 0.71 1.133 0.274 68.48 10.41 65.98 5.01 8.55 0.59 0.779 0.151 58.84 4.19 Fem ale Lavage Fluid Collagenous Peptides Total Lung Collagen Collagen Production0 Non-Collagenous Protein Synthesis"1 38.09 4.38 6.04 0.27 1.15 0.33 52.46 8.60 39.26 4.01 6.41 0.36 1.65 0.13 47.55 6.94 Significantly different (PiO.05) from the control by Dunn's or Shirley's test * P<0.01 * Mean standard error; units are presented as (tg/g control lung. Mean standard error, units are presented as mg/g controllung. 0 Mean standard error, units are presented as percent new protein. Mean standard error; units are presented as disintegrations per minute x 10'3/g control lung. 91.92 4.93** 13.71 2.81* 1.554 0.291 93.73 9.73 62.14 9.04' 7.91 0.35' 1.33 0.12 84.51 4.84 Lung Burden and Lung Biochemistry or Mice 257 Table H19 Proteinase Activity in Lavage Fluid and Lung Homogenate Supernatant Fluid of Mice at the 6-Month Interim Evaluation3 0 mg/m3 6 m g/m 3 18 m g/m 3 M ale Lavage Fluid Acid Proteinase Cathepsin D Cathepsin B Homogenate Supernatant Fluid Acid Proteinase Cathepsin D Cathepsin B Neutral Proteinase PMN Elastase Cathepsin G Macrophage Elastase Collagenase 1.27 0.078 1.006 0.24 0.038 0.239 5.83 1.07 2.27 0.46 3.56 0.80 0.634 0.039 0.446 0.014 0.207 0.058 1.65 0.656 0.992 0.47 0.321* 0.716 8.10 0.78 3.30 0.57 4.80 0.58 0.360 0.043* 0.418 0.357 0.340 0.154 Fem ale Lavage Fluid Acid Proteinase Cathepsin D Cathepsin B Homogenate Supernatant Fluid Acid Proteinase Cathepsin D Cathepsin B Neutral Proteinase PMN Elastase Cathepsin G Macrophage Elastase Collagenase 0.762 -*2 0.089 0.457 0.166 0.260 0.068 4.35 0.31 1.78 0.12 2.57 0.22 0.522 0.047 0.416 0.033 0.106 0.043 1.595 0.038** 0.998 0.016 0.571 0.063 6.95 0.61* 3.89 1.52* 3.06 + 1.01 0.535 0.039 0.347 0.066 0.188 0.058 Significantly different (P<0.05) from the control group by Dunn's or Shirley's test PiO.Ol 3 Mean standard error, units are presented as mg/hour per gram control lung. n=0; no data recorded c n = l; no standard error calculated 2.05 0.23 0.876 0.107' 0.954 0.010 7.45 0.64 _b - - 1.346 0.097 0.628 0.113 0.718 0.094' 5.77 0.61 3.12 0.06* 2.65 0.56 0.848c - 258 Talc, NTP TR 421 Table H20 Proteinase Activity in Lavage Fluid and Lung Homogenate Supernatant Fluid of Mice at the 12-Month Interim Evaluation* 0 mg/m3 6 mg/m3 18 mg/m3 M ale Lavage Fluid Acid Proteinase Cathepsin D Cathepsin B Homogenate Supernatant Fluid Acid Proteinase Cathepsin D Cathepsin B Neutral Proteinase PMN Elastase Cathepsin G Macrophage Elastase Collagenase 1.65 0.13 0.403 0.163 1.25 0.10 7.21 0.50 5.32 0.27 1.89 0.48 0.386 0.055 0.110 0.110 0.426 0.159 2.11 0.82 0.970 0.244 1.25 0.78 9.35 0.07* 7.71 0.16* 1.64 0.10 1.029 0.416 0.005 0.005 1.127 0.422 Fem ale Lavage Fluid Acid Proteinase Cathepsin D Cathepsin B Homogenate Supernatant Fluid Acid Proteinase Cathepsin D Cathepsin B Neutral Proteinase PMN Elastase Cathepsin G Macrophage Elastase Collagenase 1.94 0.526 1.50 0.17 0.263 0.41 7.88 0.24 6.40 H- 0.70 1.55 0.54 0.423 0.183 0.215 0.125 0.280 0.116 1.79 0.35 0.463b 2.14b 10.48 0.50* 8.44 0.51 2.04 0.22 0.601 0.108 0.213 0.213 0.446 0.127 Significantly different (P<0.05) from the control group by Dunn's or Shirley's test PiO.Ol * Mean standard error; units are presented as mg/hour per gram control lung. n= 1; no standard error calculated 3.25 0.28 1.796 0.306** 1.46 0.05 16.50 0.95* * 14.32 1.27* 2.18 0.39 1.088 0.271* 0.209 0.148 0.879 0.162 3.60 0.33* 1.525 0.266' 2.08 0.08 16.92 14.76 2.16 0.824 0.190 0.653 1.84*' 1.59*' 0.55 0.057 0.124 0.158 Lung Burden and Lung Biochemistry of Mice 259 Table H21 Proteinase Activity in Lavage Fluid and Lung Homogenate Supernatant Fluid of Mice at the 18-Month Interim Evaluation* 0 fflg/m1 6 mg/m1 18 mg/m1 M ale Lavage Fluid Acid Proteinase Cathepsin D Cathepsin B Homogenate Supernatant Fluid Acid Protinase Cathepsin D Cathepsin B Neutral Protinase PMN Elastase Cathepsin G Macrophage Elastase Collagnase 0.264 0.044 0.212 0.046 0.069 0.037 3.29 0.58 2.71 0.24 0.607 0.327 0.425 0.079 0.158 0.066 0.286 0.093 0.428 0.120 0.073 0.013* 0.355 0.127* 4.76 0.49 4.98 0.63* 0.053 0.053 0.548 0.022 0.242 0.061 0.306 0.041 Fem ale Lavage Fluid Acid Proteinase Cathepsin D Cathepsin B Homogenate Supernatant Fluid Acid Proteinase Cathepsin D Cathepsin B Neutral Proteinase PMN Elastase Cathepsin G Macrophage Elastase Collagenase 0.267 0.103 0.219 0.085 0.088 0.034 3.97 0.41 3.28 0.23 0.694 0.284 0.381 0.041 0.265 0.038 0.116 + 0.033 0.561 0.126 0.012 0.012 0.587 0.095* 5.57 0.26* 5.37 0.16* 0.232 0.096 0.540 0.036* 0.391 0.038 0.149 0.054 Significantly different (P<0.05) from the control group by Dunn's or Shirley's test P<0.01 1 Mean standard error, units are presented as mg/hour per gram control lung. 0384 0.066 0.051 0.035* 0.342 _ 0.057* 8.38 0.85 * 8.45 - 0.63* * 0.403 0.270 0.528 0.034 0.254 0.017 0.275 0.031 0.382 0.040 0.062 0.036 0.358 0.098* 9.03 0.88* * 9.17 0.75 ** 0.265 0.265 0.583 0.035* 0.268 0.041 0.315 0.045* 260 Talc, NTP TR 421 Table H22 Proteinase Activity in Lavage Fluid and Lung Homogenate Supernatant Fluid of Mice at the 24-Month Interim Evaluation* 0 mg/m3 6 mg/m3 18 mg/m3 M ale Lavage Fluid Acid Proteinase Cathepsin D Cathepsin B Homogenate Supernatant Fluid Acid Proteinase Cathepsin D Cathepsin B Neutral Proteinase PMN Elastase Cathepsin G Macrophage Elastase Collagenase 1.62 0.14 0.000 0.000 1.94 0.19 9.23 1.16 6.63 0.96 2.60 0.39 0.417 0.072 0.251 0.034 0.166 0.063 1.92 0.18 0.260 0 .1 5 6 1.72 0.28 13.85 1.56 10.82 0.98* 3.03 0.78 0.568 0.104 0.382 0.093 0.186 0.040 Fem ale Lavage Fluid Acid Proteinase Cathepsin D Cathepsin B Homogenate Supernatant Fluid Acid Proteinase Cathepsin D Cathepsin B Neutral Proteinase PMN Elastase Cathepsin G Macrophage Elastase Collagenase 0.854 0.077 0.194 0.089 0.708 0.118 7.83 1.11 5.10 0.67 2.73 0.47 0.454 0.096 0.172 0.063 0.421 0.293 1.012 0.149 0.114 0.114 1.000 0.365 9.76 0.56 8.04 0.95 1.71 0.57 0.646 0.143 0.341 0.082 0.314 0.162 Significantly different (P<0.05) from the control group by Dunn's or Shirley's test P<0.01 * Mean standard error, units are presented as mg/hour per gram control lung. 3.56 0.67* 1.613 0.632* 1.78 0.29 24.34 2.66* 18.75 1.73* * 5.58 1.11* 0.862 0.164* 0.341 0.106 0.521 0.250 0.998 0.212 0.402 0.146 0.596 0.305 22.54 1.29* 17.93 035 ** 4.61 1.00 0.922 0.077* 0360 0.093 0.563 0.102 261 APPENDIX I CHEMICAL CHARACTERIZATION, ANALYSIS, AND GENERATION OF CHAMBER CONCENTRATIONS Procurement and Characterization of Ta l c ....................................................................................... 