Dose-additive and dose-dependent fetal testis toxicity of a nine-phthalate mixture in the rat
Year: 2026
Type: preprint
Abstract: Phthalates (ortho-phthalic acid diesters) are used in a variety of manufacturing processes, particularly as non-covalent plasticizers in flexible polyvinyl chloride. Phthalates are male reproductive toxicants that disrupt the development of the fetal testis, resulting in malformed seminiferous cords, multinucleated germ cells (MNGs), and decreased testosterone production. We previously reported that MNGs were a sensitive, dose-dependent marker of dipentyl phthalate (DPEP) germ cell toxicity. To test whether germ cell effects of phthalates are dose-additive, we measured the induction of MNGs by a phthalate mixture. We treated timed pregnant Sprague Dawley rats with an equipotent mixture of nine phthalates, spanning a two-log dose range, from GD 17-21. We found that the phthalate mixture effects on morphogenesis of the testis, assessed through histological analysis of MNGs and seminiferous cord diameter were dose-additive. Consistent with prior results, we also found that the testosterone-reducing effect of the phthalate mixture was dose-additive and dose-dependent. Induction of MNGs had the lowest effective dose of any endpoint that we measured, with a benchmark dose (BMD 10 ) of 9.402 (6.093, 13.13) mg (∑ all phthalates)/kg/d, which is equivalent to a BMD 10 of 3.517 (2.275, 4.919) mg DPEP/kg/d, based on relative potency factors. Quantitative analysis of phthalate metabolites in dam serum showed that the metabolites were present in approximately the same relative concentrations as the parent compounds in the dosing mixture, but with lower levels of monocyclohexyl phthalate and monobenzyl phthalate. Proteomic and spatial transcriptomic analyses showed significant decreases in expression of Leydig cell genes and proteins involved in steroid hormone biosynthesis, as well as enrichment of proteins related to metabolism of lipids and amino acids. Proteomic BMD analysis produced a lowest BMDmedian of 2.156 (0.204, 27.830) mg (∑ all phthalates)/kg/d, which was within a 4.36-fold margin of the lowest apical endpoint BMD.
Authors: Joceline Helmbreck, Ziqi Zhao, Poonam Mehta, Patrick Schupp, Aditi Marehalli, Jason Scott, Matthew J. Woodcock, Andre Gomes, Cindy Su, Matthew Manwaring, Claire Gould, Thais Del Rosario Hernandez, Petra den Hollander, Susan J. Hall, Adina Badea, Sendurai Mani, Kurt D. Pennell, Daniel J. Spade
Institutions: Brown University, Rhode Island Hospital
Language: English
Subfield: Health, Toxicology and Mutagenesis
Field: Environmental Science
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W7222877455