Doses of Morpholine and Sodium Nitrite -Nitrosomorpholine and Its Application to Rats

A method was developed to monitor the in vivo formation of W-nitrosomorpholine. A/-Nitroso(2-hydroxyethyl)glycine, a major urinary metabolite of A/-nitrosomorpholine, was quantified as its methyl ester-trimethylsilyl ether derivative, using gas chromatography with nitrosamine-specific detection. When the method was applied to rats, the in vivo formation of, or exposure to, as little as 0.6 ut) of /V-nitrosomorpholine could be quantified. The method was also applicable to human urine, with a detection limit of approximately 0.5 /ug of A/-nitroso(2-hydroxyethyl)glycine per 100-ml urine sample. The formation of A/-nitrosomorpholine was measured in rats treated by gavage with a wide range of doses of morpholine and NaNO2. Depending on the dose, 0.5 to 12% of the morpholine was nitrosated. A/-Nitrosomorpholine formation showed a high degree of variability among rats treated with a given dose of morpholine and NaNO2, but the levels of A/nitrosomorpholine formed were generally in agreement with ex pectations based on in vitro studies in which dependence on morpholine concentration multiplied by nitrite concentration squared has been established. The formation of A/-nitrosomorpholine was also measured in rats administered a diet containing 50 ppm of morpholine and 1000 ppm of NaNO2, a regimen which has been previously shown to induce liver cell tumors in 58% of the animals. The mean daily formation of A/-nitrosomorpholine under these conditions was estimated to be 0.88 ±0.59 ¿¡mol/ rat (S.D.), which is high enough to account for the observed tumor incidence. The results of this study provide quantitative support for the assumption that in vivo formation of A/-nitrosomorpholine leads to tumor development.