N,N-Dimethyl-p-toluidine was nominated for toxicology and carcinogenesis studies by the National Cancer Institute based on the potential for human exposure through its use in dental materials and bone cements and the lack of toxicity and carcinogenicity data. Male and female F344/N rats and B6C3F1/N mice were administered N,N-dimethyl-p-toluidine (greater than 99% pure) in corn oil by gavage for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Escherichia coli, mouse peripheral blood, and mouse and rat liver.
Three-month study in rats
Groups of 10 male and 10 female rats were administered 0, 62.5, 125, 250, 500, or 1,000 mg N,N-dimethyl-p-toluidine/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female rats (clinical pathology study) were administered the same doses, 5 days per week for 25 days. On day 88, blood was collected from core study rats for hemoglobin and methemoglobin analyses only. All 1,000 mg/kg male and female rats and one 500 mg/kg male rat died by study day 3. Mean body weights of all surviving dosed groups of males and females were significantly less than those of the vehicle controls. Clinical findings associated with exposure to N,N-dimethyl-p-toluidine included cyanosis, abnormal breathing, and lethargy in groups administered 250 mg/kg or greater.
Methemoglobinemia appeared to be the primary hematologic toxic response, and many other lesions could be explained as secondary to methemoglobin formation including Heinz body formation; a macrocytic, hypochromic, responsive anemia; and increased hematopoietic cell proliferation in the spleen and bone marrow. In general, hematologic changes were dose-related and occurred at both evaluated timepoints in all dosed groups. Anemia was evidenced by decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts; erythrocyte macrocytosis was characterized by increases in mean cell volume and mean cell hemoglobin values; erythrocyte hypochromia was evidenced by decreases in mean cell hemoglobin concentration values; and an erythropoietic response to the anemia was characterized by substantially increased reticulocyte and nucleated erythrocyte counts. Liver weights of all surviving dosed groups of males and females were significantly greater than those of the vehicle controls. Kidney weights of all surviving dosed groups of females were significantly greater than those of the vehicle controls. There were significant decreases in left cauda epididymis and left epididymis weights in 250 mg/kg males. There was a dose-related decrease in the number of cycling females, with only four females in the 250 mg/kg group having regular cycles and females in the 125 and 250 mg/kg groups spending a significantly higher proportion of time in extended diestrus compared to the vehicle control group.
In the surviving groups of rats, there were significantly increased incidences of pigmentation in the liver of all dosed groups, hepatocyte hypertrophy in groups administered 125 mg/kg or greater, and hepatocyte necrosis in 62.5, 250, and 500 mg/kg females. In the olfactory epithelium of the nose, there were dose-related increases in the incidences and severities of degeneration in all dosed groups and significantly increased incidences of metaplasia in the 250 and 500 mg/kg groups. In the respiratory epithelium of the nose, there were significantly increased incidences of hyperplasia and squamous metaplasia in all of the groups administered 125 mg/kg or greater. The incidences of glandular hyperplasia of the nose were significantly increased in males and females administered 125, 250, or 500 mg/kg. In the spleen, there were significantly increased incidences of capsule fibrosis, congestion, mesothelial hypertrophy, and lymphoid follicle atrophy primarily in groups administered 125 mg/kg or greater. Hematopoietic cell proliferation and pigmentation were increased in severity in treated groups. In the kidney, there were significantly increased incidences of nephropathy (females), pigmentation (males and females), papillary necrosis (males and females), and mineralization (males). Other treatment-related lesions included inflammation of the forestomach in males, mesenteric lymph node atrophy in females, and bone marrow hyperplasia in males and females.
Three-month study in mice
Groups of 10 male and 10 female mice were administered 0, 15, 30, 60, 125, or 250 mg N,N-dimethyl-p-toluidine/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. All 250 mg/kg male and female mice (except for one male mouse) died before day 10, and three males and two females administered 125 mg/kg died before the end of the study. The final mean body weight of 125 mg/kg males and the mean body weight gains of 125 mg/kg males and females were significantly less than those of the vehicle controls. Clinical findings associated with administration of N,N-dimethyl-p-toluidine included abnormal breathing, thinness, lethargy, cyanosis, and ruffled fur in 125 and 250 mg/kg males and females.
Methemoglobinemia appeared to be the primary hematologic toxic response; however there were less severe erythron changes compared to the 3-month study in rats. In females, no erythron changes were detected up to 125 mg/kg. In males, inconsistent and minor decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts, and increased reticulocyte counts occurred in groups administered 60 mg/kg or greater. Methemoglobin values were minimally increased in males and females administered 30 mg/kg or greater. Heinz bodies were slightly increased in 60 mg/kg females, 125 mg/kg males and females, and the one surviving 250 mg/kg male; Heinz body formation was considered secondary to methemoglobin formation. Liver weights of all dosed groups of mice were significantly greater than those of the vehicle controls.
