title: NTP Toxicology and Carcinogenesis Studies of Nickel Sulfate Hexahydrate (CAS No. 10101-97-0) in F344 Rats and B6C3F1 Mice (Inhalation Studies).
abstract: Nickel sulfate hexahydrate is used in nickel plating, as a mordant in dyeing and printing textiles, as a blackening agent for zinc and brass, and in the manufacture of organic nickel salts. Nickel sulfate hexahydrate was nominated by the National Cancer Institute to the NTP 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 F344/N rats and B6C3F1 mice were exposed to nickel sulfate hexahydrate (greater than 98% pure) by inhalation for 16 days, 13 weeks, or 2 years. Genetic toxicology studies were conducted in L5178Y mouse lymphoma cells. 16-DAY STUDY IN RATS: Groups of five male and five female F344/N rats were exposed to 0, 3.5, 7, 15, 30, or 60 mg nickel sulfate hexahydrate/m(3) (equivalent to 0, 0.7, 1.4, 3.1, 6.1, or 12.2 mg nickel/m(3)). Rats were exposed on weekdays only, for a total of 12 exposure days during a 16-day period. Additional groups of four or five male and female F344/N rats were exposed to 0, 3.5, 15, or 30 mg nickel sulfate hexahydrate/m(3)for tissue burden studies. In the core study, two 60 mg/m(3) males, one 30 mg/m(3) female, and all 60 mg/m(3)females died before the end of the study. Final mean body weights of all exposed groups of males and females were significantly lower than those of the controls, as were mean body weight gains of male rats. Clinical findings included increased rates of respiration and reduced activity levels in rats in all exposure groups, except those exposed to 3.5 mg/m(3). Absolute lung weights of 60 mg/m(3) males and of all exposed groups of females were significantly greater than those of the controls, as were the relative lung weights of all exposed groups of males and females. Inflammation (including degeneration and necrosis of the bronchiolar epithelium) occurred in the lungs of all exposed groups of males and females. Atrophy of the olfactory epithelium occurred in the nasal passages of all exposed groups of males (except 60 mg/m(3)) and in 15, 30, and 60 mg/m(3) females. Lymphoid hyperplasia in the bronchial or mediastinal lymph nodes was observed in 30 mg/m(3) males and in 60 mg/m(3) males and females. The concentration of nickel in the lungs of all exposed groups of males and females was greater than in control animals. 16-DAY STUDY IN MICE: Groups of five male and five female B6C3F1 mice were exposed to 0, 3.5, 7, 15, 30, or 60 mg nickel sulfate hexahydrate/m(3). Mice were exposed on weekdays only, for a total of 12 exposure days during a 16-day period. Additional groups of five male and five female B6C3F1 mice were exposed to 0 or 3.5 mg nickel sulfate hexahydrate/m(3)for tissue burden studies. All core study mice exposed to 7 mg/m(3) or greater died before the end of the study; all control and 3.5 mg/m(3)mice survived to the end of the study. Final mean body weights and weight gains of 7, 15, 30, and 60 mg/m(3)males and females were significantly less than those of the controls, and clinical findings in these groups included emaciation, lethargy, and rapid respiration rates. Absolute and relative lung weights of male and female mice exposed to 7 mg/m(3) or greater were significantly greater than those of the controls. Only tissues from mice exposed to 0, 3.5, or 7 mg/m(3) were examined histopathologically. Inflammation occurred in the lungs of 3.5 and 7 mg/m(3) males and females; necrosis of the alveolar and bronchiolar epithelium was a component of the inflammation in 7 mg/m(3)males and females. In addition, atrophy of the olfactory epithelium of the nasal passages was observed in 3.5 mg/m(3) males and females. Nickel concentrations in the lungs of mice exposed to 3.5 mg/m(3) were greater than those in the controls. 13-WEEK STUDY IN RATS: Groups of ten male and ten female F344/N rats were exposed to 0, 0.12, 0.25, 0.5, 1, or 2 mg nickel sulfate hexahydrate (equivalent to 0, 0.03, 0.06, 0.11, 0.22, or 0.44 mg nickel/m(3)), 5 days per week for 13 weeks. Additional groups of six male and six female F344/N rats were exposed to 0, 0.12, 0.5, or 2 mg nic mg nickel sulfate hexahydrate/m(3)for tissue burden studies. In the core study, one 2 mg/m(3)male rat died before the end of the study; all other males and all females survived until the end of the study. Final mean body weights and body weight gains of all exposed groups were similar to those of the controls. There were no significant clinical findings noted during the study. Exposure-related increases in neutrophil and lymphocyte numbers occurred and were most pronounced in female rats. With the exception of 0.12 mg/m(3)rats, absolute and relative lung weights of all exposed groups were generally significantly greater than those of the controls. Exposure-related increases in the incidence and severity of inflammatory lesions (alveolar macrophages, chronic inflammation, and interstitial infiltration) occurred in the lungs of all exposed groups of males and females. Lymphoid hyperplasia of the bronchial and/or mediastinal lymph nodes occurred in males exposed to 0.5 mg/m(3)or greater. Atrophy of the olfactory epithelium occurred in males and females exposed to 0.5, 1, and 2 mg/m(3)and in 0.25 mg/m(3)females. The concentration of nickel in the lungs of 0.5 and 2 mg/m(3) rats was greater than that in the lungs of control animals at 4, 9, and 13 weeks for males and at 13 weeks for females. 13-WEEK STUDY IN MICE: Groups of ten male and ten female B6C3F1 mice were exposed to 0, 0.12, 0.25, 0.5, 1, or 2 mg nickel sulfate hexahydrate, 5 days per week for 13 weeks. Additional groups of up to five or six male and female B6C3F1 mice were exposed to 0, 0.12, 0.5, or 2 mg nickel sulfate hexahydrate/m(3)for tissue burden studies. In the core study, four control males, three control females, and one 0.12 mg/m(3)male died before the end of the study; the deaths were not considered to be chemical related, and all other mice survived to the end of the study. The final mean body weights and body weight gains of all exposed groups were similar to those of the controls. There were no chemical-related clinical findings. Hematology changes similar to those reported in female rats occurred in female mice, but the mice were minimally affected. The absolute and relative lung weights of 1 mg/m(3)males and 2 mg/m(3)males and females were significantly greater than those of the controls. Increased numbers of alveolar macrophages occurred in all males and females exposed to 0.5 mg/m(3)or greater. Chronic active inflammation and fibrosis occurred in 1 and 2 mg/m(3)males and females. Lymphoid hyperplasia of the bronchial lymph node and atrophy of the olfactory epithelium in the nasal passages were observed in 2 mg/m(3)males and females. Nickel concentration in the lung of 2 mg/m(3)females was significantly greater than in control animals. 2-YEAR STUDY IN RATS: Groups of 63 to 65 male and 63 to 64 female rats were exposed to nickel sulfate hexahydrate by inhalation at concentrations of 0, 0.12, 0.25, or 0.5 mg/m(3) (equivalent to 0, 0.03, 0.06, or 0.11 mg nickel/m(3)). Animals were exposed for 6 hours plus T90 (8 minutes) 5 days per week for 104 weeks. Five male and five female rats from each group were evaluated at 7 months for histopathology; an additional seven males and seven females from each group were evaluated at 7 months for nickel tissue burden in the lung and kidney; and five males and five females from each group were evaluated at 15 months for alterations in hematology, nickel tissue burden in the lung and kidney, and histopathology. Survival, Body Weights, Clinical Findings, and Hematology The survival rates of all exposed groups of males and females were similar to those of the controls. Mean body weights of 0.5 mg/m(3)female rats were slightly lower (6&percnt; to 9&percnt;) than those of the controls throughout the second year of the study; final mean body weights of all exposed groups of males and 0.12 and 0.25 mg/m(3)females were similar to those of the controls. There were no clinical findings or hematology differences that were considered to be related to nickel sulfate hexahydrate administration. Pathology Findings No exposure-related neoplasms occurred in male or female rats exposed by inhalation to nickel sulfate hexahydrate for 2 years. Increased incidences of inflammatory lung lesions were generally observed in all exposed groups of male and female rats at the end of the study. The incidences of chronic active inflammation, macrophage hyperplasia, alveolar proteinosis, and fibrosis were markedly increased in male and female rats exposed to 0.25 or 0.5 mg/m(3). Increased incidences of lymphoid hyperplasia in the bronchial lymph nodes occurred in 0.5 mg/m(3)male and female rats at the end of the 2-year study. The incidences of atrophy of the olfactory epithelium in 0.5 mg/m(3)males and females were significantly greater than those in controls at the end of the study. Tissue Burden Analyses Lung nickel burdens in exposed male and female rats were greater than those in the controls at the 7- and 15-month interim evaluations, and lung nickel burdens values increased with increasing exposure concentration. 