Development of a human biomonitoring method for assessing the exposure to ethoxyquin in the general population.

Ethoxyquin (EQ) is commonly used as an antioxidant in animal feeds. Although EQ is not permitted for usage in food products for humans within the EU, residues of EQ and its transformation products could be determined in food of animal origin. Despite its widespread use and concerns on its toxicological profile, no information about the systemic exposure to EQ in the general population is available. Hence, we developed a human biomonitoring (HBM) method for EQ. Our approach included a metabolism study with five subjects, who were administered an oral dose of 0.005 mg EQ/kg body weight. Unchanged EQ and the major metabolite 2,2,4-trimethyl-6(2H)-quinolinone (EQI) were identified as urinary excretion products of EQ. While small amounts of EQ could be determined in high concentrated samples from the metabolism study only, 28.5% of the orally applied EQ dose could be recovered as EQI. Toxicokinetic parameters were determined for EQI, the potential biomarker of exposure. In addition, an analytical method for EQI (LOQ = 0.03 µg/L) in urine based on UHPLC-MS/MS comprising enzymatic glucuronide hydrolysis and salt-assisted liquid-liquid extraction was developed, validated and applied to 53 urine samples from the general population. EQI could be quantified in 11 (21%) of the samples in levels up to 1.7 µg/L urine, proving the suitability of the developed method for the intended purpose.

Comparative xenobiotic metabolism between Tg.AC and p53+/- genetically altered mice and their respective wild types.

The use of transgenic animals, such as v-Ha-ras activated (TG:AC) and p53+/- mice, offers great promise for a rapid and more sensitive assay for chemical carcinogenicity. Some carcinogens are metabolically activated; therefore, it is critical that the altered genome of either of these model systems does not compromise their capability and capacity for metabolism of xenobiotics. The present work tests the generally held assumption that xenobiotic metabolism in the TG:AC and p53+/- mouse is not inherently different from that of the respective wild type, the FVB/N and C57BL/6 mouse, by comparing each genotype's ability to metabolize benzene, ethoxyquin, or methacrylonitrile. Use of these representative substrates offers the opportunity to examine arene oxide formation, aromatic ring opening, hydroxylation, epoxidation, O-deethylation, and a number of conjugation reactions. Mice were treated by gavage with (14)C-labeled parent compound, excreta were collected, and elimination routes and rates, as well as (14)C-derived metabolite profiles in urine, were compared between relevant treatment groups. Results of this study indicated that metabolism of the 3 parent compounds was not appreciably altered between either FVB/N and TG:AC mice or C57BL/6 and p53+/- mice. Further, expression of CYP1A2, CYP2E1, CYP3A, and GST-alpha in liver of naive genetically altered mice was similar to that of corresponding wild-type mice. Thus, these results suggest that the inherent ability of TG:AC and p53+/- mice to metabolize xenobiotics is not compromised by their altered genomes and would not be a factor in data interpretation of toxicity studies using either transgenic mouse line.

Comparative metabolism and disposition of ethoxyquin in rat and mouse. I. Disposition.

1. The biological fate of the antioxidant [3-14C]ethoxyquin (EQ) was investigated in the male F344 rat and the B6C3F1 mouse following either p.o. or i.v. administration. 2. The disposition of single doses up to 25 mg/kg was similar in the rat and mouse. About 90% of a total dose was excreted in urine and faeces within 24 h post-dosing. In contrast, no more than 60% of a higher dose of 250 mg/kg was excreted within 24 h following p.o. administration. 3. Metabolism of EQ was rapid in both the rat and mouse following either p.o. or i.v. administration. Little or no parent compound was detected in cumulative 24-h excreta. 5. EQ-derived radioactivity bioaccumulated in some tissues following repeated exposure to rat of either 25 or 250 mg/kg by gavage. However, the fold-increases in concentrations of EQ-derived radioactivity in tissues following repeated administration of the higher dose were generally less than those observed following repeated administration of the lower dose. Repeated high dose administration may overcome delayed gastric emptying (observed following single dose administration of 250 mg/kg) and/or lead to auto-induction of EQ metabolism.

