**Author details**

the HEK293-based estrogen-responsive bioreporter against a variety of EDCs and associated controls, it will be necessary to validate the performance of this assay format at the levels of scale required for commercial use. Therefore, the development of additional bioreporter cell types and their validation at scale using automated assay preparation, performance, and detection equipment is recommended as a next step in the maturation of this assay format. If autobioluminescent assays can perform reliably under these conditions while maintaining a similar level of performance to that observed from the HEK293-based estrogen-responsive

Tier 1 *in vitro* assays are the front line in EDC detection. However, the limitations of traditional assay formats, which use non-human cell lines that can obscure bioavailability data [6, 7], require the use of radioactive materials that necessitate dedicated use areas and specially trained personnel [6–8], or rely on expensive analytical equipment [8, 9], are currently incapable of handling the sheer number of compounds that must be screened. Autobioluminescent assays, such as the HEK293-based estrogen-responsive bioreporter assay presented here, are uniquely positioned to overcome the limitations of existing assay formats by autonomously generating bioluminescence in response to target chemical or chemical class bioavailability. The use of these reporter systems allows bioluminescent responses to be linked to EDC detection for reagent-free, fully automated screening at a fraction of the cost of existing assays, providing a promising route toward addressing the existing EDC compound screening backlog.

The authors acknowledge research funding provided by the U.S. National Institutes of Health under Award Numbers NIEHS-1R43ES022567-01, NIEHS-2R44ES022567-02, and

S.R., G.S., and D.C. are board members in the for-profit entity 490 BioTech.

bioreporter, they will prove a valuable tool for Tier 1 EDC detection.

**6. Conclusions**

138 Endocrine Disruptors

**Acknowledgements**

NIEHS-1R5ES023979-01.

**Conflict of interest**

**Abbreviations**

AR Androgen receptor

BPA bisphenol-A

ARE Androgen response element

Tingting Xu1 , Andrew Kirkpatrick2 , Jody Toperzer2 , Marvin Steven Furches2 , Steven Ripp1 , Gary Sayler2 and Dan Close2 \*

\*Address all correspondence to: dan.close@490biotech.com

1 Center for Environmental Biotechnology, The University of Tennessee, Knoxville, USA

2 490 BioTech, Knoxville, Tennessee, USA

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