Human skin absorption of three plasticizers: Diisononyl-1,2-cyclohexanedicarboxylate (DINCH), di(2-ethylhexyl) terephthalate (DEHTP), and di(2-ethylhexyl) adipate (DEHA).

Alternative plasticizers such as diisononyl-1,2-cyclohexanedicarboxylate (DINCH), di(2-ethylhexyl) terephthalate (DEHTP), and di(2-ethylhexyl) adipate (DEHA) are progressively replacing phthalates in many consumer and professional products because of adverse effects on reproduction associated with some phthalates. Human exposures to these phthalate substitutes can occur through ingestion, skin absorption and inhalation. Skin uptake can lead to greater concentration at the target organs compared to ingestion because the skin exposure route bypasses the first-pass effect. Skin absorption studies are almost absent for these alternative plasticizers. We therefore wanted first, to characterize skin absorption of a mixture containing DINCH, DEHA and DEHTP in vitro using a flow-through diffusion cell system with ex vivo human skin, quantifying their respective monoester metabolites (mono-isononyl-cyclohexane-1,2-dicarboxylate (MINCH), mono-2-ethylhexyl adipate (MEHA), mono-2-ethylhexyl terephthalate (MEHTP), respectively); second, to validate these results by exposing five human volunteers to this mixture on their forearm and quantifying the corresponding urinary metabolites (including the monoesters and their oxidation products). Our study showed that two of these alternative plasticizers, DEHTP and DINCH, did not permeate skin showing as quantifiable metabolite levels in vitro and only traces of DEHA were quantified as its monoester metabolite, MEHA. Permeation coefficient (Kp) 0.06 and 55.8*10-7 cm/h for neat and emulsified DEHA, respectively, while the permeation rate (J) remained low for both (0.005 and 0.001 µg/cm2/h, respectively). Participants exposed to a mixture of these three plasticizers did not have noteworthy urinary concentrations of their respective metabolites after 24 hours post-application. However, the alternative plasticizer mixture was completely absorbed after six hours post-application on the forearms of the human volunteers, and the urinary elimination curves showed a slight increase after 24 hours post-application. Further studies on skin absorption of these substances should follow the urinary elimination kinetics of these metabolites more than 24 hours post-application. We also recommend quantifying the parent compounds in the in vitro diffusion experiments.

Human skin absorption of three phthalates.

Population studies reveal widespread exposure to phthalates. Understanding their absorption, distribution, metabolism, and excretion is vital to reduce exposure. However, data on skin absorption remain limited. We thus aim to characterize the skin permeation of three phthalates in a mixture, neat or in emulsion; di(2-ethylhexyl) phthalate (d4-DEHP), dibutyl phthalate (d4-DBP), and diethyl phthalate (d4-DEP), by comparing in vitro human skin (800 µm) permeation (24 hours) results using flow-through diffusion cells with urine results obtained from volunteers exposed to the same mixture applied to a forearm (40 cm2). Metabolites were analyzed in receptor fluids and urine. Phthalates crossed the skin barrier and metabolized into monoesters before elimination. Increased permeation was observed for phthalates in emulsion compared to neat substances, with polyethylene glycol (PEG) in the receptor fluid enhancing emulsion permeation, but not affecting neat substances. In vitro results mirrored in vivo findings: DEP showed rapid permeation (J: ∼2 ug/cm2/h) and urinary excretion peaking at six hours post-application, whereas DBP exhibited slower kinetics (J: ∼0.1 ug/cm2/h), with a urinary peak at 15-17 hours post-application. DEHP had minimal permeation (J: ∼0.0002 ug/cm2/h) with no observable urinary peak. These findings underscore the importance of comprehending phthalate skin absorption for effective exposure mitigation strategies.

Human biomonitoring and toxicokinetics as key building blocks for next generation risk assessment.