262 Generation and Monitoring of Chamber Co ncentratio ns............................................................... 264 Figure II Infrared Absorption Spectrum of T a l c ........................................................................................... 266 Figure 12 Fluid Bed G e n e ra to r............................................................................................................... 267 Figure 13 Aerosol Dilution/Delivery System ..........................................................................................268 Figure 14 Talc Chronic Exposure S ystem ...............................................................................................269 Figure 15 Talc Aerosol Filter Concentrations in the 6 mg/m3 Rat C h am b er.................................... 270 Figure 16 Talc Aerosol Filter Concentrations in the 18 mg/m3 Rat C h a m b e r.................................... 271 Figure 17 Talc Aerosol Filter Concentrations in the 6 mg/m3 Mouse C ham ber..................................272 Figure 18 Talc Aerosol Filter Concentrations in the 18 mg/m3 Mouse C h am b er............................ 273 Table II Summary of Aerosol Size Measurements for the 6 and 18 mg/m3 Rat Chambers ................................................................................ 274 Table 12 Summary of Aerosol Size Measurements for the 6 and 18 mg/m3 Mouse Chambers ........................................................................... 275 262 Talc, NTP TR 421 CHEMICAL CHARACTERIZATION, ANALYSIS, AND GENERATION OF CHAMBER CONCENTRATIONS P r o c u r e m e n t an d C h a r a c ter iza tio n o f T alc Talc was obtained from Walsh and Associates (North Kansas City, MO) in two lots (W101882 and B5415). Lot W101882 was used from the beginning of the 2-year studies through 26 January 1986. Lot B5415 was used in the 2-year studies from 27 January 1986 to the end of the studies. The talc was extensively characterized by the analytical chemistry laboratory, Midwest Research Institute (MRI; Kansas City, MO) and McCrone Associates (Norcross, GA). Reports on analyses performed in support of the talc studies are on file at the National Institute of Environmental Health Sciences. The two lots of the chemical, a finely powdered white solid, were identified as talc by infrared spectroscopy. All spectra were consistent with those expected for the structure and with the literature spectra of talc (Sadtler Standard Spectra), as shown in Figure II. Lot W101882 was divided into three subbatches, which were analyzed separately. Each subbatch was characterized by elemental analyses, Karl Fischer water analysis, spark source mass spectrometry, and microscopic analyses. Microscopic analysis of each lot consisted of polarized light microscopy (PLM) and transmission electron microscopy (TEM). For PLM the sample was mounted in refractive index liquids and the optical parameters were determined. Dispersion staining has the advantage that small quantities of asbestos can easily be detected since the optical properties are interpreted from bright colors seen on a black background. The colors seen are the results of differences in refractive index dispersion for a liquid and a solid. TEM was performed by sonically dispersing approximately 0.1 g of talc in a solution of 0.001% methyl cellulose in particle-free water. A drop of the suspension was placed on a carbon coated 200-mesh copper grid, and 20 grid openings were examined. The detection limit was 0.1% by weight. No asbestos fibers were detected in any of the subbatches by polarized light microscopy or transmission electron microscopy. Elemental analyses of hydrogen, magnesium, and silicon for all three subbatches of the lot were in agreement with the theoretical values for talc. The major impurities were 0.7% aluminum and 1.0% iron. Karl Fischer water analysis indicated approximately 0.2% absorbed water. Spark source mass spectrometry for the three subbatches also indicated approximately 0.1% phosphorus, 0.5% fluorine, and 0.05% calcium, while the remaining elemental impurities were less than 0.01%. A special study was performed on this lot to determine if the sample met the American Society for Testing and Materials standard specifications for magnesium silicate. Results indicated that lot W101882 met the standard specifications. Automated scanning electron microscopic analysis demonstrated that the talc was virtually free of silica. In the analysis a sample of talc is suspended in methylcellulose. Under computer control the particles are located, and maximum, minimum, and average diameters are determined; then a chemical analysis is performed. Of the 1,466 particles that were examined, one was identified as silica, 1,241 were talc, 136 were of tremolite type composition, 77 were mixed silicates, one was possibly zircon, and 10 were not identified. The single silica particle had an average diameter of 3.9 pm. Lot B5415 was characterized by elemental analyses, Karl Fischer water analysis, spark source mass spectrometry, and microscopic analyses using the same methods described for lot W101882. Elemental analyses values were similar to results obtained for lot W101882. The major impurities present were 0.1% calcium, 0.5% aluminum, and 1% iron. Karl Fischer water analysis indicated 1.2% absorbed water. Spark source mass spectrometry also indicated 0.04% phosphorus, >0.5% aluminum, 0.03% sodium, Chemical Characterization and Dose Formulation 263 0.35% flu orin e, and all