title: NTP Toxicology and Carcinogenesis Studies of Nickel Subsulfide (CAS No. 12035-72-2) in F344 Rats and B6C3F1 Mice (Inhalation Studies).
abstract: Nickel subsulfide is used in the manufacture of lithium batteries and is a major component in the refining of certain nickel ores. Nickel subsulfide was nominated as part of a class study of nickel compounds, for which there was little information on the toxic and carcinogenic effects of inhalation exposure. Male and female F334/N rats and B6C3F1 mice were exposed to nickel subsulfide (at least 97% pure; the mean value for the mass median aerodynamic diameter at each exposure concentration ranged from 2.0 to 2.2 mm by inhalation 6 hours per day, 5 days per week, for 16 days, 13 weeks, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, and mouse peripheral blood samples were analyzed for frequency of micronucleated normochromatic erythrocytes. 16-DAY STUDY IN RATS: Groups of five male and five female F344/N rats were exposed to atmospheres containing 0, 0.6, 1.2, 2.5, 5, or 10 mg nickel subsulfide/m(3) (equivalent to 0, 0.44, 0.88, 1.83, 3.65, and 7.33 mg nickel/m(3)) 6 hours per day, 5 days per week for a total of 12 exposure days during a 16-day period. Additionalmgroups of three male and three female rats were exposed to 0, 0.6, 2.5, or 10 mg/m(3) for tissue burden studies. One male exposed to 10 mg nickel subsulfide/m(3) in the core study died on day 14; all other rats survived until the end of the study. Final mean body weights and mean body weight gains of males exposed to 5 or 10 mg nickel subsulfide/m(3) and females exposed to 2.5, 5, or 10 mg/m(3) were significantly lower than those of the controls. Clinical findings of toxicity on day 5 of the study included labored respiration in 10 mg/m(3) males and 5 and 10 mg/m(3) females and dehydration in 5 and 10 mg/m(3) females. Absolute and relative lung weights of 2.5, 5, and 10 mg/m(3) males and all exposed groups of females were significantlymgreater than those of the controls, as was the absolute lung weight of 1.2 mg/m(3) males. Inflammation of the lung and atrophy of the nasal olfactory epithelium occurred in all exposed mgroups. The concentrations of nickel in the lungs of exposedmgroups of rats increased with exposure concentration (males, 7 to 67 mg nickel/g lung; females, 9 to 77 mg/g lung). 16-DAY STUDY IN MICE: Groups of five male and five female B6C3F1 mice were exposed to atmospheres containing 0, 0.6, 1.2, 2.5, 5, or 10 mg nickel subsulfide/m(3) for 6 hours per day, 5 days per week for a total of 12 exposure days during a 16-day period. Additional groups of three male and three female mice were exposed to 0, 0.6, 2.5, or 10 mg/m(3) for tissue burden studies. All male and female mice exposed to 10 mg nickel subsulfide/m(3) in the core study died before the end of the study; the death of one female was accidental. One control male, one control female, and one 1.2 mg/m(3) male also died before the end of the study. Final mean body weights and mean body weight gains of 5 mg/m(3) males were significantly lower than those of the controls. Clinical findings at day 5 included labored respiration in 10 mg/m(3) males and females. The absolute lung weight of 5 mg/m(3) males, the absolute and relative lung weights of 10 mg/m(3) males and 5 mg/m(3) females, and the relative lung weight of 10 mg/m(3) females were significantly greater than those of the controls. Inflammation of the lung occurred in 2.5, 5, and 10 mg/m(3) male and female mice, fibrosis of the lung occurred in 5 mg/m(3) males and females, and lymphoid hyperplasia of the bronchial lymph nodes and atrophy of the nasal olfactory epithelium occurred in 1.2, 2.5, 5, and 10 mg/m(3) males and females. Nickel concentrations in the lung of exposed male and female mice were greater than those of the controls (males, 10 to 20 mg nickel/g lung; females, 8 to 20 mg/g lung) 13-WEEK STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were exposed to atmospheres containing 0, 0.15, 0.3, 0.6, 1.2, or 2.5 mg nickel subsulfide/m(3) (equivalent to 0, 0.11, 0.22, 0.44, 0.88, and 1.83 mg nickel/m(3)) 6 hours per day, 5 days per week for 13 weeks. Additional groups of 18 male and 18 female female rats were exposed to 0, 0.15, 0.6, or 2.5 mg/m(3) for tissue burden studies. All core study rats survived until the end of the study. Final mean body weights and mean body weight gains of 2.5 mg/m(3) males were significantly lower than those of the controls; final mean body weights of all other exposure groups were similar to those of the controls. Chemical-related clinical findings included labored respiration in 2.5 mg/m(3) males and females during weeks 2 through 7. In general, neutrophil and erythrocyte counts, hematocrit values, and hemoglobin concentrations were minimally increased in exposed rats. Absolute and relative lung weights of all exposed groups were significantly greater than those of the controls. Increases