Food contact chemicals (FCCs) can modulate normal biological functions by interfering with gene expression, i.e., which genes get turned on or off. Nuclear receptors play a key role in regulating gene expression. The retinoid-X receptor (RXR), in particular, facilitates DNA binding and builds heterodimers (a molecular complex formed by two different subunits) with one-third of human nuclear receptors (such as thyroid hormone receptors). Because RXRs are involved in so many pathways, disrupting them can lead to wide-ranging biological effects such as changes in development, metabolism, and endocrine function.
Brenda J. Mengeling from University of California and co-authors screened 57,277 environmental chemicals and identified 104 candidates predicted to interact with RXR using machine learning (NR-Toxpred model). In their article published in Scientific Reports on July 18, 2025, they describe their findings that 102 of the 104 chemicals were confirmed to fit in the RXR-ligand binding domain by using in silico toxicology tools, namely molecular docking and single-point free energy calculations (MM-PBSA).
Cluster analysis revealed a dominant group of 58 butylphenols, several of which are FCCs. Mengeling and co-authors then selected six of the 58 compounds, all included in the Food Packaging Forum’s (FPF’s) Food Contact Chemicals database (FCCdb), for further in vitro and in vivo hazard analysis. The six selected tert-butylphenols included five predicted RXR agonists and one predicted inactive compound. In vitro Tox21 high-throughput screening confirmed that the potency of tert-butylphenols to activate RXR decreased as the size of the molecule decreased. Specifically, if the R-group in the C4 position of the butylphenol molecule decreased the activation was less potent. These findings were in line with the machine learning in silico predictions.
Following the computational and cell-based assessments, African clawed frog, Xenopus laevis, served as in vivo model to further verify the six FCCs’ disruptive effect on RXR function. Specifically, the scientists investigated the disruption of thyroid hormone signaling where RXR serves as a binding partner of the thyroid hormone (nuclear) receptor. Three compounds, 2,6 di-tert-butyl-4-ethylphenol (CAS 4130-42-1), 4-(butan-2-yl)-2,6-di-tert-butylphenol (CAS 17540-75-9), and 2,4,6-tris(tert-butyl)phenol (2,4,6-TTBP, CAS 732-26-3), potentiated thyroid hormone signaling at nanomolar concentrations, confirming their activity as RXR agonists. The bulky ethyl, butan-2-yl, and tert-butyl at the C4 position of these three active compounds, respectively, “appear to enhance steric complementarity within the RXR ligand binding domain pocket.”
In contrast, the structurally similar but inactive compounds butylated hydroxytoluene (BHT, CAS 128-37-0) and 2,6-di-tert-butylphenol (CAS 128-39-2) lacked sufficient steric bulk and hydrophobic interactions, resulting in a poor fit within the RXR ligand-binding domain. 2,6-di-tert-butyl-4-[(dimethylamino)methyl]phenol (CAS 88–27-7), which has a bulky tertiary amine instead of a branched alkane, did not potentiate thyroid hormone signaling.
The study elegantly demonstrates that in silico approaches can help predict in vivo binding capacity and biological activity of chemicals. Since the three most active compounds can migrate from food contact materials according to the Database on Migrating and Extractable Food Contact Chemicals (FCCmigex), they have “a major potential for human exposure [which] highlights the need for further evaluation of their toxicological effects and possible regulatory action”, the authors concluded.
Tert-butylphenols are synthetic antioxidants commonly used to inhibit the oxidation of polymers and other polymeric materials, such as cured printing inks, thereby slowing their aging process. Research demonstrates that they are present in and migrate from food contact materials (FCMs) (FPF reported), leading to their presence in foodstuffs, including baby foods (FPF reported). Even “eco-friendly” labeled bio-based food contact articles are not free of these compounds, e.g., 2,4-di-tert-butylphenol (2,4-DTBP, CAS 96-76-4) was identified in some of such materials (FPF reported). 2,4-di-tert-butylphenol (CAS 96-76-4) and 7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione (CAS 82304-66-3) have been further detected in recycled plastics, specifically post-consumer high-density polyethylene (HDPE) milk bottle flakes (FPF reported).
Some of these chemicals have been linked to adverse health effects, such as endocrine disruption or reproductive toxicity. 4-tert-butylphenol (CAS 98-54-4) is a recognized substance of very high concern (SVHC) due to its endocrine-disrupting properties and other butylphenols are suspected endocrine disrupters (e.g., 2,4-DTBP, BHT).
Three of the six selected tert-butylphenols by Mengeling and co-authors have been measured in human samples according to the Database on Food Contact Chemicals Monitored in Humans (FCChumon): BHT, 2,4,6-TTBP, and 2,6 di-tert-butyl-4-ethylphenol.
Reference
Mengeling, B. J. (2025). “An in silico to in vivo approach identifies retinoid-X receptor activating tert-butylphenols used in food contact materials.” Scientific Reports. DOI: 10.1038/s41598-025-09244-z
