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 α (hERα) 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 17β-estradiol (E2β) at 5 ng g−1, 17α-ethynylestradiol (EE2) at 5 ng g−1, diethylstilbestrol (DES) at 10 ng g−1, zearalenone at 1.25 µg g−1or equol at 200 µg g−1. All of these blank and low estrogen spiked feed samples fulfilled the CCα and CCβ criterions, meaning that all 20 blank feed samples gave a signal below the determined decision limit CCα and were thus classified as compliant, and at least 19 out of the 20 spiked samples gave a signal above this CCα (β=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.