ECE2022 Eposter Presentations General Endocrinology (15 abstracts)
The development of a GLP protocol for the measurement of 17β-estradiol and testosterone in the H295R steroidogenesis assay, Test No 456
Carol Murray 1 , Angela McIntyre 1 , Connor McKeever 1 , Rebecca White 1 , Rachel McBrinn 1 , Victoria Light 2 , Susan Brown 1 , Aron Robinson 1 , Graeme Clark 1 & Tilly Bingham 3
1Concept Life Sciences, Dundee, United Kingdom; 2Concept Life Sciences, Bradford, United Kingdom; 3Concept Life Sciences, Chapel-en-le-Frith, United Kingdom
Regulators are concerned about the potential for environmental chemicals such as agrochemicals and their metabolites to perturb hormone systems. This has led to recommendations for the testing of potential endocrine disrupting chemicals1. The Steroidogenesis H295R assay is an in vitro cell model used to investigate compound effects on steroid hormone biosynthesis, specifically 17β-estradiol (E2) and testosterone (T). The human H295R adreno-carcinoma cell line expresses genes that encode for all the key enzymes for steroidogenesis and thus forms one of the required OECD in vitro tests (TG456) for the assessment of potential endocrine disrupting chemicals2. Although it is possible to assess hormone levels with ELISA we elected to perform the TG456 assay with LC-MS/MS hormone detection, avoiding the test item interference issues reported for immunoassay-based readouts. We describe herein the implementation of a robust GLP bioanalytical method for the detection of testosterone and 17β-estradiol in the steroidogenesis assay, to LLOQ levels of 10 pg/ml for each hormone. This method has been used to correctly identify inducers and inhibitors of T and E2 production while remaining unresponsive to a negative chemical. We present the impact this has on assay performance with respect to the proficiency items and discuss the benefits of the optimised bioanalytical protocol on Test No 456 performance.
References: 1. Guidance for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC)No 1107/2009. European Chemical Agency (ECHA) and European Food Safety Authority (EFSA) with thetechnical support of the Joint Research Centre (JRC)Niklas Andersson, Maria Arena, Domenica Auteri, Stefania Barmaz, Elise Grignard, Aude Kienzler, Peter Lepper, Alfonso Maria Lostia, Sharon Munn, Juan Manuel Parra Morte, Francesca Pellizzato, Jose Tarazona, Andrea Terron and Sander Van der Linden DOI https://doi.org/10.2903/j.efsa.2018.5311
2. Test No. 456: H295R Steroidogenesis Assay, https://www.oecd.org/env/test-no-456-h295r-steroidogenesis-assay-9789264122642-en.htm
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