Validation and application of a robust yeast estrogen bioassay for the screening of estrogenic activity in animal feed

Previously we described the construction and properties of a rapid yeast bioassay stably expressing human estrogen receptor alpha (hER alpha) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to estrogens. In the present study this yeast estrogen assay was validated as a qualitative screening method for the determination of estrogenic activity in animal feed. This validation was performed according to EC Decision 2002/657. Twenty blank animal feed samples, including milk replacers and wet and dry feed samples, were spiked with 17 beta-estradiol (E2 beta) at 5 ng g(-1), 17 alpha-ethynylestradiol (EE2) at 5 ng g(-1), diethylstilbestrol (DES) at 10 ng g(-1), zearalenone at 1.25 mu g g(-1) or equol at 200 mg g(-1). All of these blank and low estrogen spiked feed samples fulfilled the CC alpha and CC beta criterions, meaning that all 20 blank feed samples gave a signal below the determined decision limit CC alpha and were thus classified as compliant, and at least 19 out of the 20 spiked samples gave a signal above this CC alpha (beta = 5%) and were thus classified as suspect. The method was specific and estrogens in feed were stable for up to 98 days. In this study we also present long-term performance data and several examples of estrogens found in the routine screening of animal feed. This is the first successful example of a developed, validated and applied bioassay for the screening of hormonal substances in feed.