Using targeted fetal rat testis genomic and endocrine alterations to predict the effects of a phthalate mixture on the male reproductive tract

Administration of phthalates in utero disrupts gene expression and hormone levels in the fetal rat testis, which are key events in an Adverse Outcome Pathway (AOP) for the Phthalate Syndrome. These measures can be used to predict the postnatal adverse effects of phthalate esters (PEs) on male rat sexual differentiation. Here, pregnant rats were exposed to dibutyl (DBP)- and diisononyl (DINP) phthalate on gestational days 14 to 18 individually and as a mixture (DBP,250 mg/kg/d; DINP, 750 mg/kg/d; and DBP 250 mg/kg/d plus DINP 750 mg/kg/d). We found that each PE reduced testosterone production (T Prod) and related gene transcripts by about 50 % and that they acted in a dose additive manner, reducing T Prod and gene expression by 75 % as a mixture. Based upon effects on T Prod, DINP was 0.33 times as potent as DBP and thus the DBP + DINP mixture was predicted to be equivalent to 500 mg DBP/kg/d. Logistic regression models of T Prod predicted that the adverse effects of the DBP + DINP mixture group versus the DBP and DINP individual treatments would reduce anogenital distance (AGD) by 27 % versus 10 %, increase hypospadias in 18 % versus < 1 %, induce epididymal agenesis in 46 % versus 10 %, and increase areolae/ nipples in 4.8 % versus < 0.1 % of the, respectively. These predictions were highly consistent with effects from previously published dose response studies on the male reproductive effects of DBP. In summary, these results support the use of this New Approach Method to predict the detrimental effects of PEs and PE mixtures, replacing or reducing the need to run long-term, resource and animal use intensive extended one-generation reproduction studies for this class of chemicals.