Prioritization Based on Potency and Efficacy Measures

We used three measures to represent the potency and/or efficacy of each E2- and P4-up chemical: a) the maximal fold change (MFC) in hormone concentration; b) the lowest effective concentration (LEC) at which the tested chemical produced a significant hormone increase; and c) the adjusted maximal mean Mahalanobis distance (adj.maxmMd). The adj.maxmMd, described by Haggard et al. (2018), is a unitless statistical value reflecting a chemical’s magnitude of steroidogenic pathway disruption for 11 hormones across the tested chemical concentration range; it controls for correlation and covariance among the hormone analytes (Haggard et al. 2018).

To find the MFC, a proxy for efficacy, we used the raw hormone data found in the Haggard et al. (2018) Supplemental Data File 3. Using the raw hormone data, we first calculated the fold change relative to the average of the DMSO solvent control duplicates from the same plate at each of the tested concentrations. We then used Haggard et al. (2018) Supplemental Data Files 4 and 7 to identify the chemical concentrations with significant hormone increases. Following OECD hit-call logic, we identified the chemical concentrations with consecutive significant hormone increases or a significant hormone increase at the highest tested noncytotoxic chemical concentration. Of these chemical concentrations, the highest fold change was chosen as the MFC.

We also identified the LEC, a proxy for potency, using the raw hormone data from the Haggard et al. (2018) Supplemental Data File 3. As with the MFC, we used the Haggard et al. 2018 Supplemental Data Files 4 and 7 to identify the chemical concentrations with consecutive significant hormone increases, or significance at the highest tested noncytotoxic chemical concentration, and then selected the lowest chemical concentration from these as the LEC. Furthermore, we compared the LEC values with the potency values calculated using the tcpl: the AC50 and the AC10. We compiled the AC50 and AC10 values for each hormone-chemical pair using the ToxCast and Tox21 Summary Files for invitroDBv3.2 (U.S. EPA 2019a). Tcpl calculated AC50s only for chemicals it identified as active, and AC10s for chemicals that were able to fit a model even if their hit-call was not active. We conducted a Pearson’s correlation analysis to assess the relationship between the LEC and the AC50 and AC10 values.

Last, the adj.maxmMd calculated by Haggard et al. (2018) was used to take both potency and efficacy into account and identify chemicals with a clear response above assay noise considering data for 11 hormones (excluding DHEA and pregnenolone owing to their frequent detection below the lower LOQ). The adj.maxmMd for each chemical tested can be found in the Haggard et al. (2018) Supplemental Data File 11.

We defined E2- and P4-up chemicals with higher potency and efficacy as active on the basis of the following criteria: MFC≥1.5, LEC≤33μM, and adj.maxmMd >0. These active chemicals were the focus of subsequent analysis because they are less likely to be false-positives (based on an MFC≥1.5, as suggested by the OECD interlaboratory analysis) and more likely to be active at environmentally relevant concentrations (by limiting to more potent chemicals). An adj.maxmMd >0 is also less likely to result in a false-positive for steroidogenic pathway disruption, per Haggard et al. (2018). We defined the excluded chemicals as borderline active. We also removed the following synthetic or endogenous steroid hormones because they can interfere with steroidogenesis by acting as substrates: 17-methyltestosterone, 17α-estradiol, 17α-ethinylestradiol, 17α-hydroxyprogesterone, 17β-estradiol, 4-androstene-3,17-dione, 5α-dihydrotestosterone, androsterone, dehydroepiandrosterone, equilin, estrone, P4, and testosterone propionate.

We classified the active chemicals into three groups corresponding to their potency and efficacy by ranking them on the basis of the average of their MFC percentile rank and LEC percentile rank. We considered those chemicals in the top 25% of the rank as having higher (compared with the other chemicals in the ranking) efficacy and potency, chemicals in the middle 25–75% as having intermediate potency and efficacy, and chemicals in the bottom 25% as having lower potency and efficacy.