o th er im pu rities w ere less than 0.03% . M icroscop ic an alyses u sin g P L M and T E M d etected n o asb estos fibers. C o m p a ra tiv e purity a n a ly ses o f th e tw o lo ts used in th e se stu d ie s w ere c o n d u c te d d u e to p ro b lem s w ith the gen eration o f inhalation con centration s. F our sam ples o f talc w ere used, tw o sam p les each from lots W 101882 and B 5415. Sam p les A and B w ere from lot W 101882, sam p le C w as from lo t B 5415, and sam p le D w as a frozen reference from lot B 5415 that had b een stored at M R I. A n alyses p erform ed inclu d ed elem en tal analyses, m icroscop ic analyses (P L M , T E M , d eterm in ation o f p article size d istrib ution , and asp ect ratios), X -ray diffraction, and therm ogravim etric analysis (T G A ). PLM and T E M analyses w ere perform ed on sam ples C and D . A nalysis by P L M follow ed the procedu res described earlier, T E M follow ed the sam e p rocedu re described earlier excep t th e talc w as son ically dispersed in a so lu tio n o f 90% isop rop an ol in p article-free w ater. T h e d eterm in ation s o f particle size d istrib ution and asp ect ratios w ere perform ed on all four sam ples. U sin g T E M for b oth analyses, selected area diffraction (S A D ) patterns w ere used to confirm that th e particles b ein g m easured w ere talc. T h e p article size w as taken as th e average o f tw o d iam eters 90 to each o th er and asp ect ratios w ere taken as th e ratio o f the tw o diam eters. T h erm ogravim etric analysis (T G A ) w as p erform ed on sam p les A , B, and C on a D u P on t 910 d ifferen tial scann ing calorim eter (D S C ) w ith calcium oxalate m on oh yd rate used as a calibrating standard, at an initial tem perature o f 50 C w ith a program m ed m axim um tem p eratu re o f 1,100 C, at a rate o f 20 C per m inu te. E lem en tal analyses for h ydrogen, m agn esiu m , and silicon for all four sam p les w ere in agreem en t w ith th eo retica l valu es. P L M and T E M d etected n o a sb esto s fibers in any o f th e sam p les. T h e resu lts for p article siz e d istrib u tio n an d a sp ect ratios in d icated that th ere w ere o n ly m in o r d ifferen ces in p article s iz e b e tw e e n th e sa m p le s an d m o r e th an 75% o f th e p a rticles w er e in th e 1.0 to 3 .0 zm ran ge. M o r e than 90% o f th e talc p articles had asp ect ratios b etw een 1 and 1.4, and less than 1% had ratios greater than 3:1. X -ray d iffraction con firm ed that all four sa m p les w ere prim arily talc w ith sm all q u a n tities o f ch lorite and d olo m ite. T herm ogravim etric analysis indicated that sam ples A , B , and C w ere sim ilar. A m ain p eak at 912 C in all th ree sa m p les cau sed by th e lo ss o f ch em ica lly co m b in ed w ater w as eq u al to a lo ss o f 4.7% by w eigh t. A m in or p eak at 590 C in all th ree sa m p les m ay rep resen t th e lo ss o f C 0 2 from d o lo m ite and am o u n ted to a loss o f 0.7% by w eigh t w hich is eq u ivalen t to 1.5% d o lo m ite. Size R anee tum i 0.5-1.0 1.0-1.5 1.5-2.0 2.0-2.5 2 5 -3 .0 3.0-3.5 3.5--4.0 4.0-4.5 4.5-5.0 5.0-5.5 5.5-6.0 6.0-6.5 Size Distribution Analysis of Talc Samples _____ (% of Total Particles Counted)______ T alc A 5.88 15.69 26.47 20.59 11.76 5.88 3.92 2.94 2.94 1.96 1.96 - Talc B 2.97 9.90 26.73 17.82 18.81 12.87 5.94 1.98 0.99 0.99 0.99 - Talc C 1250 19.23 24.04 ' 21.15 10.58 4.81 2.88 1.92 0.96 0.96 0.96 - Talc D 1.94 11.65 26.21 23.30 8.74 7.77 5.83 4.85 3.88 291 1.94 0.97 T h e m oistu re co n ten t o f th e bulk ch em ical w as reanalyzed every 4 m on th s a t th e study laboratory by d eterm in in g th e w eigh t loss follow in g h eatin g at 120 C for 16 hours. T h e results in dicated that the m o istu re co n ten t o f th e talc w as sim ilar b etw een th e tw o lo ts and did n ot ch a n g e d u rin g th e 2-year stud ies. B ulk ch em ical stability stu d ies w ere not perform ed on talc b ecau se th e physical and chem ical 264 Talc, N TP TR 421 p ro p erties o f talc in d icate that it sh o u ld b e sta b le o v er a w id e ran ge o f tem p eratu res. T h e co m p o u n d w as stored in tightly sealed p lastic bags at 25 C. G eneration and M onitoring of Cham ber C oncentrations Aerosol Generation System: T a lc a e r o s o l w a s g e n e r a te d fr o m o n e 4 -in c h , flu id b e d g e n e r a t o r ( F B G ) . F ig u re 12 sh o w s th e sc h e m a tic o f th e F B G w ith th e gravity fee d an d c o lle c tin g pan c o lle c tio n sy stem s. T h e F B G bed con tained type 316 stain less steel pow der (H oegan aes C orporation, R iverton, N J), co n sistin g o f irregularly sh ap ed p articles 125 to 180 in d iam eter. T h e sta in less stee l p ow d er w as clean ed prior to use. T h e clea n in g system used a 4-in ch F B G w ith dry, filtered air flow in g through at a f lo w r a te o f 8 0 ft3/m in . T h e h ig h f lo w r a te t h r o u g h t h e b e d r e m o v e d t h e f in e s t s t a in le s s s t e e l p a r tic le s . T h e c le a n in g sy ste m w as run fo r 2 4 h o u r s to e n su r e th at a ll th e "fines" w e r e re m o v ed . F o llo w in g clean in g o f th e bed m aterial, talc w as m ixed w ith th e stain less stee l p ow d er at approxim ately 1 to 2.5 g o f talc p er 5 0 0 g bed m aterial. T h e co n cen tra tio n o f talc in th e bed m aterial w as o n e m eth o d used to adjust exp osu re con cen tration s in th e cham ber. D u rin g th e tim e period o f N ovem b er 1985 to January 1986, w h en difficulty in m ain tain in g target co n cen tra tio n s w as exp erien ced , h igher load in gs w ere used in an effort to m aintain target con cen tration s. F or gen eration o f th e talc aero so l, flu id ization o f th e bed m aterial m ixed w ith talc occurred w hen com p ressed air ( 2 0 0 L p m ) w as in jected in to th e bed through a p orou s m etal d istrib u tion p late w hich su p p o rts th e b ed . T h e m o tio n o f th e b ed relea sed th e m uch sm a ller talc p a rticles in to th e air; th