Evaluation of a hypothesized Sertoli cell-based adverse outcome pathway for effects of diisononyl phthalate on the...

Exposure of pregnant rats to some phthalates during the masculinization programming window (MPW) can lower fetal testis testosterone production and adversely affect development of the fetal male reproductive tract. Some of the effects in rats are androgen-dependent, while others also occur in mice without lower testosterone production. An adverse outcome pathway (AOP) network has been proposed for these developmental effects that includes both androgen-dependent and androgen-independent pathways, the latter of which includes a short list of putative molecular initiating events (MIEs) including peroxisome proliferator activated receptor (PPAR) activation, and effects on Sertoli cells in the developing testes as early key events (KEs) (PMID 34314370). Data from peer-reviewed literature, publicly cited toxicology reports, and EPA's Toxicity Forecaster (ToxCast) were evaluated in the context of this hypothesized Sertoli cell-based AOP and exposure to diisononyl phthalate (DINP). Each of the fifteen identified studies underwent a risk of bias (RoB) assessment, which revealed a high risk of bias for all but one study endpoint. In vitro evidence in kidney, liver, and fibroblast-like cell lines indicates that the DINP metabolites mono-isononyl phthalate (MINP) and mono-hydroxyisononyl phthalate (MHINP) activate PPARα/γ and that mouse PPARα/γ are more sensitive than human PPARα/γ. However, DINP did not activate PPARα-related genes in rat fetal testes at high maternal dosages (PMID 22112501), and it remains unknown whether PPARs are expressed in fetal Sertoli cells. Overall, there is insufficient evidence to evaluate whether PPAR activation in the developing male reproductive tract is causally linked to the KEs in the hypothesized AOP. Regarding the KEs, no in vivo studies were identified that examined the effects of DINP on Sertoli cell proliferation or cytoskeleton; a single in vitro study found no effect of DINP on Sertoli cell proliferation. There was limited and conflicting evidence for the effects of DINP on tubulogenesis, but strong in vivo evidence for increased multinucleated germ (MNG) cells. No evidence was found concerning germ cell apoptosis. For the adverse outcomes (AOs), there was limited in vivo evidence for testicular dysgenesis following altered tubulogenesis, and impaired spermatogenesis following increased MNGs. There was strong evidence against reduced fertility, but this is not a sensitive endpoint in rats given their robust sperm production and excess capacity. In conclusion, following in utero DINP exposure, while PPAR activation (MIE) is plausible, linkage to effects on Sertoli cells and downstream AOPs is lacking. The sparse evidence currently available is insufficient to support the applicability of the hypothesized Sertoli cell-based AOP to DINP.