in the number of alveolar macrophages, interstitial infiltrates, or incidences of chronic inflammation of the lung occurred in all groups exposed to nickel subsulfide concentrations of 0.3 mg/m(3) or greater; the severity of these lesions generally increased with increasing exposure concentration. Increases in the number of alveolar macrophages were observed in 0.15 mg/m(3) males and females. Lymphoid hyperplasia of the bronchial and mediastinal lymph nodes was observed in rats exposed to 0.3 mg/m(3) or greater. Most 0.6, 1.2, and 2.5 mg/m(3) males and females had atrophy of the nasal olfactory epithelium, and the severity generally increased with increasing exposure concentration. Nickel concentrations in the lung increased with exposure concentration and were greater than those in the controls in rats exposed for 13 weeks (males, 5 to 18 mg nickel/g lung; females, 5 to 17 mg/g lung). 13-WEEK STUDY IN MICE: Groups of 10 male and 10 female B6C3F1 mice were exposed to atmospheres containing 0, 0.15, 0.3, 0.6, 1.2, or 2.5 mg nickel subsulfide/m(3) for 6 hours per day, 5 days per week for 13 weeks. Additional groups of six male and six female mice were exposed to 0, 0.15, 0.6, or 2.5 mg/m(3) for tissue burden studies. Final mean body weights of all exposure groups were similar to those of the controls. No chemical-related clinical findings were observed. Lymphocyte counts in 1.2 and 2.5 mg/m(3) males were minimally greater than that of the controls. Hemoglobin concentrations and erythrocyte counts in 0.3, 0.6, 1.2, and 2.5 mg/m(3) females were minimally greater than those of the controls. Absolute and relative lung weights of 1.2 and 2.5 mg/m(3) males and females were significantly greater than those of the controls. An increase in alveolar macrophages was present in mice from the 0.3 mg/m(3) and higher exposure groups. Chronic inflammation and fibrosis were observed in the lung of 1.2 and 2.5 mg/m(3) males and females. Interstitial infiltrates of lymphocytes were observed in mice exposed to 0.6, 1.2, or 2.5 mg/m(3). Lymphoid hyperplasia of the bronchial lymph nodes was observed in groups exposed to 1.2 or 2.5 mg/m(3). Atrophy of the nasal olfactory epithelium occurred in 0.6, 1.2, and 2.5 mg/m(3) males and females, and incidences and severity generally increased with increasing exposure concentration. At 13 weeks, nickel concentrations in the lungs of exposed mice were greater than those of the controls (males, 3 to 17 g nickel/g lung; females, 6 to 23 mg/g lung), and these concentrations increased with increasing exposure concentration. 2-YEAR STUDY IN RATS: Survival, Body Weights, Clinical Findings, and Hematology: Groups of 63 male and 63 female F344/N rats were exposed to 0, 0.15, or 1 mg nickel subsulfide/m(3) (equivalent to 0, 0.11, or 0.73 mg nickel/m(3)) by inhalation for 6 hours per day, 5 days per week for 104 weeks. Survival of exposed males and female rats was similar to that of the controls. Mean body weights of males and females exposed to 0.15 mg/m(3) were similar to those of the controls. Mean body weights of rats exposed to 1 mg/m(3) were lower than those of the controls throughout the second year of the study. Chemical-related clinical findings included rapid and shallow breathing following exposure periods. Hematocrit values and hemoglobin concentrations in 1 mg/m(3) males and females and the erythrocyte count in 1 mg/m(3) males were mildly greater than those in the controls. Pathology Findings: In general, the absolute and relative lung weights of exposed males and females were significantly greater than those of the controls at 7 and 15 months. There were exposure-related increases in the incidences of alveolar/bronchiolar adenoma in males, alveolar/bronchiolar carcinoma in males and females, and alveolar/bronchiolar adenoma or carcinoma (combined) in males and females at 2 years. Nonneoplastic lung lesions generally observed in exposed males and females included fibrosis; chronic active inflammation; focal alveolar epithelial hyperplasia, macrophage hyperplasia, and proteinosis; bronchial lymphoid hyperplasia; and interstitial inflammation. At 2 years, there were significant exposure-related increases in the incidences of benign pheochromocytoma, malignant pheochromocytoma, and benign or malignant pheochromocytoma (combined) in males and of benign pheochromocytoma in females. The incidence of adrenal medulla hyperplasia in 1 mg/m(3) females was significantly greater than that of the controls At 2 years, the incidences of chronic active inflammation of the nose in 1 mg/m(3) females and of olfactory epithelial atrophy in 1 mg/m(3) males and females were significantly greater than those of the controls. The incidences of lymphoid hyperplasia of the bronchial lymph node in exposed males at 7 and 15 months and in exposed males and females at 2 years were significantly greater than those of the controls. Incidences of macrophage hyperplasia in the bronchial lymph node of exposed males at 15 months and exposed males and females at 2 years were greater than those of the controls. Tissue Burden Analyses: Nickel concentrations in the lungs of exposed rats were greater than those of the controls at 7 months (males, 6 to 9 mg nickel/g lung; females, 6 to 9 mg/g lung) and 15 months (males, 4 to 3 mg nickel/g lung; females, 4 to 7 mg/g lung). 