e larger, h eavier sta in less ste e l p articles w ere retain ed in th e bed. A K r-85 disch arger w as p laced ab ove th e bed to red u ce th e p article charges. T h e a ero so lized talc p articles w ere m ixed w ith d ilu tin g air ( 2 0 0 L pm ) to ach ieve the desired con cen tration s and w ere then delivered to th e exp osu re cham bers (F ig u r e s 13 a n d 14). A s th e ta lc p o w d e r w a s r e m o v e d fro m th e b ed , th e b ed m a te r ia l w a s c o n tin u a lly drained from th e F B G through an overflow port located at th e sid e o f th e gen erator. A s sp en t bed m aterial w as drained from th e gen erator, fresh talc-con tain in g bed m aterial w as con stan tly added in to the gen erator from a h opp er located ab ove the generator. S tain less steel m ulti-tiered w hole-bod y exp osu re cham bers (H 2 0 0 0 , Lab P roducts, Inc.) w ere used to exp o se th e rats in this study w h ile the sm aller H 1000 cham bers w ere used for th e m ice. F low rates t h r o u g h t h e c h a m b e r s w e r e 1 2 2 ft3/m in . T o r e d u c e t h e s p a tia l v a r ia tio n o f a e r o s o l c o n c e n t r a t io n a n d to in crease th e uniform ity o f m ixing, th e a erosol w as d ilu ted u sing a d ilu tor p rior to its in trod u ction in to th e cham ber. A lso , anim al cages w ere rotated w eekly to redu ce th e variation o f con cen tration s o f talc a ero so ls that th e rod en ts w ere exp osed to during th e 2-year stud ies. Aerosol Concentration Monitoring: A e r o s o l c o n c e n t r a tio n s in e a c h e x p o s u r e c h a m b e r w e r e m o n ito r e d by co llec tin g filter sam p les for three, 2-h ou r periods during each 6-h ou r exp osu re day. T h e background co n cen tra tio n o f to ta l su sp en d ed p articles in each co n tro l ch am ber w as m o n ito red each ex p o su re day by co llectin g o n e 6-h ou r filter sam ple. O vernight filter sam ples for total su sp en d ed p articles w ere collected from the 18 m g/m 3 cham bers m onthly. A ll filter sam ples w ere taken at a flow rate o f 3 L /m inute. E ach filter w as w eigh ed b efore and after th e sam p le w as collected , and th e aerosol m ass con cen tration s w ere c a lc u la t e d b y d iv id in g t h e m a s s in c r e m e n t ( m g ) by t h e v o lu m e s a m p le d ( m 3); t h e m e a n s a n d s ta n d a r d d eviation s for each cham ber w ere calculated for each exp osu re day. W eekly m ean exp osu re c o n c e n t r a t io n s fo r t h e 2 -y e a r s t u d ie s a r e p r e s e n te d in F ig u r e s 15 th r o u g h 18. T h e c o n c e n t r a t io n s d u r in g n o n - e x p o s u r e h o u r s in t h e 1 8 m g /m 3 c h a m b e r s r a n g e d fr o m 0 .0 2 t o 1.1 m g /m 3. A R A M -S co n tin u ou s a erosol m on itor w as used to m on itor th e stability o f th e a erosol con cen tration s and to d eterm in e th e n eed to adjust th e a ero so l gen eration system during exp osu res. T h e R A M -S w as used to m on itor each cham ber for at least 5 m inu tes at th e b egin ning, m iddle, and end o f th e filter sam p lin g p eriod . A 2 L /m in u te flow rate through th e R A M -S w as ach ieved u sing an in tern al p um p in th e d evice. B oth R A M -S and filter sam ples w ere taken at o n e p oin t o f th e cham bers ab ove th e anim al cage. A Y -shap ed p rob e w as used, allow in g sim u ltan eou s filter sam pling and R A M -S aerosol m ass Chemical Characterization and Dose Formulation 265 m o n ito r o p e r a tio n . T h e o v era ll tem p o ra l v a riation in ch am b er co n ce n tr a tio n s in th e 2-year stu d ie s w ere 33% and 27% relative standard d eviation (R S D ) for the m ou se 6 and 18 m g/m 3 cham bers. T h e variations w ere 31% and 36% R S D for th e rat 6 and 18 m g/m 3 cham bers. A t least a p ortion o f this variability m ay b e ascribed to the period w hen talc gen eration p rob lem s w ere en cou n tered (N ovem b er 1985 th rou gh F ebruary 1986). In a d d itio n , a p o rtio n o f th e variability for th e 18 m g/m 3 rat ch am b er m ay be ascribed to the tim e w hen higher con cen tration s w ere b ein g generated (S ep tem b er through N ovem ber, 1984). D urin g the period o f N ovem ber 5, 1985, through January 27, 1986, d ifficulties w ere experienced m ain tain in g th e required exp osu re levels o f talc for th e lifetim e and 2-year exp osu re stu d ies. C on cen tration s o f a erosolized talc w ere significantly b elow target. A ttem p ts w ere m ade to in crease the flow o f talc in to th e gen erator and raise th e con centration ; h ow ever, th e talc-lad en stain less steel bed m aterial fed in to th e g en era to r less freely than it had prior to N o v em b er 1985. T h e r e w er e n o o b ser v a b le c h e m ic a l ch a n g es in e ith e r th e talc or th e sta in le ss s te e l b ed m a teria l an d n o m a lfu n ctio n s in th e gen eration system w hich cou ld be p in p oin ted as the underlying cau se for th e p oor flow characteristics o f th e bed m aterial. O n January 27, 1986, th e gen erator w as restarted w ith a new batch o f talc. A fter a sta b iliza tio n p eriod o f 3 w eek s, th e flow p rop erties o f th e bed m aterial sh ow ed significant im provem ent. It w a s a ls o o b ser v ed d u rin g F eb ru ary 1 9 8 6 , th at w h e n th e ra tio o f ta lc to b ed m a teria l w as in crea sed ab ove 1.6 g talc p er 5 0 0 g bed m aterial, th e bed began to sh ow th e p o o r flow p rop erties ch aracteristic o f th e p reviou s b atch o f talc. W h en th e bed load in g w as redu ced b elo w 1.6 g talc p er 5 0 0 g b ed m aterial, th e flow p rop erties stabilized. T his in dicated that th e bed has a m axim um load in g lim it w hich m ust not b e exceeded. By M arch 1986, the generator had stabilized and cham ber target con cen tration s w ere achieved. T h e exact cau se o f th ese gen eration p rob lem s w as never resolved. In N o v e m b e r 1 9 8 4 it w as n o tic ed th at th e R A M -S m o n ito r in d ica ted an o