NATIONAL TOXICOLOGY PROGRAM Technical Report Series No. 278 TOXICOLOGY AND CARCINOGENESIS STUDIES OF 2,6=XYLIDINE (2,6-DIMETHYLANILINE) (CAS NO. 87-62-7) IN CHARLES RIVER CD RATS (FEED 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 agen- cies 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 chemical 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. All NTP toxicology and carcinogenesis studies are subjected to a comprehensive audit before being pre- sented for public review. This Technical Report has been reviewed and approved by the NTP Board of Scientific Counselors’ Peer Review Panel in public session; the interpretations described herein rep resent the official scientific position of the NTP. These studies are designed and conducted to characterize and evaluate the toxicologic potential, in- cluding 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, Springfeld, VA 22161 (703-487-4650). Single copies of this Technical Report are available without charge while supplies last from the NTP Public Information Office, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709 (919-541-3991). Special Note: This Technical Report underwent peer review in public session and was approved by the NTP Board of Scientific Counselors’ Technical Reports Review Subcommittee on September 22, 1982 [see pages 7-81. Thereafter, the NTP adopted the policy that the experimental data and labora- tory records from all NTP toxicology and carcinogenesis studies not yet printed and distributed would be audited. [A summary of the data audit is presented in Appendix G.1 Consequently, printing and distribution of this Technical Report have been delayed, and the format differs from that of Technical Reports that underwent peer review more recently. The categories of evidence of carcinogenicity adopted by the NTP in June 1983 were not used to evaluate these data. This final Technical Report supersedes all previous &afts of this report which have been distributed. 2,6-Xylidine, NTP TR 278 NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF 2,6=XYLIDINE (2,6-DIMETHYLANILINE ) (CAS NO. 87-62-7) IN CHARLES RIVER CD RATS (FEED STUDIES) Mary Kornreich, Ph.D., and Charles A. Montgomery, Jr., D.V.M., Study Scientists NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 January 1990 NTP TR 278 NIH Publication No. 90-2534 U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service National Institutes of Health CONTENTS PAGE ABSTRACT ................................................................ 3 CONTRIBUTORS ............................................................ 5 PEERREVIEWPANEL ........................................................ 6 SUMMARY OF PEER REVIEW COMMENTS ......................................... 7 I. INTRODUCTION ........................................................ 9 II. MATERIALS AND METHODS .............................................. 13 III. RESULTS ............................................................. 25 IV. DISCUSSION AND CONCLUSIONS ........................................... 47 V . REFERENCES ......................................................... 53 APPENDIXES APPENDIX A SUMMARY OF LESIONS IN MALE CD RATS IN THE TWO-YEAR FEED STUDY OF 2.0-XYLIDINE ................................................ 59 APPENDIX B SUMMARY OF LESIONS IN FEMALE CD RATS IN THE TWO-YEAR FEED STUDY OF2.&XYLIDINE ................................................ 85 APPENDIX C MUTAGENICITY OF 2. &XYLIDINE IN SALMONELLA TYPHlMURlUM .............. 111 APPENDIX D CHEMICAL CHARACTERIZATION OF 2.6-XYLIDINE ....................... 115 APPENDIX E CHARACTERIZATION OF FORMULATED DIETS ......................... 125 APPENDIX F ANALYSIS OF FORMULATED DIETS .................................. 129 APPENDIX G AUDIT SUMMARY ............................................... 137 2.6.Xylidine. NTP TR 278 2 2,6-XYLIDINE (2,6-DIMETHYLANILINE) CAS NO.87-62-7 ABSTRACT 2,6-Xylidine is a chemical intermediate used principally in the production of dyes. It is also a com- ponent of tobacco smoke, a degradation product of aniline-based pesticides, and a metabolite of cer- tain drugs, particularly the xylide group of local anesthetics. The National Toxicology Program (NTP) sponsored single-administration, 2-week, and 13-week studies of 2,6-xylidine by gavage in F344/N rats. The U.S.Environmental Protection Agency (EPA) sponsored short-term gavage studies and 10-week range-finding feed studies in Charles River CD rats (a Sprague Dawley-derived strain). A carcinogenesis study of 2,6-xylidine was initiated by the EPA, which designed and monitored the study during the 2-year exposure period. The NTP then assumed responsibility for the study, con- ducting terminal kill, necropsy, histopathologic evaluation, data analysis, and report preparation. Oral LD50 values of 1.2-1.3 g/kg were calculated for F344/N and Charles River CD rats administered single doses of 2,6-xylidine. Marginally toxic effects occurred in the hepatic, renal, and hematopoietic systems of dosed rats in the single-.administration, 2-week, 10-week, and 13-week studies. The 56 male and 56 female Charles River CD rats used in the 104-week carcinogenesis studies were the offspring of animals fed diets containing 0, 300, 1,000, or 3,000 ppm 2,6-xylidine before breeding, during pregnancy, and through the lactation period. The concentrations of 2,6-xylidine offered to ani- mals in the 104-week studies were the same as those given to their parents. During most of the 2-year studies, high dose male and female rats showed a reduction (greater than 10%) in body weight gain. Survival in high dose male rats was significantly reduced (P<O.OOl) rela- tive to that in controls. Survival also was reduced in the 1,000-ppm group. There was no significant relationship between concentration and mortality in female rats, but mortality was high for all groups of female rats during the second year of the study. The epithelium of the nasal cavity was the primary site of compound-related neoplastic and nonneo- plastic lesions. The incidences of both papillomas and carcinomas of the nasal cavity were signifi- cantly increased in high dose male and female rats. Carcinomas or adenocarcinomas (combined) occurred in 28/56 high dose males, 24/56 high dose females, and 1/56 mid dose females. Papillary ade- nomas occurred in 10156 high dose males, 2/56 mid dose males, and 6/56 high dose females. None occurred in the other groups. The carcinomas were highly invasive and frequently destroyed the nasal turbinates and nasal septum. Metastasis to the brain was present in 5/56 male and 7/56 female high dose rats. Malignant mesenchymal tumors were observed in the nasal cavity. Rhabdomyosarcomas occurred in two high dose male rats and two high dose female rats. These rare malignant tumors have not been previously reported at this site in Sprague Dawley rats. Malignant mixed tumors having features of adenocarcinomas and rhabdomyosarcomas were reported in one high dose male and one high dose 3 2,6-Xylidine, NTP TR 278 female rat. One undifferentiated sarcoma was seen in a high dose female rat. The nonneoplastic le- sions observed in the nasal cavity included acute inflammation, epithelial hyperplasia, and squamous metaplasia. The incidences of subcutaneous tissue fibromas were increased in high dose male and female rats (male: control, 0156; low dose, 1/56; mid dose, 2/56; high dose, 4/56; female: 0156; 2/56; 1/56; 4/56) and were dose related. Subcutaneous fibrosarcomas were observed in three high dose females, one high dose male, one mid dose female, one low dose male, and one control female. A significant dose-related increase occurred in the incidence of female rats with neoplastic nodules of the liver (0156; 1/56; 2/56; 4/55). This increase was significant