Table 1.

Summary Table for key event relationships in the proposed AOP for disruption of testosterone-mediated male sexual development.

Key Event Relationships (KERs)	Evidence
KER1	Active monoesters (MEHP, MBP) interact with phospholipase A2 (PLA2) proteins (e.g., calcium-dependent phospholipase A2 (cPLA2) (Six and Dennis, 2000).
	cPLA2 is responsible for intracellular arachidonic acid (AA) release from cell membranes (Kudo and Murakami, 2002).
	The C2 domain of the CPLA2 C-terminus binds to calcium ions (Ca++), targeting the head group of the phosphatidylcholine (PC) phospholipid membranes in the endoplasmic reticulum. This causes the activated enzyme to cleave the phospholipid and release arachidonic acid (AA) (Clewell et al., 2009).
	In platelets, MEHP inhibits the PLA2 release of AA, which can be metabolized into eicosanoids (Labow et al., 1988).
	DEHP inhibits cPLA2 in vivo (Kim et al., 2004).
	Active monoesters inhibit the release of AA that is incorporated through the plasma in Leydig cells (Clewell et al., 2009).
KER2	Protein kinase A (PKA) and cyclic adenosine monophosphate (c-AMP) are intermediate signaling molecules between luteinizing hormone (LH) and testosterone production. However, the signaling cascade that leads to the release of AA is independent of the c-AMP/PKA cascade (Clewell et al., 2009; Cooke et al., 1991; Kudo and Murakami, 2002; Moraga et al., 1997; Six and Dennis, 2000; Wang and Stocco, 1999).
	MBP disrupts the LH pathway prior to up-regulation of steroidogenic acute regulatory protein (StAR) gene transcription, and independent of PKA/c-AMP pathway, suggesting phthalates work through AA pathway rather than PKA/c-AMP (Didolkar and Sundaram, 1989; Lin, 1985; Wang and Stocco, 1999; Wang et al., 2000).
	Phthalates inhibit AA release (see evidence for KER1).
	Phthalates inhibit transcription of key transport and steroid metabolism genes (e.g., scavenger receptor class B member 1 (SRB1), StAR, cholesterol side-chain cleavage enzyme (P450scc), cytochrome P450 17A1 (CYP17)) in fetal rat testes (Gaido et al., 2007; Gray et al., 1982; Lehmann et al., 2004) and in rodent Leydig cells (Balbuena et al., 2013; Clewell et al., 2013; Wang and Stocco, 1999).
KER3	Phthalates disrupt testosterone signaling independent of androgen receptor (AR) (Parks et al., 2000).
	Active phthalates reduce testosterone production in rat testes in vivo and in rodent Leydig cells in vitro (Balbuena et al., 2013; Clewell and Clewell, 2008; Clewell et al., 2009; Clewell et al., 2013; Gaido et al., 2007; Gray et al., 1982; Lehmann et al., 2004; Wang and Stocco, 1999).
KER4	Inhibition of fetal testosterone synthesis leads to hypospadias and feminization of male rat demonstrated by delayed testes descent (cryptorchidism), vaginal pouch development (Foster, 2005, 2006; Gray et al., 2009; Rider et al., 2009b).