ff-sc a le rea d in g ( > 1 0 V w h ich is e q u iv a le n t t o 2 0 m g /m 3) fo r t h e 1 8 m g /m 3 rat c h a m b e r . R e a s o n a b l e a g r e e m e n t w a s fo u n d b e t w e e n R A M -S readings and filter sam p les in th e o th er ch am bers. In vestigation s o f th is d iscrep ancy in dicated that th e airflow through th e critical orifice co n trollin g flow through th e filter w as reduced. E valu ation o f th e previously co llected pressure drop associated w ith this orifice and o n e having nearly id en tical n om in al flow revealed that th e flow to th e sam p lin g filter o f th e high level rat ch am ber drop ped sign ifican tly o n S ep tem b er 24, 1984. T h ese data su ggest that th e sam p lin g orifice had b eco m e partially clo g g ed . In o rd er to o b ta in a co rrectio n factor to reca lcu la te th e ch am b er co n ce n tr a tio n d ata, th e filter pressure drop and exp osu re cham ber pressure drop data w ere retrieved and used to d eterm in e th e actual pressure d rop across th e sam p lin g filter for the tim e period o f S ep tem b er 24 through N ovem b er 14, 1984. A grou p o f 18 filters from different lots o f th e type used to sam p le th e talc exp osu re cham bers w ere tested to d eterm in e th e pressure drop across them as a fu n ction o f th e flow through th e filter. T h ese data in dicated that valu es for flow cou ld b e calcu lated from th e pressure d rop data. T h e relation sh ip b etw een pressure drop and filter flow rate w as used to recalcu late th e sam pling filter flow for each day. W hen th e cham ber sam pling orifice flow rate w as taken in to accou n t, th e best estim ate o f th e correction factor is 2.06. T h is factor has b een used to m ultip ly th e originally recorded cham ber con cen tration s for th ose dates. T h e corrected valu es are reported. A ero so l size d istrib ution w as d eterm in ed o n ce a m onth for each cham ber u sing a cascad e im pactor op erated at a flow rate o f 15 L /m in u te. S tain less steel disks coated w ith a p iezo n grease w ere u sed as im p a cto r su b stra tes and th e a m o u n t o f ta lc c o lle c te d o n ea c h sta g e w as d e ter m in ed by th e d iffe r e n ce in stage w eigh t b efore and after the sam p le w as collected . T h e m ass m edium aerodynam ic d iam eter and th e geom etric standard d eviation w ere calculated from th e m ass data, effective cu to ff d iam eter o f each sta g e , a n d im p a c to r flo w rate. T h e re su lts a re p r e se n te d in T a b le s II a n d 12. MICROMETERS I Talc, NTP TR 421 Figure II Infrared Absorption Spectrum of Talc ABSCISSA E XPANSIO N 1 SUPPR ESSIO N SAM PLE: Talc Lot W 101 882 Batch 02 Subbatch A ORDINATE E X P A N S IO N 1 % T 0-100 ABS - REM A R K S Trimmer comb in reference beam SCAN TIM E RESPONSE SLIT PRO G RAM 24 min 1 6 REP. SCAN TIM E DRIVE OPERATOR - SING LE BEAM - - PRE SAMPLE CHOP - A Clark D A TE 11/9/82 SOLVENT C O NCENTRATIO N 1 % in KBr CELL PATH REFERENCE 154N Chemical Characterization and Dose Formulation AEROSOL FEED HOPPPER 267 COMPRESSED AIR ORIFICE METER DISTRIBUTION PLATE ROTAMETER COMPRESSED AIR COLLECTING RAN READ OUT BALANCE Figure 12 Fluid Bed Generator 268 Talc, NTP TR 421 TO C h a m b e r vented ENCLOSURE E>0 I--- EXHAUST Figure 13 Aerosol Dilutlon/Delivery System Chemical Characterization and Dose Formulation 269 Figure 14 Talc Chronic Exposure System 272 Talc, NTP TR 421 O X 0) 4) <y u a m 0 ft X w Figure 17 Talc Aerosol Filter Concentrations in the 6 mg/m1 Mouse Chamber Chemical Characterization and Dose Formulation 273 (gin/3m) -OUO0 o N O O O co M i> t> o 4> t- 3 n 0 . X w o * O w o Figure 18 Tale Aerosol Filter Concentrations in the 18 mg/m3 Mouse Chamber 274 Talc, NTP TR 421 Table II Summary of Aerosol Size Measurements for the 6 and 18 mg/mJ Rat Chambers D ate 6 m g/m 1 M ass M edian A erodynam ic D ia m e te r (p.m) G eom etric Standard D eviation D ate 18 m g/m J M ass M edian A erodynam ic D ia m e te r Oim) G eom etric S ta n d a r d D eviation 9 July 1984 6 August 1984 4 September 3 October 1984 31 October 1984 27 November 1984 4 January 1985 25 January 1985 25 February 1985 19 March 1985 22 April 1985 13 June 1985 9 July 1985 9 August 1985 3 September 1985 30 September 1985 28 October 1985 2 December 1985 18 December 1985 3 January 1986 8 January 1986 13 January 1986 24 February 1986 24 March 1986 22 April 1986 23 May 1986 23 May 1986 27 May 1986 16 June 1986 30 June 1986 28 July 1986 25 August 1986 22 September 1986 20 October 1986 Mean standard deviation Z3 2.6 2.8 2.6 2.9 2.5 2.6 2.5 2.6 2.8 2.9 3.0 2.8 2.7 2.7 2.3 2.6 3.1 3.0 1.8 3.6 3.1 2.9 3.4 3.2 2.4 2.9 2.3 2.7 2.2 2.5 Z1 2.5 2.7 2.7 0.4 . 2.6 1.7 1.8 1.8 1.8 1.8 1.8 1.7 1.8 1.8 1.7 1.9 1.8 1.9 1.5 1J 1.4 1.7 1.7 2.8 1.9 1.8 2.2 1.9 2.3 1.9 1.9 1.9 2.7 2.4 Z3 2.5 2.0 2.3 1.9 0.4 25 June 1984 1 August 1984 27 August 1984 26 September 1984 24 October 1984 20 November 1984 24 December 1984 14 January 1985 19 February 1985 15 March 1985 12 April 1985 8 May 1985 10 June 1985 5 July 1985 1 August 1985 26 August 1985 23 September 1985 21 October 1985 25 November 1985 17 December 1985 30 December 1985 3 January 1986 8 January 1986 18 February 1986 17 March 1986 14 April 1986 19 May 1986 2 June 1986 17 June 1986 15 July 1986 11 August 1986 9 September 1986 6 October 1986 3.6 3.0 3.2 2.9 3.2 3.0 2.8 2.9 2.8 3.1 3.1 2.9 3.0 3.5 3.1 2.9 2.6 2.7 4.0 3.3 3.7 4.0 3.8 3.2 3.6 4.0 3.2 3.2 3.3 3.4 3.1 2.9 2.7 3.2 0.4 2.0 1.8 1.9 1.8 1.9 1.9 1.8 1.8 1.8 2.0 1.8 1.9 1.9 1.8 1.9 1.9 1.6 1.5 2.1 1.9 1.8 2.2 1.9 2.1 1.9 2.0 1.8 2.1 1.9 2.0 1.9 1.9 2.3 1.9 0.2 Chemical Characterization and Dose Formulation 275 T a b l e 12 Summary or Aerosol Size Measurements Tor the 6 and 18 mg/m3 Mouse Chambers Date 6 mg/m3 Mass Median Aerodynamic Diameter (pm) Geometric Standard Deviation Date 18 mg/m3 Mass Median Aerodynamic Diameter (fim) Geometric Standard Deviation 18 June 1984 16 July 1984 14 August 1984 18 September 1984 10 October 1984 7 November 1984 4 December 1984 7 January 