Table 1.

H295R Steroidogenesis Assay Methodology Comparison

Design Phase	Aspect	OECD TG 456	HT-H295R
Cell culture	Plate format	24-well, but OECD TG specifies other plate formats can be used (eg, 48-well)	96-well
	Experimental timeline	24 h acclimatization of cells, followed by 48 h chemical exposure, terminated at sample collection.	Overnight acclimatization of cells, followed by 48 h pre-stimulation with forskolin, followed by 48 h chemical exposure, terminated at sample collection.
	Cell passage	5–10	5–10
	Target cell confluency	50%–60%	50%–60%
	Replicates	Triplicate technical replicates	Duplicate technical replicates
		Triplicate biological replicates	Most of the library had 1 biological replicate; approximately 16% was screened with 2–3 biological replicates.
Viability testing	Viability measures	Live/Dead® or MTT assay	MTT assay
	Cell viability threshold	≥80%	≥70%
Hormone detection	Baseline stimulation	Nonea	Cells are pre-stimulated for 48 h in medium containing 10 μM forskolin.
	Minimum basal production	500 pg/ml or ≥ 5-fold method detection limit (MDL) for T and 40 pg/ml or ≥ 2.5-fold MDL for E2.	Following forskolin stimulation, DMSO-exposed H295R demonstrated 2.19 ± 0.32 ng/ml and 1.57 ± 0.36 ng/ml for T and E2, respectively. This is ≥ 5-fold the LLOQ for both hormones.b
	Accuracy	Within 30% of nominal concentrations.	98.1%–101.7% recovery for 13 hormones (Karmaus et al. 2016).b
	Precision	Variation between replicate samples should be ≤ 25%.	Percent relative standard deviation for controls ranging from 3.3 to 10.0% during assay optimization for the 13 hormones measured (Karmaus et al. 2016).b
Steroid hormone data analysis	Analysis	Normally distributed data: an analysis of variance (ANOVA) with differences from vehicle control evaluated using a Dunnett’s test. Non-normally distributed data: Kruskal–Wallis test followed by a Mann–Whitney U test.	Initial data analysis used the ToxCast data pipeline (tcpl) (Filer et al., 2017) to enable standardization of the data with other HT data and a first look at the data. Data analysis for comparison to the OECD reference chemicals involved use of an ANOVA with differences from vehicle control evaluated by Dunnett’s test (new in this work).
	Criteria for positive	Two consecutive concentrations and/or maximum non-cytotoxic concentration significantly different from control.	See Karmaus et al. (2016) for a description of the tcpl analysis employed for a first analysis. For the ANOVA approach presented here: 2 consecutive concentrations and/or maximum non-cytotoxic concentration significantly different from control.

A summary of the OECD TG 456 requirements versus the performance of the HT-H295R assay (as currently implemented).

^a22-R-Hydroxycholesterol has been suggested as a medium supplement (20–40 μM) to increase basal E2 production as needed, but it is not part of the standard protocol. Further, the OECD validation report (2008) noted that, “during the qualifying experiments it was only expected that the laboratory showed conformance with the performance criteria for E2 induction after exposure to the stimulator forskolin.”

^bNote these are reported performance results and not criteria for acceptance of the HT-H295R assay data.