Cumulative effects of in utero administration of mixtures of reproductive toxicants that disrupt common target tissues via diverse mechanisms of toxicity

Summary Although risk assessments are typically conducted on a chemical‐by‐chemical basis, the 1996 Food Quality Protection Act required the US Environmental Protection Agency to consider cumulative risk of chemicals that act via a common mechanism of toxicity. To this end, we are conducting studies with mixtures of chemicals to elucidate mechanisms of joint action at the systemic level with the goal of providing a framework for assessing the cumulative effects of reproductive toxicants. Previous mixture studies conducted with antiandrogenic chemicals are reviewed briefly and two new studies are described. In all binary mixture studies, rats were dosed during pregnancy with chemicals, singly or in pairs, at dosage levels equivalent to approximately one‐half of the ED50 for hypospadias or epididymal agenesis. The binary mixtures included androgen receptor (AR) antagonists (vinclozolin plus procymidone), phthalate esters [di(n‐butyl) phthalate (DBP) plus benzyl n‐butyl phthalate (BBP) and diethyl hexyl phthalate (DEHP) plus DBP], a phthalate ester plus an AR antagonist (DBP plus procymidone), a mixed mechanism androgen signalling disruptor (linuron) plus BBP, and two chemicals which disrupt epididymal differentiation through entirely different toxicity pathways: DBP (AR pathway) plus 2,3,7,8 TCDD (AhR pathway). We also conducted multi‐component mixture studies combining several ‘antiandrogens’. In the first study, seven chemicals (four pesticides and three phthalates) that elicit antiandrogenic effects at two different sites in the androgen signalling pathway (i.e. AR antagonist or inhibition of androgen synthesis) were combined. In the second study, three additional phthalates were added to make a 10 chemical mixture. In both the binary mixture studies and the multi‐component mixture studies, chemicals that targeted male reproductive tract development displayed cumulative effects that exceeded predictions based on a response‐addition model and most often were in accordance with predictions based on dose‐addition models. In summary, our results indicate that compounds that act by disparate mechanisms of toxicity to disrupt the dynamic interactions among the interconnected signalling pathways in differentiating tissues produce cumulative dose‐additive effects, regardless of the mechanism or mode of action of the individual mixture component.