1985 4 February 1985 1 March 1985 29 March 1985 23 April 1985 22 May 1985 21 June 1985 23 July 1985 15 August 1985 9 September 1985 7 October 1985 4 November 1985 9 December 1985 19 December 1985 3 January 1985 8 January 1986 20 January 1986 3 March 1986 31 March 1986 28 April 1986 Mean standard deviation 3.9 3.4 3.5 3.3 3.1 3.3 3.0 3.4 3.2 2.9 3.1 3.6 3.1 3.3 3.4 3.5 2.6 2.7 2.5 3.4 3.6 3.9 4.0 3.7 3.0 2.9 3.2 3.3 0.4 1.8 1.9 1.8 1.8 1.9 1.8 1.8 1.6 1.8 1.9 1.8 1.8 2.0 1.8 1.8 1.8 1.3 1.5 1.5 1.6 2.0 2.0 2.1 1.8 2.1 2.1 4.7 1.9 0.6 25 June 1984 23 July 1984 20 August 1984 10 September 1984 17 October 1984 19 November 1984 12 December 1984 7 January 1985 8 February 1985 7 March 1985 5 April 1985 2 May 1985 29 May 1985 26 June 1985 29 July 1985 20 August 1985 16 September 1985 14 October 1985 12 November 1985 16 December 1985 3 January 1986 8 January 1986 10 February 1986 13 March 1986 7 April 1986 5 May 1986 3.6 3.7 3.5 3.9 3.8 3.5 3.3 3.4 3.6 3.6 3.5 3.6 3.5 3.7 3.5 3.8 3.3 2.8 4.1 3.8 3.6 5.0 3.3 3.1 3.4 3.3 3.6 0.4 2.0 1.9 1.8 2.0 1.9 1.7 1.9 1.8 1.9 1.9 1.9 1.8 2.2 20 1.9 1.9 1.8 1.7 2.1 2.0 1.9 2.0 2.4 2.5 20 22 2 0 + 0.2 J 276 Talc, NTP TR 421 277 APPENDIX J INGREDIENTS, NUTRIENT COMPOSITION, AND CONTAMINANT LEVELS IN NIH-07 RAT AND MOUSE RATION Table J1 Table J2 Table J3 Table J4 Ingredients of NIH-07 Rat and Mouse R a tio n .......................................................................... Vitamins and Minerals in NIH-07 Rat and Mouse R a tio n .................................................... Nutrient Composition of NIH-07 Rat and Mouse R a tio n ....................................................... Contaminant Levels in NIH-07 Rat and Mouse R a tio n ......................................................... 278 278 279 280 278 Table J 1 Ingredients of NIH-07 Rat and Mouse Ration* Ingredients*1 Percent by Weight Ground # 2 yellow shelled com Ground hard winter wheat Soybean meal (49% protein) Fish meal (60% protein) Wheat middlings Dried skim milk Alfalfa meal (dehydrated, 17% protein) Com gluten meal (60% protein) Soy oil Dried brewer's yeast Dry molasses Dicalcium phosphate Ground limestone Salt Premixes (vitamin and mineral) 24.50 23.00 12.00 10.00 10.00 5.00 4.00 3.00 2.50 2.00 1.50 1.25 0.50 0.50 0.25 * NCI, 1976; N1H, 1978 Ingredienls were ground to pass through a U.S. Standard Screen No. 16 before being mixed. Talc NTP TR 421 Table J2 Vitamins and Minerals in NIH-07 Rat and Mouse Ration* Amount Source Vitam ins A 3 -a-Tocopheryl acetate Choline Folic acid Niacin -Pantothenic add Riboflavin Thiamine Bn Pyridoxine Biotin M inerais Iron Manganese Zinc Copper Iodine Cobalt * Per ton (2,000 lb) of finished product 5,500,000 IU 4,600,000 IU 2.8 g 20,000 IU 560.0 g 2-2 g 30.0 g 18.0 g 3.4 g 10.0 g 4,000 Mg 17 g 140.0 mg 120.0 g 60.0 g 16.0 g 4.0 g 1.4 g 0.4 g Stabilized vitamin A palmilate or acetate D-activated animal sterol Menadione Choline chloride -Caldum pantothenate Thiamine mononitrate Pyridoxine hydrochloride -Biotin Iron sulfate Manganous oxide Zinc codde Copper sulfate Calcium iodate Cobalt carbonate Feed Analyses Table J3 Nutrient Composition of NIH-07 Rat and Mouse Ration N u trien t M ean Standard D eviation Protein (% by weight) Crude fat (% by weight) Crude fiber { % by weight) Ash ( % by weight) Am ino Acids {% o f total diet) Arginine Cystine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan TVrosine Valine E ssential Fatty Acids (% o f total diet) Linoleic Linolen ic V itam ins Vitamin A (IU/kg) Vitamin D (IU/lcg) o-Tocopherol (ppm) Thiamine (ppm) Riboflavin (ppm) Niacin (ppm) Pantothenic acid (ppm) Pyridoxine (ppm) Folic acid (ppm) Biotin (ppm) Vitamin B12 (ppb) Choline (ppm) M inerals Calcium (%) Phosphorus (%) Potassium ( % ) Chloride (%) Sodium (%) Magnesium (%) Sulfur (%) Iron (ppm) Manganese (ppm) Zinc (ppm) Copper (ppm) Iodine (ppm) Chromium (ppm) Cobalt (ppm) 22.22 0.72 5.59 0.55 3.36 0.30 6.55 0.23 1.308 0.606 0.306 0.084 1.150 0.047 0.576 0.024 0.917 0.029 1.946 0.055 1.270 0.058 0.448 0.128 0.987 0.140 0.877 0.042 0.236 0.176 0.676 0.105 1.103 0.040 2.393 0.258 0.280 0.040 9,846 2,839 4,450 1,382 37.95 9.41 20.77 2.01 7.92 0.87 103.4 26.59 29.54 3.60 9.55 3.48 2.25 0.73 0.254 0.042 38.45 22.01 3,089 328.69 1.17 0.09 0.92 0.03 0.883 0.078 0.526 0.092 0.313 0.390 0.168 0.010 0.280 0.064 360.5 100 9 2 0 6.01 54.72 5.67 11.06 250 3.37 0.92 1.79 0.36 0.681 0.14 Range 21.1-23.5 4.7-6.4 2.7-3.8 6.1-7.0 1.210-1.390 0.181-0.400 1.060-1.210 0.531-0.607 0.881-0.944 1.850-2.040 1.200-1.370 0.306-0.699 0.665-1.110 0.824-0.940 0.107-0.671 0.564-0.794 1.050-1.170 1.830-2570 0.210-0.320 5,600-15,000 3,000-6,300 22.5-48.9 17.0-23.0 6.10-9.00 65.0-150.0 23.0-34.0 5.60-14.0 1.80-3.70 0.19-0.32 10.6-65.0 2,400-3,430 1.06-1.41 0.87-0.99 0.772-0.971 0380-0.635 0.258-0.371 0.151-0.181 0.208-0.420 255.0-523.0 81.70-99.40 46.10-64.50 8.090-15.39 1-52-4.13 1.04-2.09 0.490-0.780 279 Number o f Sam ples 13 13 13 13 8 8 8 8 8 8 8 8 8 8 8 8 8 7 7 13 4 8 13 8 8 8 8 8 8 8 8 13 13 6 8 8 8 8 8 8 8 8 6 8 4 280 Table J4 Contaminant Levels in NIH-07 Rat and Mouse Ration M ean Standard D eviation* Range C ontam inants Arsenic (ppm) Cadmium (ppm) Lead (ppm) Mercury (ppm) Selenium (ppm) Aflatoxins (ppb) Nitrate nitrogen (ppm)b Nitrite nitrogen (ppm)" BHA (ppm)c B H T (ppm )c Aerobic plate count (CFU/g)d Coliform (MPN/g)e Coliform (MPN/g)f E c o lL (MPN/g)* Total nitrosamines (ppb)h N-Nitrosodimethylamine (ppb)h N-Nitrosopyrrolidine (ppb)" Pesticides (ppm) a-BHC1 0-BHC y-BHC 5-BHC Heptachlor Aldrin Heptachlor epoxide DDE DDD DDT HCB Mirex Methoxychlor Dieldrin Endrin Telodrin Chlordane Toxaphene Estimated PCBs Ronnel Ethion Trithion Diazinon Methyl parathion Ethyl parathion Malathion1 Endosulfan I Endosulfan II Endosulfan sulfate 0.72 0.19 < 0.1 0.57 0.31 <0.05 0.35 0.08 <5.0 12.56 4.47 0.14 0.11 2.54 1.05 2.39 1.33 39,523 39,878 3.72 1.79 9.46 14.11 3.08 0.28 6.99 4.13 5.67 3.79 1.32 0.73 < 0.01 < 0.02 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 <0.05 < 0.01 < 0.01 < 0.01 <0.05 < 0.1 < 0.2 < 0.01 < 0.02 <0.05 < 0.1 < 0.02 < 0.02 0.09 0.07 < 0.01 < 0.01 <0.03 0.33-0.94 0.14-132 0.21-0.44 2.80-18.0 <0.10-030 <2.00-5.00 <1.00-4.00 3,400-130,000 <3.00-9.00 <3.00-43.0 <3.0-4.00 1.80-16.00 0.80-15.00 1.00-3.40 0.05-0.28 Talc NTP TR 421 N um ber of Samples 13 13 13 13 13 13 13 13 13 13 13 11 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 Feed Analyses Table J4 Contaminant Levels in NIH-07 Rat and Mouse Ration (continued)* * For values less than the limit of detection, the detection limit is given for the mean. b Sources of contamination: alfalfa, grains, and fish meal c Sources of contamination: soy oil and fish meal d CFU = colony forming unit * MPN = most probable number f Includes two high values of 39 and 43 MPN/g obtained from lots milled 15 March 1984 and 9 May 1984, respectively. e One lot milled 17 October 1984 contained 4.00 MPN/g; all other lots contained 3.00 MPN/g h All values were corrected for percent recovery. ' BHC = hexachlorocyclohexane or benzene hexachloride. 1 Seven lots contained more than 0.05 ppm. 281 282 m 283 APPENDIX K SENTINEL ANIMAL PROGRAM M e t h o d s .............................................................................................................................................................................. T able K1 Murine Virus Antibody Determinations for Rats and Mice in the 2-Year and Lifetime Inhalation Studies of Talc ......................................................... 284 286 284 Talc, NTP TR 421 SENTINEL ANIMAL PROGRAM M ethods Rodents used in the Carcinogenesis Program of the National Toxicology Program are produced in optimally clean facilities to eliminate potential pathogens that may affect study results. The Sentinel Animal Program is part of the periodic monitoring of animal health that occurs during the toxicologic evaluation of chemical compounds. Under this program, the disease state of the rodents is monitored via serology on sera from extra (sentinel) animals in the study rooms. These animals and the study animals are subject to identical environmental conditions. The sentinel animals come from the same production source and weanling groups as the animals used for the studies of chemical compounds. Rats Prior to the beginning of the lifetime study, five male and five female F344/N rats of each sex were sacrificed and serum samples were taken for serological evaluation by Microbiological Associates (Bethesda, MD). Serum samples were also taken from selected rats for serology testing at each of the interim evaluations: three male and three female rats at 6 months; eight male and nine female rats at 12 and 18 months; 11 male and 17 female rats at 24 months; and 15 male and 15 female rats at the terminal sacrifice (male, 113 weeks; female, 122 weeks). Blood collected from each animal was allowed to clot and the serum was separated. The serum was cooled on ice and shipped to Microbiological Associates (Bethesda, MD) for determination of antibody titers. The following tests were performed: Method of Analysis ELISA CARB (cilia-associated respiratory bacillus) Mycoplasma arthritidis Mycoplasma pulmonis PVM (pneumonia virus of mice) RCV/SDA (rat coronavirus/sialodacryoadenitis virus) Sendai Time of Analysis Study termination (males only) 12, 18, 24 months, study termination 12, 18, 24 months, study termination 6, 12, 18, 24 months, study termination Study initiation, 6, 12, 18, 24 months, study termination 6, 12, 18, 24 months, study termination Hemagglutination Inhibition H-l (Toolan's H-l virus) KRV (Kilham rat virus) PVM Sendai Study initiation, 6, 12, 18, 24 months, study termination Study initiation, 6, 12, 18, 24, study termination Study initiation Study initiation Immunofluorescence Assay KRV RCV RCV/SDA 24 months (males only) 24 months (males only) 28 months (males only) Sentinel Animal Program 285 Mice Prior to the beginning of the 2-year study, five male and five female B6C3F, mice were sacrificed and serum samples were taken for serological evaluation by Microbiological Associates (Bethesda, MD). Serum samples for serology testing were also taken from control males and females at each of the interim evaluations (four males and four females at 6 months; 12 males and 12 females at 12 months) and at the terminal sacrifice (IS males and IS females). (Samples were inadvertently omitted for mice evaluated after 18 months of exposure on 4-5 December, 1985.) Blood collected from each animal was allowed to clot and the serum was separated. The serum was cooled on ice and shipped to Microbiological Associates (Bethesda, MD) for determination of antibody titers. The following were performed: Method of Analysis Complement Fixation LCM (lymphocytic choriomeningitis virus) Mouse adenoma virus Time of Analysis Study initiation, 6, 12, 24 months Study initiation ELISA Ectromelia virus GDVII (mouse encephalomyelitis virus) Mouse adenoma virus MHV (mouse hepatitis virus) M. arthritidis M. pulmonis PVM Reovirus 3 Sendai 6, 12, 24 months Study initiation, 6, 12, 24 months 6, 12, 24 months Study initiation, 6, 12, 24 months 6, 12, 24 months 6, 12, 24 months 6, 12, 24 months 6, 12, 24 months 6, 12, 24 months Hemagglutination Inhibition Ectromelia virus K (papovirus) MVM (minute virus mice) PVM Polyoma virus Reovirus 3 Sendai Study initiation 12, 24 months Study initiation, 6, 12, 24 months Study initiation Study initiation, 6, 12, 24 months Study intitiation Study initiation Immunofluorescence Assay EDIM (Epizootic diarrhea of infant mice) Reovirus 3 6, 12, 24 months 24 months 286 Talc, NTP TR 421 T a b l e K1 Murine Virus Antibody Determinations for Rats and Mice in the 2-Year and Lifetime Inhalation Studies of Talc Interval (m onths) Incidence o f Antibody in Sentinel A nim als Positive Serologic Reaction for R ats 6 months 12 months 18 months 24 months (males) (females) 28 months 30 months M ice 6 months 12 months 24 months 0/6 0/17 0/17 1/11 9/11 6/11 13/17 13/17 15/15 3/15 15/15 1A5 0/8 0/24 2/30 7/30 21/30 None positive None positive None positive