Supplemental File 11

Supplemental Table 11. OECD Reference Chemical Performance in HT H295R versus OECD inter-laboratory results and literature-reported results. Chemical identifiers (chemical name and casn) are provided for the 25 reference chemicals that overlapped between high-throughput (HT) H295R screening and the OECD inter-laboratory validation study (Hecker et al., 2011). Trilostane, glyphosate, and human chorionic gonadotrophin were not screened in the HT H295R assay. The adjusted maxmMd value, quadrants of the steroid synthesis pathway affected (progestagens (P), glucocorticoids (G), androgens (A), and/or estrogens (E)), and the number of steroid hormones affected using the ANOVA-based logic described in the main text are also provided. The OECD inter-laboratory results for estradiol (E2) and testosterone (T) are summarized along with a brief overview of additional information from the reported literature for activity in the H295R assay (if other in vitro assay data are referenced, the assay type is provided). Only 2 of the 25 chemicals with overlapping data were reported as negative for effects on both E2 and T: ethylene dimethanesulfonate and benomyl. NA indicates that no concentration-response screening data were available (only single concentration screening available).

# Chemical identifiers Results from HT H295R assay OECD Inter-laboratory and literature-reported Chemical name casn Adjusted maxmMd Quadrants # Steroid results of steroid hormones biosynthesis affected pathway affected 1 Mifepristone 84371-65-3 27 P 2 Used pharmacologically as an abortifacient with antiprogestagen, antiglucocorticoid, and antiandrogen properties. Moderate induction of E2 (2 to 4-fold induction) and T (equivocal) synthesis (Hecker, et al., 2011). Strong modulation of glucocorticoid pathway in H295R cells as a GR antagonist (Asser et al., 2014). 2 Prochloraz 67747-09-5 15 P,G,A,E 11 Imidazole fungicide used as an H295R assay control for inhibition. Strong inhibition of T (~0.1- fold) and moderate inhibition of E2 synthesis (0.5 to 0.25-fold) (Hecker, et al., 2011; Nielsen et al., 2012). Strong inhibition of glucocorticoid synthesis (Winther et al., 2013). 3 Genistein 446-72-0 15 P,G,A 8 Phytoestrogen. Strong induction of E2 (≥ 20-fold) and minor induction of T (0.25 to 0.5-fold reduction) synthesis (Hecker, et al., 2011). Moderate to strong effects on progestagens, corticosteroids, and androstenedione (Hasegawa et al., 2013; Nielsen, et al., 2012). 4 Ketoconazole 65277-42-1 15 P,G,A,E 11 Imidazole fungicide. Moderate to strong inhibition of E2 and T synthesis (0.5 to >0.1-fold) (Hecker, et al., 2011; Nielsen, et al., 2012). Strong inhibition of glucocorticoid synthesis (Winther, et al., 2013). 5 Danazol 17230-88-5 8.6 - 13 P,G,A,E 11 Used pharmacologically as a synthetic androgen that decreases estrogen production. Moderate to strong inhibition of E2 synthesis (0.5 to >0.1- fold) (Hecker, et al., 2011). Inhibition of glucocorticoid and progestagen synthesis has been reported in in vitro assays other than H295R, with possible inhibition of cAMP activation (Lambert et al., 1985; Olsson et al., 1986a; Olsson and Hillensjo, 1987; Olsson et al., 1986b; Rabe et al., 1983). 6 Finasteride 98319-26-7 10 P,G,A 6 Used pharmacologically as a 5α-reductase inhibitor. Moderate inhibition of T synthesis (0.25 to 0.5-fold) and equivocal effects on E2 (Hecker, et al., 2011). Moderate inhibition of progestagens and glucocorticoids in porcine adrenal cells (Jager et al., 1996) 7 Fenarimol 60168-88-9 10 P,G,A,E 11 Pyrimidine fungicide has been reported to inhibit CYP19A1 (Vinggaard et al., 2000), but possibly at concentrations approaching cytotoxicity (Sanderson et al., 2002). Equivocal effects on E2 and T synthesis (Hecker, et al., 2011). 8 Letrozole 112809-51-5 9.5 P,G,A,E 7 Used pharmacologically as a non-steroidal CYP19A1 inhibitor. Moderate (0.25 to 0.5-fold) and potent (0.001 µM) inhibition of E2 synthesis; moderate inhibition of T synthesis at high concentrations (100 µM) (Hecker, et al., 2011). 9 Spironolactone 52-01-7 6.4 P,G,A 8 Mineralocorticoid receptor antagonist. Moderate to strong inhibition of T synthesis (0.1 to o.5- fold) with no effects on E2 synthesis (Hecker, et al., 2011). Strong inhibition of cortisol synthesis in H295R (Ye et al., 2009) and strong induction of pregnenolone (Lesouhaitier et al., 2001). 10 Forskolin 66575-29-9 5.3 P,G,A,E 11 Used as a control for induction. Low to moderate induction (<2 to <4-fold) of T synthesis and strong induction of E2 synthesis (2 to >20-fold) (Hecker, et al., 2011). Forskolin moderately induced progestagens, glucocorticoids, and androgens (Strajhar et al., 2017). 11 Bisphenol A 80-05-7 3.4 – 4.2 P,G,A,E 5 to 7 Moderate increase in E2 (2 to <4-fold) and minor to moderate decrease in T (0.25 to 0.5-fold) synthesis (Hecker, et al., 2011). Moderate induction of E2, estrone, and progesterone; moderate inhibition of corticosterone and cortisol; and, strong inhibition of androstenedione and T (Zhang et al., 2011). 12 Butylparaben 94-26-8 3.5 P 2 Equivocal induction of T and moderate induction (<2 to <4-fold) of E2 (Hecker, et al., 2011). Moderate induction of progesterone synthesis (~4-fold) and no effect on E2 or T synthesis (Taxvig et al., 2008). 13 Atrazine 1912-24-9 2.6 P,G,A,E 9 Minor induction of T (<2-fold) and minor to strong induction of E2 synthesis (<2 to ≥ 20-fold) (Hecker, et al., 2011). Strong induction of E2 and estrone synthesis and moderate induction of progesterone synthesis (Tinfo et al., 2011). 14 Prometon 1610-18-0 2.5 P,G,E 9 No effect on T synthesis and moderate to strong induction of E2 synthesis (2 to ≥20-fold) (Hecker, et al., 2011). Moderate induction of E2 synthesis (Higley et al., 2010). 15 Piperonyl butoxide 51-03-6 1.8 P,G,E 6 Minor inhibition of T (>0.5-fold) and no effect on E2 synthesis (Hecker, et al., 2011). 16 Molinate 2212-67-1 1.7 G,E 5 No effect on T synthesis (1/5 labs observed a minor effect) and minor to moderate induction of E2 (<2 to <4-fold) synthesis (Hecker, et al., 2011). 17 Di(2-ethylhexyl) phthalate 117-81-7 1.1 P,G,E 9 No effect on T synthesis and moderate induction of E2 (2 to <4-fold) (Hecker, et al., 2011). Moderate inhibition of T synthesis has been reported previously (Desdoits-Lethimonier et al., 2012). 18 Aminoglutethimide 125-84-8 0.98 P,G,A 6 Used pharmacologically to inhibit aromatase activity and decrease steroid synthesis via CYP11A1 inhibition. Moderate to strong inhibition of T (0.5 to ≤ 0.1-fold inhibition) and moderate inhibition of E2 (0.5 to >0.25-fold) synthesis (Hecker, et al., 2011). Moderate inhibition of E2 and strong inhibition of T synthesis (Higley, et al., 2010). 19 Tricresyl phosphate 1330-78-5 0.065 P,G,A,E 7 Equivocal effects on T and E2 synthesis (Hecker et al., 2011). Minor induction of T and moderate induction of E2 have been reported (Liu et al., 2012). 20 Dimethoate 60-51-5 0.054 None 0 No effect on T synthesis and equivocal effects on E2 synthesis (Hecker, et al., 2011). 21 Benomyl 17804-35-2 <0 P,G,A 4 No effects on T or E2 synthesis (Hecker, et al., 2011). Benomyl decreased expression of CYP17 gene and protein in H295R cells, and decreased DHEA production (Robitaille et al., 2015). 22 Nonoxynol-9 26027-38-3 <0 G,E 4 Minor to moderate inhibition (<0.5 to 0.5-fold) of T synthesis with no effects detected on E2 synthesis (Hecker, et al., 2011). 23 Ethylene dimethanesulfonate 4672-49-5 <0 P,G,A,E 6 No effects on T and E2 synthesis (Hecker, et al., 2011). 24 Flutamide 13311-84-7 NA NA NA Used pharmacologically as a nonsteroidal anti- androgen via selective antagonism of the androgen receptor. Negative for effects on T and equivocal effects on E2 synthesis (Hecker, et al., 2011). 25 2,4-Dinitrophenol 51-28-5 NA NA NA Minor to moderate inhibition (<0.5 to 0.5-fold) of T synthesis with no effects detected on E2 synthesis (Hecker, et al., 2011).