In the surviving groups of mice, there were significantly increased incidences of bronchiolar epithelium degeneration, bronchiolar epithelium regeneration, and peribronchiolar chronic active inflammation in the lung of 125 mg/kg groups, and histiocytic infiltrates of the alveoli in 125 mg/kg females. In the nose, there were significantly increased incidences of glandular hyperplasia and olfactory epithelium metaplasia in the 125 mg/kg groups and olfactory epithelium degeneration in 60 mg/kg females and 125 mg/kg males and females. In the thymus, the incidences of thymocyte necrosis in the 125 mg/kg groups were significantly increased. In the liver, the severities of cytoplasmic vacuolization of the hepatocytes were increased in dosed groups of males and females.
Two-year study in rats
Groups of 50 male and 50 female rats were administered 0, 6, 20, or 60 mg N,N-dimethyl-p-toluidine/kg body weight in corn oil by gavage, 5 days per week for 104 or 105 weeks. Additional groups of 10 male and 10 female rats (clinical pathology study) were administered the same doses for 86 days. Survival of 60 mg/kg males was significantly less than that of the vehicle controls. Mean body weights of 60 mg/kg males and females were more than 10% less than those of the vehicle controls after week 61 and week 33, respectively. Clinical findings included signs of pallor in 60 mg/kg females and hyperactivity and boxing behavior in 20 mg/kg females and 60 mg/kg males and females.
The hematology findings at the 3-month timepoint were consistent with those in the 3-month study in rats which indicated that methemoglobinemia was the primary hematologic toxic response. In the 20 and 60 mg/kg groups, there were dose-related decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts. There were similar trends toward erythrocyte macrocytosis and hypochromia and increased erythropoiesis as seen in the 3-month study. While the magnitudes of the erythron decreases were not sufficient to classify the responses as anemias, the patterns of the erythron changes were identical to those in the 3-month study.
In the liver of 60 mg/kg males and females, there were significantly increased incidences of hepatocellular carcinoma and hepatocellular adenoma or hepatocellular carcinoma (combined). Numerous nonneoplastic liver lesions occurred in dosed males and females primarily in the 20 and 60 mg/kg groups.
In the nose, there were significantly increased incidences of transitional epithelium adenoma and transitional epithelium adenoma or carcinoma (combined) in 60 mg/kg males; transitional epithelium adenoma also occurred in female rats administered 6 or 60 mg/kg. In the nose, there were significantly increased incidences of nonneoplastic lesions in the olfactory, respiratory, and transitional epithelia of dosed rats. These lesions occurred with the greatest incidence and severity in the 60 mg/kg groups. The incidences of inflammation and nerve atrophy were significantly increased in males and females administered 60 mg/kg.
There were increased incidences of follicular cell adenoma or carcinoma (combined) of the thyroid gland in all dosed groups of males, and an increased incidence of follicular cell adenoma in 20 mg/kg females.
In the spleen, there were significantly increased incidences of hematopoietic cell proliferation in all dosed groups of males and females. The incidences of congestion and mesothelial hypertrophy of the capsule were significantly increased in 60 mg/kg males and all dosed groups of females. There were also significantly increased incidences of capsular fibrosis and atrophy of the lymphoid follicle in the 60 mg/kg groups. The incidences of pigmentation were significantly increased in all dosed groups of males and in 60 mg/kg females.
In all dosed groups of female rats, there were significantly increased incidences of nephropathy. Although the incidences of this lesion were not significantly increased in dosed males, the severities increased with increasing dose in both males and females. The incidences of pigmentation of the kidney were significantly increased in all dosed groups of males and in 60 mg/kg females.
In the forestomach of males, there were significantly increased incidences of hyperplasia and ulcer in the 20 and 60 mg/kg groups and inflammation in the 60 mg/kg group. In the bone marrow of 20 and 60 mg/kg males and 60 mg/kg females, there were significantly increased incidences of hyperplasia. In the mesenteric lymph node of 20 and 60 mg/kg males, there were significantly increased incidences of histiocytic cellular infiltrates.
Two-year study in mice
Groups of 50 male and 50 female mice were administered 0, 6, 20, or 60 mg N,N-dimethyl-p-toluidine/kg body weight in corn oil by gavage, 5 days per week for 105 weeks. Survival of 60 mg/kg females was significantly less than that of the vehicle control group. Mean body weights of 60 mg/kg males and females were more than 10% less than those of the vehicle controls after week 89 and week 65, respectively.
