Table 3.
Assessment of biological plausibility and empirical evidence in support of the KERs in AOP2.
| KER (Adjacency of KEs) WoE Conclusion |
WoE Rationale |
|---|---|
| Biological Plausibility WoE | |
| KE 1 leads to KE 2 (Direct) Moderate |
Established mechanistic basis that SF-1 regulates StAR, Cyp11a1, and Cyp17 genes. Coup-TFII is a known transcriptional repressor and steroidogenesis genes have SF-1 and COUP-TFII overlapping binding sites in the promoter. Limited biological and mechanistic evidence that COUP-TFII displaces SF-1 from binding to target genes leading to down regulation of genes critical for steroidogenesis (van den Driesche et al., 2012). Some evidence based on dose response studies with COUP-TF1 which shares a conserved binding domain with COUP-TFII (Shibata et al., 2003). |
| KE2 leads to KE5 (Direct) High |
Established biological knowledge of the steroidogenic process and that enzymatic disruption impacts male hormones (DHT) levels (Hasegawa et al., 2000; Hu et. al., 2002; Barsoum and Yao 2006; Miller and Walter 2007; Turcu and Auchus 2015). |
| KE5 leads to AO1(Indirect) High |
Established biological knowledge across a range of chemicals (flutamide, finasteride) and clinical outcomes that disruption of DHT (reduced production or interference with receptor binding) causes hypospadias (Kim et al., 2002, Turcu and Auchus, 2015). Established mechanistic basis that the importance of testosterone in external genital development is exclusively as a precursor for DHT; testosterone does not play a role. (Hib and Ponzio, 1995, Tian and Russell, 1997). |
| KE5 leads to KE7 (Direct) Moderate |
Strong but limited biological evidence that MAFB (serving as an indicator of urethral tube closure) is androgen dependent (sexually dimorphic expression, co-localization with AR receptor expression, and AR dependent expression) (Suzuki et al., 2014). |
| KE7 leads to AO2(Direct) High |
Established biological knowledge based on Mafb-/- mice with hypospadias phenotype (Suzuki et al., 2014). Limited mechanistic basis: transcription factor MAFB expressed in the mesenchymal cell population of the genital tubercle adjacent to the urethral plate (cell population critical for urethral tube formation and closure) (Nishida et al., 2008, Miyagawa et al., 2009, Suzuki et al., 2014). |
| KE2 leads to AO2 (Indirect) Moderate |
Male Cyp11a1 KO mice have external female genitalia which demonstrates a direct link with the development of male external genitalia and a male like urethra (Hu et al., 2002). |
| Empirical Evidence WoE | |
| KE1 leads to KE2* (Direct) Moderate |
Dose and incidence concordance well supported in a single study on DBP (Table 4). Dose, incidence concordance challenged when combining data across multiple studies for DBP (Supplemental Table 6). Pattern of observations within the broader dataset supports a relationship between KEs (Supplemental Table 7). |
| KE2* leads to KE5** (Direct) Moderate |
Dose and incidence concordance well supported in a single study on DPP (Supplemental Table 5). Dose and incidence concordance supported when combining data across multiple studies for DBP (Supplemental Table 6). A dependent change in both events is observed in the same study for a number of LMWPs (Supplemental Table 1) |
| KE5** leads to AO2 (Indirect) Moderate |
Dose and incidence concordance well supported in a single study on DPP (Supplemental Table 5). Data lacking to support concordance when combining data across multiple studies for DBP (Supplemental Table 6). Association between severity of DHT impact (as measured by severity of AGD reduction) and hypospadias incidence has been demonstrated (Supplemental Table 7). A dependent change in both events is observed in the same study for a number of LMWPs (Supplemental Table 1). |
| KE5** lead to KE7 (Direct) Low |
Data on markers of urethral tube disruption (KE7) were not found. Pattern of observations within the broader dataset supports the KER (Supplemental Table 7) |
| KE7 leads to AO2 (Direct) Low |
Data on markers of urethral tube disruption (KE7) were not found in. Pattern of observations within the broader dataset supports the KER (Supplemental Table 7). |
*The WoE assessment focused on data measuring steroidogenesis gene transcription/protein expression. **The WoE assessment relied on AGD as a surrogate measure for DHT during the MPW. Testosterone was not used as a surrogate for KE2 or KE5 in this WoE assessment. KE1 = sustained Coup-TFII expression; KE2 = decreased steroidogenic biosynthetic protein expression (StAR, CYP11A1, CYP17); KE5 = decreased DHT; KE7 = inhibition of urethral tube closure; AO2 = hypospadias; KER = key event relationships; WoE = weight of evidence.