Prenatal phthalate mixture exposure increases early childhood internalising problems via maternal oxidative stress

Abstract Prenatal phthalate exposure has been linked to internalising problems in children. Maternal oxidative stress is a plausible biological mechanism underlying this association as it is induced by phthalates and is associated with early internalising problems, but this pathway has not yet been empirically tested. In the Barwon Infant Study, a population-based birth cohort of 1,074 Australian children, we investigated the potential mixture effect of prenatal phthalate exposure on maternal oxidative stress and assessed whether oxidative stress mediates the relationship between the prenatal phthalate mixture and early internalising problems. Concentrations of phthalate metabolites and nucleic acid oxidation biomarkers were measured in third-trimester maternal urine, and phthalate daily intakes estimated. Internalising problems were assessed at ages 2 and 4 years using the parent-report Child Behavior Checklist and Strengths and Difficulties Questionnaire, respectively. Applying weighted quantile sum regression with repeated holdout validation, we found that higher phthalate mixture exposure was associated with increased maternal oxidative stress (0.22 standard deviations per interquartile range increase in mixture, 95% CI: 0.13, 0.31), with dimethyl phthalate and diethyl phthalate as main contributors. Bayesian kernel machine regression yielded comparable results. For all child outcomes, counterfactual mediation analyses across holdout datasets revealed evidence of mediating effects, suggesting that oxidative stress is a pathway through which prenatal phthalate mixture exposure increases early-childhood internalising problems. These findings highlight the heightened risk associated with co-exposure to multiple phthalates during pregnancy and the potential to mitigate this harm not only through improved chemical regulation but also by monitoring and reducing maternal oxidative stress. Competing Interest Statement The authors have declared no competing interest. Funding Statement The establishment work and infrastructure for the Barwon Infant Study was provided by the Murdoch Children's Research Institute, Deakin University and Barwon Health. Subsequent funding was secured from the National Health and Medical Research Council of Australia (NHMRC), Minderoo Foundation, NHMRC and EU partnership grant for the ENDpoiNT consortium, The Shepherd Foundation, The Jack Brockhoff Foundation, Scobie & Claire McKinnon Trust, Shane O'Brien Memorial Asthma Foundation, Our Women Our Children's Fund Raising Committee Barwon Health, Rotary Club of Geelong, Australian Food Allergy Foundation, Geelong Medical and Hospital Benefits Association, Vanguard Investments Australia Ltd, Percy Baxter Charitable Trust, Perpetual Trustees, Gwyneth Raymond Trust, and William and Vera Ellen Houston Memorial Trust. In-kind support was provided by the Cotton On Foundation and CreativeForce. The Florey Institute of Neuroscience and Mental Health acknowledges the strong support from the Victorian Government and in particular the funding from the Operational Infrastructure Support Grant. The study sponsors were not involved in the collection, analysis, and interpretation of data; writing of the report; or the decision to submit the report for publication. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Barwon Health Human Research Ethics Committee (HREC 10/24) of Barwon Health gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability Barwon Infant Study (BIS) data requests are considered on scientific and ethical grounds by the BIS Steering Committee. If approved, data are provided under collaborative research agreements.