In the liver, there were significantly increased incidences of hepatocellular adenoma in 20 and 60 mg/kg females and hepatocellular carcinoma in 60 mg/kg males and all dosed female groups. The increased incidences of hepatocellular adenoma and carcinoma in the dosed groups were primarily due to increased incidences of animals with multiple hepatocellular neoplasms. The incidences of hepatoblastoma were significantly increased in 20 mg/kg males and 60 mg/kg males and females. In all dosed groups of males and females, there were significantly increased incidences of hepatocyte hypertrophy, and the incidences of eosinophilic focus were significantly increased in the 20 and 60 mg/kg males and females. There were significantly increased incidences of fatty change and necrosis in 60 mg/kg females.
In the lung of 20 and 60 mg/kg female mice, there were significantly increased incidences of alveolar/ bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined). There were also significantly increased incidences of alveolar epithelium hyperplasia in 20 mg/kg females; bronchiolar epithelium regeneration, bronchus epithelium regeneration, and bronchus necrosis in 60 mg/kg females; and alveolar infiltrates of histiocytes in 60 mg/kg males and females.
In the forestomach of 20 and 60 mg/kg female mice, there were significantly increased incidences of squamous cell papilloma and squamous cell papilloma or carcinoma (combined). There were significantly increased incidences of epithelium hyperplasia in 20 and 60 mg/kg females, and inflammation and ulcer in 60 mg/kg females.
In the nose, there were significantly increased incidences of nonneoplastic lesions of the olfactory and respiratory epithelia in 60 mg/kg males and 20 and 60 mg/kg females. There were significantly increased incidences of nerve atrophy in 60 mg/kg males and females and of inflammation in 60 mg/kg females. The incidences of atrophy of the olfactory lobe were significantly increased in 60 mg/kg males and females. In 60 mg/kg females, the incidences of regenerative hyperplasia of the nasolacrimal duct and necrosis of the vomeronasal organ were significantly increased.
In the spleen, there was a significantly increased incidence of atrophy in 60 mg/kg females. There were also significantly increased incidences of bone marrow hyperplasia in all dosed groups of females and mesenteric lymph node atrophy in 60 mg/kg females.
Genetic toxicology
N,N-Dimethyl-p-toluidine was tested in two independent bacterial gene mutation studies; both studies gave negative results in S. typhimurium or E. coli tester strains, with and without exogenous metabolic activation. In vivo, no significant increases in the frequencies of micronucleated erythrocytes were observed in peripheral blood of male or female B6C3F1/N mice treated with N,N-dimethyl-p-toluidine by gavage for 3 months. Furthermore, no increases in micronucleated reticulocytes were observed in male B6C3F1/N mice treated with N,N-dimethyl-p-toluidine for 4 days. Results of DNA damage (comet) studies yielded mixed results. No increases in DNA damage (measured as percent tail DNA) were seen in liver cells or blood leukocytes of male B6C3F1/N mice administered N,N-dimethyl-p-toluidine by gavage once daily for 4 days. However, a small but significant increase in DNA damage was seen in liver cells of male Sprague-Dawley rats administered 60 mg/kg N,N-dimethyl-p-toluidine once daily for 4 days.
Conclusions
Under the conditions of these 2-year oral gavage studies, there was clear evidence of carcinogenic activity of N,N-dimethyl-p-toluidine in male F344/N rats based on increased incidences of hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined), and increased incidences of nasal cavity neoplasms (primarily nasal cavity transitional epithelium adenoma). The increased incidences of thyroid gland follicular cell neoplasms may have been related to treatment. There was clear evidence of carcinogenic activity of N,N-dimethyl-p-toluidine in female F344/N rats based on increased incidences of hepatocellular carcinoma and hepatocellular adenoma or carcinoma (combined). The occurrence of nasal cavity transitional epithelium adenoma was considered to be related to treatment. There was clear evidence of carcinogenic activity of N,N-dimethyl-p-toluidine in male B6C3F1/N mice based on increased incidences of hepatocellular adenoma (multiple), hepatocellular carcinoma, and hepatoblastoma. There was clear evidence of carcinogenic activity of N,N-dimethyl-p-toluidine in female B6C3F1/N mice based on increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatoblastoma and increased incidences of alveolar/ bronchiolar neoplasms (primarily adenoma). The increased incidences of forestomach squamous cell papilloma in female mice were considered to be related to treatment.
Administration of N,N-dimethyl-p-toluidine resulted in increased incidences of nonneoplastic lesions of the liver and nasal cavity in male and female rats and mice; the kidney in male and female rats; the spleen and bone marrow in male and female rats and female mice; the lung in male and female mice; the forestomach in male rats and female mice; the mesenteric lymph node in male rats and female mice; and the olfactory lobe in male and female mice.
N,N-Dimethyl-p-toluidine also caused hematologic toxicity and increases in methemoglobin levels in male and female rats and mice (as measured at 3 months).