Hepatic Metabolism of Methyl Anthranilate and Methyl N-Methylanthranilate as Food Flavoring Agents in Relation to Allergenicity in the Guinea Pig

To assess the safety of the food flavoring agents, methyl anthranilate (MA) and methyl N-methylanthranilate (MNMA), their relationships with metabolism and allergenicity were examined in guinea pigs. Both MA and MNMA were hydrolyzed to anthranilic acid (AA) and N-methylanthranilic acid (N-methylAA), respectively, by guinea pig liver microsomes. These hydrolytic activities at 1000 μM were 320 and 35 nmol/min/mg protein, respectively. Moreover, MNMA was N-demethylated to MA by the liver microsomes; the oxidative activity at 1000 μM of MNMA was 2.8 nmol/min/mg protein. The N-demethylase activity for N-methylAA at 1000 μM in the liver microsomes was 3.9 nmol/min/mg protein. Kinetic analysis indicated that the Vmax/Km values of MA and MNMA hydrolyses were 15- and 7.4-fold greater in guinea pig liver microsomes than in the cytosol, respectively, suggesting that these hydrolytic activities were predominantly localized in the microsomes. Liver microsomal activities for the hydrolysis of these flavoring esters were markedly inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and bis(p-nitrophenyl)phosphate but not by physostigmine. These hydrolytic activities were suppressed by aspirin, a substrate of carboxylesterase, in a concentration-dependent manner. The N-demethylations of MNMA and N-methylAA were inhibited by SKF 525-A, a nonselective inhibitor of cytochrome P450. At the same time as the metabolic study described above, skin reactions in guinea pigs were investigated using MA, MNMA, N-methylAA, and AA. All compounds examined elicited positive skin reactions, although MNMA and AA exhibited relatively greater sensitizing properties. These results may provide useful information about metabolism in the toxicologic evaluations of MA and MNMA.