2-YEAR STUDY IN MICE: Survival, Body Weights, Clinical Findings, and Hematology: Groups of 80 male and 80 female B6C3F1 mice were exposed to 0, 0.6, or 1.2 mg nickel subsulfide/m(3) (equivalent to 0, 0.44, or 0.88 mg nickel/m(3)) by inhalation for 6 hours per day, 5 days per week for 105 weeks. Survival of exposed male and female mice was similar to that of the controls. Mean body weights of 0.6 and 1.2 mg/m(3) males and females were less than those of the controls throughout the second year of the study. Chemical-related clinical findings in male and female mice included labored respiration following exposure periods. The hematocrit value and the segmented neutrophil, monocyte, lymphocyte, and total leukocyte counts in 1.2 mg/m(3) females were greater than those in the controls. Pathology Findings: Absolute and relative lung weights of exposed males and females were generally significantly greater than those of the controls at 7 and 15 months. The incidence of alveolar/bronchiolar carcinoma in 0.6 mg/m(3) females and the incidences of alveolar/bronchiolar adenoma or carcinoma (combined) in 0.6 mg/m(3) males and 0.6 and 1.2 mg/m(3) females were significantly less than those of the controls. In general, the incidences of chronic active inflammation; bronchialization (alveolar epithelial hyperplasia), macrophage hyperplasia and proteinosis; interstitial infiltration; and fibrosis in exposed groups of males and females were greater than those of the controls at 7 and 15 months and at 2 years. The incidences of atrophy of the nasal olfactory epithelium and inflammation of the nose in exposed mice were also generally greater than those of the controls. At 2 years, the incidences of degeneration of olfactory epithelium in exposed females were significantly less than that of the controls. The incidences of lymphoid hyperplasia of the bronchial lymph node in 1.2 mg/m(3) males at 15 months, in 0.6 and 1.2 mg/m(3) females at 15 months, and in 0.6 and 1.2 mg/m(3) males and females at 2 years were significantly greater than those of the controls. The incidences of macrophage hyperplasia in 1.2 mg/m(3) males at 7 and 15 months, in 0.6 and 1.2 mg/m(3) females at 15 months, and in 0.6 and 1.2 mg/m(3) males and females at 2 years were significantly greater than those of the controls. Tissue Burden Analyses: Nickel concentrations in the lungs of exposed mice were greater than those of the controls at 7 months (males, 10 to 11 mg nickel/g lung; females, 10 to 14 mg/g lung) and 15 months (males, 12 to 20 mg nickel/g lung; females, 15 to 26 mg/g lung). GENETIC TOXICOLOGY: Nickel subsulfide was considered to be equivocal in the Salmonella gene mutation assay overall. Sporadic weakly positive and equivocal responses were obtained in strain TA100 with and without S9 metabolic activation enzymes; all other strain/activation combinations gave negative results. No increase in the frequency of micronucleated erythrocytes was observed in peripheral blood samples from male or female mice exposed to nickel subsulfide by inhalation for 13 weeks. CONCLUSIONS: Under the conditions of these 2-year inhalation studies, there was clear evidence of carcinogenic activity of nickel subsulfide in male F344/N rats based on increased incidences of alveolar/bronchiolar adenoma, carcinoma, and adenoma or carcinoma (combined) and on increased incidences of benign, malignant, and benign or malignant (combined) pheochromocytoma of the adrenal medulla. There was clear evidence of carcinogenic activity of nickel subsulfide in female F344/N rats based on increased incidences of alveolar/bronchiolar carcinoma and alveolar/bronchiolar adenoma or carcinoma (combined) and an increased incidence of benign pheochromocytoma of the adrenal medulla. There was no evidence of carcinogenic activity of nickel subsulfide in male or female B6C3F1 mice exposed to 0.6 or 1.2 mg/m(3). Exposure of male and female rats to nickel subsulfide by inhalation for 2 years resulted in inflammation, hyperplasia, and fibrosis in the lung; inflammation and atrophy of the olfactory epithelium in the nose; and hyperplasia in the adrenal medulla (females). Exposure of male and female mice to nickel subsulfide by inhalation for 2 years resulted in inflammation, bronchialization, hyperplasia, and fibrosis in the lung and inflammation and atrophy of the olfactory epithelium in the nose. Synonyms: Heazlewoodite, nickel subsulphide, nickel sulfide (3:2), a-nickel sulfide (3:2) crystalline, nickel sulphide, nickel tritadisulphide, trinickel disulfide