Possible mutagenic constituents in nitrite-treated soy sauce.

Soy sauce was heated with 100, 500, 1000 or 2000 ppm sodium nitrite for 30 min at 80 degrees C and pH 3. The reaction mixtures were extracted with dichloromethane followed by ethyl acetate. After removal of the solvents, the extracts were subjected to analysis (gas chromatograph-thermal energy analyser and gas chromatograph-mass spectrometer) and Ames mutagenicity tests. N-Nitrosodimethylamine and N-nitrosodiethylamine were found in the dichloromethane extract of the soy sauce treated with 2000 ppm nitrite at levels of 10 and 120 micrograms/ml, respectively. N-Nitrosoproline was identified in the ethyl acetate extract of the same sample at a level of 0.5 microgram/ml. Both extracts exhibited dose-related mutagenicity in Salmonella typhimurium strain TA100 with S-9 mix. The dichloromethane extract showed much higher mutagenicity than did the ethyl acetate extract. The samples obtained from soy sauce treated with 100, 500 and 1000 ppm nitrite were not mutagenic, but N-nitrosodiethylamine was detected by thermal energy analysis in the soy sauce treated with 1000 ppm nitrite. The addition of 10,000 ppm L-ascorbic acid, along with 2000 ppm nitrite, to soy sauce prevented the formation of mutagenic materials or detectable nitrosamines.