Metabolism of xenobiotics in the incubated hen's egg: investigations with ethyl 4-hydroxybenzoate.

It is well known that in vitro preparations of the chick embryo can functionalise and conjugate selected model substances. Based on this biochemical knowledge an ex vivo model was developed to study xenobiotic metabolism in the incubated hen's egg. The xenobiotic is injected into the yolk sac, i.e. the nutritional compartment, on day 6 and metabolites are identified in the excretion medium of the embryonic kidneys (allantoic fluid) on day 11. During this developmental period the embryo lacks or has very limited sensitivity. Thus the model is in accordance with the 3R concept. In the present investigation ethyl 4-hydroxybenzoate was chosen as a test substance. Concentrations of this paraben up to 24 mg/egg did not affect embryo viability. After inoculation of 6 mg/egg, 4-hydroxybenzoic acid and 4,4'-dihydroxy-L-ornithuric acid were identified in their free form. The 4-hydroxybenzoic acid was also eliminated in its conjugated form (glucuronide and/or sulphate). No unchanged paraben was excreted. 4,4'-dihydroxy-L-ornithuric acid [2,5-bis-(4-hydroxybenzoylamino)pentanoic acid] is a new metabolite. The structure of this amino acid conjugate was elucidated by synthesis and spectral methods (MS, 1H and 13C NMR).