Citation:

Garnovskaya, M., M. Feshuk, W. Stewart, K. Friedman, R. Thomas, AND C. Deisenroth. Evaluation of a High-throughput H295R Homogenous Time Resolved Fluorescence Assay for Androgen and Estrogen Steroidogenesis Screening. TOXICOLOGY IN VITRO. Elsevier Science Ltd, New York, NY, 92:105659, (2023). https://doi.org/10.1016/j.tiv.2023.105659

Impact/Purpose:

The Organisation for Economic Co-operation and Development (OECD) validated H295R test guideline assay (TG 456) evaluates the effect of test substances on the synthesis of 17β-estradiol (E2) and testosterone (T). A high-throughput (HT)-H295R alternative in 96-well format has previously been employed by the Agency to detect alterations in 11 steroid hormones using similar analytical techniques specified in TG 456. In order to maximize screening throughput, increase the speed of data generation, and reduce overall cost, existing commercial assay technology was employed to measure E2 and T levels, as well as cytotoxicity, in the H295R cell line. The resulting homogenous time resolved fluorescence assay platform (hereby H295R-HTRF) was evaluated against a training set of 36 chemicals derived from the OECD inter-laboratory validation study, EPA OCSPP Guideline 890.1200 (aromatase assay), and azole fungicides active in the HT-H295R assay. Scaling the assay from the low- (TG 456) or moderate- (HT-H295R) throughput formats to a 384-well platform enabled the technical design considerations necessary to more accurately assess steroid hormone analytes in a miniaturized high-throughput format that generally maintained equivalent or improved performance over the guideline assay. Implementation of the H295R-HTRF assay could prove useful to ongoing efforts in the ToxCast program and EDSP to predict disruption of steroidogenesis across the TSCA and EDSP universe of chemicals.

Description:

The H295R test guideline assay evaluates the effect of test substances on synthesis of 17β-estradiol (E2) and testosterone (T). The objective of this study was to leverage commercial immunoassay technology to develop a more efficient H295R assay to measure E2 and T levels in 384-well format. The resulting Homogenous Time Resolved Fluorescence assay platform (H295R-HTRF) was evaluated against a training set of 36 chemicals derived from the OECD inter-laboratory validation study, EPA guideline 890.1200 aromatase assay, and azole fungicides active in the HT-H295R assay. Quality control performance criteria were met for all conditions except E2 synthesis inhibition where low basal hormone synthesis was observed. Five proficiency chemicals were active for both the E2 and T endpoints, consistent with guideline classifications. Of the nine OECD core reference chemicals, 9/9 were concordant with outcomes for E2 and 7/9 for T. Likewise, 9/13 and 11/13 OECD supplemental chemicals were concordant with anticipated effects for E2 and T, respectively. Of the 10 azole fungicides screened, 7/10 for E2 and 8/10 for T exhibited concordant outcomes for inhibition. Generally, all active chemicals in the training set demonstrated equivalent or greater potency in the H295R-HTRF assay, supporting the sensitivity of the platform. The adaptation of HTRF technology to the H295R model provides an efficient way to evaluate E2 and T modulators in accordance with guideline specifications.

URLs/Downloads:

Record Details: