Abstract
Nitrapyrin, a nitrification inhibitor, produces liver tumors in B6C3F1 mice. In a 2-year oncogenicity study, increased incidence of mice with hepatocellular tumors was observed following exposure to 125 (females only) or 250 mg/kg/day (males and females) nitrapyrin in the diet. Previous data was generated in male mice to support a mode-of-action (MoA) characterized by constitutive androstane receptor (CAR) nuclear receptor (NR) activation, increased hepatocellular proliferation, and subsequent hepatocellular foci and tumor formation. Uncertainty as to the relevance of this MoA for females remained given the increased sensitivity to tumor formation in female mice. A targeted MoA study was conducted to evaluate CAR activation and hepatic responses in female mice treated with the female carcinogenic dose of nitrapyrin for 4 days. Nitrapyrin induced a treatment-related increase in hepatocellular hypertrophy and hepatocellular proliferation. Nitrapyrin also induced a dose-related increase in the Cyp2b10/CAR-associated transcript and liver weights. Nitrapyrin-induced liver weights and Cyp2b10 gene expression for both males and females were compared to data generated from three other established CAR activators; methyl isobutyl ketone, phenobarbital, and sulfoxaflor. The response observed in female mice following exposure to nitrapyrin was within range of the degree of change observed in mice following exposure to tumorigenic doses of other CAR activators. Consistent with the liver MoA in male mice, these data support a CAR-mediated mode of action for nitrapyrin-induced liver tumors in female mice, with the understanding that a focused approach minimizing animal use can bridge male and female datasets when sex-specific carcinogenic differences are observed.
Highlights
Nitrapyrin (2-chloro-6-(trichloromethyl) pyridine; CAS Number 1929-82-4; Figure 1) is the active ingredient in N-SERVETM.1 nitrogen stabilizer
Based on previous MoA studies in male B6C3F1 mice, constitutive androstane receptor (CAR) activation was supported by a 370.7-fold increase in Cyp2b10 gene expression and a 3.5-fold increase in panlobular hepatocellular proliferation following 4 days of exposure to the male carcinogenic dose of nitrapyrin (250 mg/kg/day)
Following 4 days of exposure to 125 mg/kg/day nitrapyrin in female B6C3F1 mice, the relative liver weight changes (12.6% increase compared to control), panlobular hepatocellular proliferation (1.4-fold increase compared to control), and increased Cyp2b10 gene expression (11.4-fold compared to control) indicate female mice did not attain a hepatic response of a similar magnitude as male mice exposed to nitrapyrin over the same duration, albeit the dose in male mice was two times higher
Summary
Nitrapyrin (2-chloro-6-(trichloromethyl) pyridine; CAS Number 1929-82-4; Figure 1) is the active ingredient in N-SERVETM. nitrogen stabilizer. Two separate dietary chronic toxicity/2-year carcinogenicity studies were performed with a total of five dose levels of nitrapyrin up to 250 mg/kg/ day in B6C3F1 mice. 75 mg/kg/day, and in a second carcinogenicity study, male and female mice were given 0, 125 or 250 mg/kg/day nitrapyrin in the diet. At 75, 125, and 250 mg/kg/day, males and females had increased absolute and relative liver weights. Microscopic evaluation revealed hepatocellular hypertrophy and altered tinctorial properties at 125 and 250 mg/kg/day in males and females, while single cell necrosis was identified only in males at 250 mg/kg/day. An increased incidence of mice with hepatocellular tumors (adenomas and/or carcinomas) were observed following exposure to 125 (females only) or 250 mg/kg/day (males and females) nitrapyrin in the diet. Tumor incidence data have been published (LaRocca et al, 2017)
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