Asser, L., Hescot, S., Viengchareun, S., Delemer, B., Trabado, S., and Lombes, M. (2014). Autocrine positive regulatory feedback of glucocorticoid secretion: Glucocorticoid receptor directly impacts H295R human adrenocortical cell function. Mol Cell Endocrinol 395(1-2), 1-9.

Desdoits-Lethimonier, C., Albert, O., Le Bizec, B., Perdu, E., Zalko, D., Courant, F., Lesne, L., Guille, F., Dejucq-Rainsford, N., and Jegou, B. (2012). Human testis steroidogenesis is inhibited by phthalates. Hum Reprod 27(5), 1451-9.

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Lesouhaitier, O., Chiappe, A., and Rossier, M. F. (2001). Aldosterone increases T-type calcium currents in human adrenocarcinoma (H295R) cells by inducing channel expression. Endocrinology 142(10), 4320-30.

Liu, X., Ji, K., and Choi, K. (2012). Endocrine disruption potentials of organophosphate flame retardants and related mechanisms in H295R and MVLN cell lines and in zebrafish. Aquat Toxicol 114-115, 173-81.

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Rabe, T., Kiesel, L., and Runnebaum, B. (1983). Inhibition of human placental progesterone synthesis by danazol in vitro. Fertil Steril 40(3), 330-3.

Robitaille, C. N., Rivest, P., and Sanderson, J. T. (2015). Antiandrogenic mechanisms of pesticides in human LNCaP prostate and H295R adrenocortical carcinoma cells. Toxicol Sci 143(1), 126-35.

Sanderson, J. T., Boerma, J., Lansbergen, G. W., and van den Berg, M. (2002). Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol Appl Pharmacol 182(1), 44-54.

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Winther, C. S., Nielsen, F. K., Hansen, M., and Styrishave, B. (2013). Corticosteroid production in H295R cells during exposure to 3 endocrine disrupters analyzed with LC-MS/MS. Int J Toxicol 32(3), 219-27.

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Zhang, X., Chang, H., Wiseman, S., He, Y., Higley, E., Jones, P., Wong, C. K., Al-Khedhairy, A., Giesy, J. P., and Hecker, M. (2011). Bisphenol A disrupts steroidogenesis in human H295R cells. Toxicol Sci 121(2), 320-7.