Combined toxicokinetic and in vivo genotoxicity study on Alternaria toxins

Abstract

The objective of this project was to gain information on toxicokinetic behaviour and in vivo genotoxicity of alternariol (AOH) in NMRI mice. Therefore, commercially available AOH was purified using a preparative HPLC-method. Initial dose-range finding proved AOH to be nontoxic after single or repeated oral application of up to 2000 mg/kg (limit dose). Subsequently, an in vivo oral toxicokinetic study (OECD guideline 417) was performed with 200 and 1000 mg/kg radiolabelled AOH. The study revealed low systemic absorption, with about 90 % of the total dose excreted via faeces and up to 9 % via urine. Blood levels did not exceed 0.06 % of the administered dose during the first 24 h after administration. Thus, target organ toxicity would most likely be restricted to the gastrointestinal tract. Metabolism of AOH was then investigated in a toxicokinetic study with non-radiolabelled AOH. Three dosage schemes were used: 200 and 2000 mg/kg (single dose) and 3 x 2000 mg/kg (0, 24 and 45 h). Whole blood (LC-MS/MS analysis) and urine (GC-MS in SIM mode) were analysed for AOH and its hydroxy-metabolites. Four metabolites (8-hydroxy-AOH, 4-hydroxy-AOH, 10-hydroxy-AOH, 2-hydroxy-AOH) were detected and ID was confirmed by NMR and mass spectrometry. Results also pointed to low systemic absorption, but mean blood levels (0.5 µM AOH, 3 h after the last of three applications) was considered sufficient to justify performance of a combined bone marrow micronucleus test (OECD 474) and in vivo alkaline comet assay (stomach, gut, liver). Therefore, 3 x 2000 mg/kg AOH were applied in corn oil (at 0, 24, and 45 h) and animals were sacrificed 48 h after the first application. The micronucleus assay revealed no toxic or genotoxic effect of AOH in bone marrow and the comet assay with liver tissue also did not indicate systemic genotoxicicity.