Comparative metabolism and disposition of ethoxyquin in rat and mouse. II. Metabolism.

1. The major pathways of ethoxyquin (EQ) metabolism in both the rat and mouse are O-deethylation and conjugation to endogenous substrates. 2. The two major EQ-derived metabolites excreted in rat urine were in the form of sulphate conjugates, 1,2-dihydro-6-hydroxy-2,2,4-trimethylquinoline sulphate, and 1,2,3,4-tetrahydro-3,6-dihydroxy-4-methylene-2,2-dimethylquinoline sulphate. The latter apparently arises from an intramolecular rearrangement of the 3,4-epoxide of ethoxyquin. 3. Mouse urine contained one major glucuronide, 1,2-dihydro-6-hydroxy-2,2,4-trimethylquinoline glucuronide as well as one major sulphate conjugate, 1,2-dihydro-6-hydroxy-2,2,4-trimethylquinoline sulphate. 4. EQ-derived radioactivity was excreted in rat bile, mainly as GSH conjugates, with little unchanged EQ present. Two of the biliary metabolites are glutathione conjugates of ethoxyquin 3,4-epoxide; the third appears to be a conjugate of either ethoxyquin 7,8-epoxide or 2,2,4-trimethylquinol-6-one.

Ovine urinary metabolites of ethoxyquin.

Metabolites of ethoxyquin (EQ, 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) in the urine of sheep and rats were separated and identified by gas chromatography-mass spectrometry (GC-MS). Sheep were given diets containing EQ or EQ.HCl (0.5% of total diet) and urine samples were collected for the first 24 h and for another 24-h period after 12 d of feeding. Rats were given EQ/corn oil (0.08 g EQ/d/rat) orally for 7 d and urine samples were collected at ambient temperature for a 24-h period following 6 d of dosing. The urine samples were extracted with ethyl acetate at pH 5, and the concentrated extracts were analyzed by GC-MS. Ethoxyquin was identified in all sheep urine samples collected during the first 24 h of feeding, and EQ and hydroxylated EQ were identified in all urine samples collected after 12 d of feeding. In contrast, EQ, hydroxylated EQ, and dihydroxylated EQ were identified in urine collected from rats fed EQ for 7 d.

Promotion by ascorbic acid, sodium erythorbate and ethoxyquin of neoplastic lesions in rats initiated with N-butyl-N-(4-hydroxybutyl) nitrosamine.

The promoting effects of ascorbic acid, sodium erythorbate and ethoxyquin on two-stage urinary bladder carcinogenesis in F344 rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) at a dose of 0.05% in the drinking water were examined. Administration of 5% sodium erythorbate in the diet significantly increased the incidences of preneoplastic lesions, papilloma and cancer of the urinary bladder, whereas administration of 5% ascorbic acid in the diet did not. Administration of 0.8% ethoxyquin also increased the incidence of neoplastic lesions. Administrations of 5% sodium L-ascorbate and 5% sodium erythorbate caused increases in the pH, the sodium content and crystals of MgNH4PO4 in the urine. These results show that sodium erythorbate and ethoxyquin promote urinary bladder carcinogenesis, while ascorbic acid does not.

Studies on the metabolism of the antioxidant ethoxyquin, 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline in the rat.

1. The metabolism of ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline) in rat has been investigated. Urinary metabolites were identified by combined g.l.c.-mass spectrometry. 2. The major metabolic reaction was de-ethylation which gave rise to 6-hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline and an oxidation product, 2,2,4-trimethyl-6-quinolone. Other reactions were hydroxylation to four different hydroxylated metabolites and one dihydroxylated metabolite. A total of 95% of the dose (100 mg/kg) was accounted for.