2-YEAR STUDY IN MICE: Groups of 80 male and 80 female mice were exposed to nickel sulfate hexahydrate by inhalation at concentrations of 0, 0.25, 0.5, or 1 mg/m(3) (equivalent to 0, 0.06, 0.11, or 0.22 mg nickel/m(3)). Animals were exposed for 6 hours plus T90 (8 minutes) 5 days per week for 104 weeks. Five male and five female mice from each group were evaluated at 7 months for histopathology; five males and five females from each group were evaluated at 7 months for nickel tissue burden in the lung and kidney; five males and five females from each group were evaluated at 15 months for alterations in hematology and histopathology; and five males and five females from each group were evaluated at 15 months for nickel tissue burden in the lung and kidney. Survival, Body Weights, Clinical Findings, and Hematology The survival rates of all exposed groups of males and females were similar to those of the controls. The mean body weights of 1 mg/m(3)males and of all exposed groups of females were lower than those of the controls during the second year of the study. There were no clinical findings or hematology differences considered to be related to chemical exposure. Pathology Findings Inflammatory lesions of the lung generally occurred in all exposed groups of male and female mice at the end of the 2-year study. These lesions included macrophage hyperplasia, chronic active inflammation, bronchialization (alveolar epithelial hyperplasia), alveolar proteinosis, and infiltrating cells in the interstitium. Incidences of macrophage hyperplasia and/or lymphoid hyperplasia occurred in the bronchial lymph nodes of most of the 1 mg/m(3)males and females and in some 0.5 mg/m(3)females at the end of the 2-year study. Atrophy of the olfactory epithelium was observed in 0.5 and 1 mg/m(3)males and in all exposed groups of females at the end of the 2-year study. Tissue Burden Analyses At the 7- and 15-month interim evaluations, lung nickel burden parameters measured in control and exposed groups were below the limit of detection. Absolute lung weights of 0.5 and 1 mg/m(3)lung burden study females were significantly greater than those of the controls at 15 months. GENETIC TOXICOLOGY: Nickel sulfate hexahydrate (500 to 800 g/mL) was tested for induction of trifluorothymidine resistance in L5178Y mouse lymphoma cells. A positive response was observed in the absence of S9. The test was not performed with S9. CONCLUSIONS: Under the conditions of these 2-year inhalation studies, there was no evidence of carcinogenic activity of nickel sulfate hexahydrate in male or female F344/N rats exposed to 0.12, 0.25, or 0.5 mg/m(3) (0.03, 0.06, or 0.11 mg nickel/m(3)). There was no evidence of carcinogenic activity of nickel sulfate hexahydrate in male or female B6C3F1 mice exposed to 0.25, 0.5, or 1 mg/ m3 (0.06, 0.11, or 0.22 mg nickel/m(3)). Exposure of rats to nickel sulfate hexahydrate by inhalation for 2 years resulted in increased incidences of chronic active inflammation, macrophage hyperplasia, alveolar proteinosis, and fibrosis of the lung; lymphoid hyperplasia of the bronchial lymph node; and atrophy of the olfactory epithelium. Exposure of mice to nickel sulfate hexahydrate by inhalation for 2 years resulted in increased incidences of chronic active inflammation, bronchialization (alveolar epithelial hyperplasia), macrophage hyperplasia, interstitial infiltration, and alveolar proteinosis of the lung; lymphoid and macrophage hyperplasia of the bronchial lymph node; and atrophy of the olfactory epithelium. Synonyms: Blue salt; hexahydrate, nickel (2+) salt; nickel monosulfate hexahydrate; nickel (2+) sulfate hexahydrate; nickel (II) sulfate hexahydrate; nickel sulphate hexahydrate; nickelous sulfate hexahydrate; nickelous sulphate hexahydrate; single nickel salt, sulfuric acid