KRV Sendai RCV Sendai RCV/SDA Sendai RCV/SDA Sendai RCV/SDA None positive MHV Reovirus 3 M . a rth ritid is EDIM NATIONAL TOXICOLOGY PROGRAM TECHNICAL REPORTS PRINTED AS OF SEPTEMBER 1993 TR No. CHEMICAL 201 2,3,7,8-Tetrachlorodibenzo-p-dioxin (Dermal) 206 l,2-Dibromo-3-chloropropane 207 Cytembena 208 FD & C Yellow No. 6 209 23.7,8-Tetrachlorodibenzo-p-dioxin (Gavage) 210 1,2-Dibromoethane 211 C.l. Acid Orange 10 212 Di(2-ethylhexyl)adipate 213 Butyl Benzyl Phthalate 214 Caprolactam 215 Bisphenol A 216 11-Aminoundecanoic Acid 217 Di(2-Ethylhexyl)phthalate 219 2,6-Dichloro-p-phenylenediamine 220 C.l. Acid Red 14 221 Locust Bean Gum 222 G I. Disperse Yellow 3 223 Eugenol 224 Tara Gum 225 D & C Red No. 9 226 C.l. Solvent Yellow 14 227 Gum Arabic 228 Vinylidene Chloride 229 G uar Gum 230 Agar 231 Stannous Chloride 232 Penlachloroethane 233 2-Biphenylamine Hydrochloride 234 Allyl Isothiocyanate 235 Zearalenone 236 D-Mannitol 237 1,1,1,2-Tetrachloroethane 238 Ziram 239 Bis(2-chk>ro-l-Methylethyl)ether 240 Propyl Gallate 242 Diallyl Phthalate (Mice) 243 Trichlorethylene (Rats and Mice) 244 Polybrominated Biphenyl Mixture 245 Melamine 246 Chrysotile Asbestos (Hamsters) 247 L-Ascorbic Acid 248 4,4 -Methylenedianiline Dihydrochloride 249 Amosite Asbestos (Hamsters) 250 Benzyl Acetate 251 2,4- & 2,6-Toluene Diisocyanate 252 Geranyl Acetate 253 Allyl Isovalerate 254 Dichloromethane (Methylene Chloride) 255 1,2-Dichlorobenzene 257 Diglycidyl Resorcinol Ether 259 Ethyl Acrylate 261 Chlorobenzene 263 1,2-Dichloropropane 266 Monuron 267 1,2-Propylctie Oxide 269 Telone II* (1,3-Dichloropropene) 271 HC Blue No. 1 272 Propylene TR No. CHEMICAL 273 Trichloroethylene (Four Rat Strains) 274 Tris(2-ethylhexyl)phosphate 275 2-Chloroethanol 276 8-Hydroxyquinoline 277 Tremolite 278 2,6-Xylidine 279 Amosite Asbestos 280 Crocidolite Asbestos 281 HC Red No. 3 282 Chlorodibromomethane 284 Diallylphthalate (Rats) 285 C l. Basic Red 9 Monohydrochloride 287 Dimethyl Hydrogen Phosphite 288 1,3-Butadiene 289 Benzene 291 Isophorone 293 HC Blue No. 2 294 Chlorinated Trisodium Phosphate 295 Chrysotile Asbestos (Rats) 296 Tetrakis(hydroxymethyl) phosphonium Sulfate & Tetralcis(hydroxymethyl) phosponium Chloride 298 Dimethyl Morphoiinophosphoramidate 299 C.l. Disperse Blue 1 300 3-Chloro-2-melhylpropene 301 o-Phenylphenol 303 4-Vinylcyclohexene 304 Chlorendic Acid 305 Chlorinated ParafTms (C23, 43% chlorine) 306 Dichloromethane (Methylene Chloride) 307 Ephedrine Sulfate 308 Chlorinated Pariflins (C 12, 60% chlorine) 309 Decabromodiphenyl Oxide 310 Marine Diesel Fuel and JP-5 Navy Fuel 311 Tetrachloroethylene (Inhalation) 312 n-Butyl Chloride 313 Mirex 314 Methyl Methacrylate 315 Oxytetracycline Hydrochloride 316 l-Chloro-2-methylpropene 317 Chlorpheniramine Maleate 318 Ampicillin Trihydrate 319 1,4-Dichlorobenzene 320 Rotenone 321 Bromodichloromethane 322 Phenylephrine Hydrochloride 323 Dimethyl Methylphosphonate 324 Boric Acid 325 Pentachloronitrobenzene 326 Ethylene Oxide 327 Xylenes (Mixed) 328 Methyl Carbamate 329 1,2-Epoxybutane 330 4-Hexylresorcinol 331 Malonaldehyde, Sodium Salt 332 2-Mercaptobenzothiazole 333 iV-Phenyl-2-naphthylamine 334 2-Amino-5-nitrophenol 335 C.l. Acid Orange 3 M J . S . GOVERNMENT P R I N T I N G O F F I C E : 1 9 9 3 3 0 0 - 9 7 0 / 0 0 0 0 2 NATIONAL TOXICOLOGY PROGRAM TECHNICAL REPORTS PRINTED AS OF SEPTEMBER 1993 (CONT.) TR No. CHEMICAL 336 Penidllin VK 337 Nitrofurazooe 338 Erythromycin Stearate 339 2-Amino-4-nitrophcnol 340 Iodinated Glycerol 341 Nitrofurantoin 342 Dichiorvoe 343 Benzyl Alcohol 344 Tetracycline Hydrochloride 345 Rootarsooe 346 Chloroethane 347 D-Limonene 348 a-Mcthyldopa Scsquihydrate 349 Pentachlorophenol 350 T ribromomethane 351 p-Chloroaniline Hydrochloride 352 N-Methylolacrylamide 353 2,4-Dichloro^henol 354 D im etharine 355 Diphen nydramine Hydrochloride 356 Furosemide 357 Hydrochlorothiazide 358 O'diratoodn A 359 ii-Methcstypsoralen 360 N.N -Dimethylaniline 36'i Hexachloroethane 362 4-Vinyl-l-Cydohexene Diepoxide 363 Bromoethane (Ethyl Bromide) 364 Rhodamine 6G ( C l. Basic Red 1) 365 Pentaerythritol Tetranitrate 366 Hydroquinone 367 Phenylbutazone 368 Nalidixic A dd 369 Alpha-Methylbenzyl Alcohol 370 Benzofuran 371 Toluene 372 33-Dimethoxybenzidine Dihydrochloride 373 Sucdnic Anhydride 374 Glyddol 375 Vinyl Toluene 376 Allyl Gtyddyl Ether 377 o-Chlorobenzalmalononitrile TR No. CHEMICAL 378 Benzaldhyde 379 Z-Chloroacetophenonc 380 Epinephrine Hydrochloride 381 d-Carvone 382 Furfural 385 Methyl Bromide 386 Tetranitromethane 387 Amphetamine Sulfate 388 Ethylene Thiourea 389 Sodium Azide 390 3,3 ' -Dimethylbenzidine Dihydrochloride 391 TrU(2-chloroethyl) Phosphate 392 Chlorinated Water and Chioraminated Water 393 Sodium Fluoride 394 Acetaminophen 395 Probenecid 396 Monochloroacetic Add 397 C l. Direct Blue 15 398 Polybrominated Biphenyls 399 Titanocene Dichloride 401 2,4-Diaminophenol Dihydrochloride 402 Furan 403 Resorcinol 405 C l. A dd Red 114 406 y-Butyrolactone 407 C l. Pigment Red 3 408 Mercuric Chloride 409 Quercetin 410 Naphthalene 411 C l. Pigment Red 23 412 4,4-Diamino-2^-Stilbenedisulfonic Add 413 Ethylene Glycol 414 Pentachloroanisoie 415 Polysorbate 80 416 o-Nitroanisoie 417 p-Nitrophenol 418 /-Nitroaniline 419 HC Hellow 4 427 Turmeric Oleoresin 434 13-Butadiene 443 Oxazepam These NTP Technical Reports are available for sale from the National Technical Information Service, U.S. Department o f Commerce, 5285 Pori Royal Road, Springfield, VA 22161 (703-487-4650). Single copies of this Technical Report are available without charge (and while supplies last) from the NTP Central Data Management, N1EHS, P.O. Bax 12233, MD AO-01, Research Triangle Park, NC 27709. DEPARTMENT OF HEALTH & HUMAN SERVICES Public Health Service National Toxicology Program Central Data Management P.O. Box 12233, MD A0-01 Research Triangle Park, NC 27709 Official Business Penalty for Private Use - $300 SPECIAL FOURTH-CLASS RATE POSTAGE AND FEES PAID DHHS/NIH Permit No. G-763 NIH Publication No. 93-3152 September 1993