Human health risk assessment is historically built upon animal testing, often following Organisation for Economic Co-operation and Development (OECD) test guidelines and exposure assessments. Using combinations of human relevant in vitro models, chemical analysis and computational (in silico) approaches bring advantages compared to animal studies. These include a greater focus on the human species and on molecular mechanisms and kinetics, identification of Adverse Outcome Pathways and downstream Key Events as well as the possibility of addressing susceptible populations and additional endpoints. Much of the advancement and progress made in the Next Generation Risk Assessment (NGRA) have been primarily focused on new approach methodologies (NAMs) and physiologically based kinetic (PBK) modelling without incorporating human biomonitoring (HBM). The integration of toxicokinetics (TK) and PBK modelling is an essential component of NGRA. PBK models are essential for describing in quantitative terms the TK processes with a focus on the effective dose at the expected target site. Furthermore, the need for PBK models is amplified by the increasing scientific and regulatory interest in aggregate and cumulative exposure as well as interactions of chemicals in mixtures. Since incorporating HBM data strengthens approaches and reduces uncertainties in risk assessment, here we elaborate on the integrated use of TK, PBK modelling and HBM in chemical risk assessment highlighting opportunities as well as challenges and limitations. Examples are provided where HBM and TK/PBK modelling can be used in both exposure assessment and hazard characterization shifting from external exposure and animal dose/response assays to animal-free, internal exposure-based NGRA.

Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure Experiments.

BACKGROUND: Chemicals are not required to be tested systematically for their neurotoxic potency, although they may contribute to the development of several neurological diseases. The absence of systematic testing may be partially explained by the current Organisation for Economic Co-operation and Development (OECD) Test Guidelines, which rely on animal experiments that are expensive, laborious, and ethically debatable. Therefore, it is important to understand the risks to exposed workers and the general population exposed to domestic products. In this study, we propose a strategy to test the neurotoxicity of solvents using the commonly used glycol ethers as a case study. OBJECTIVE: This study aims to provide a strategy that can be used by regulatory agencies and industries to rank solvents according to their neurotoxicity and demonstrate the use of toxicokinetic modeling to predict air concentrations of solvents that are below the no observed adverse effect concentrations (NOAECs) for human neurotoxicity determined in in vitro assays. METHODS: The proposed strategy focuses on a complex 3D in vitro brain model (BrainSpheres) derived from human-induced pluripotent stem cells (hiPSCs). This model is accompanied by in vivo, in vitro, and in silico models for the blood-brain barrier (BBB) and in vitro models for liver metabolism. The data are integrated into a toxicokinetic model. Internal concentrations predicted using this toxicokinetic model are compared with the results from in vivo human-controlled exposure experiments for model validation. The toxicokinetic model is then used in reverse dosimetry to predict air concentrations, leading to brain concentrations lower than the NOAECs determined in the hiPSC-derived 3D brain model. These predictions will contribute to the protection of exposed workers and the general population with domestic exposures. RESULTS: The Swiss Centre for Applied Human Toxicology funded the project, commencing in January 2021. The Human Ethics Committee approval was obtained on November 16, 2022. Zebrafish experiments and in vitro methods started in February 2021, whereas recruitment of human volunteers started in 2022 after the COVID-19 pandemic-related restrictions were lifted. We anticipate that we will be able to provide a neurotoxicity testing strategy by 2026 and predicted air concentrations for 6 commonly used propylene glycol ethers based on toxicokinetic models incorporating liver metabolism, BBB leakage parameters, and brain toxicity. CONCLUSIONS: This study will be of great interest to regulatory agencies and chemical industries needing and seeking novel solutions to develop human chemical risk assessments. It will contribute to protecting human health from the deleterious effects of environmental chemicals. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/50300.

Repeated Human Exposure to Semivolatile Organic Compounds by Inhalation: Novel Protocol for a Nonrandomized Study.

BACKGROUND: Semivolatile organic compounds (SVOCs) comprise several different chemical families used mainly as additives in many everyday products. SVOCs can be released into the air as aerosols and deposit on particulate matter during use by dispersion, evaporation, or abrasion. Phthalates are SVOCs of growing concern due to their endocrine-disrupting effects. Human data on the absorption, distribution, metabolism, and excretion (ADME) of these compounds upon inhalation are almost nonexistent. OBJECTIVE: The goal of this study is to develop a method for repeated inhalation exposures to SVOCs to characterize their ADME in humans. METHODS: We will use diethylhexyl phthalate (DEHP), a major indoor air pollutant, as a model SVOC in this novel protocol. The Swiss official Commission on Ethics in Human Research, Canton de Vaud, approved the study on October 14, 2020 (project-ID 2020-01095). Participants (n=10) will be repeatedly exposed (2 short daily exposures over 4 days) to isotope-labeled DEHP (DEHP-d4) to distinguish administered exposures from background exposures. DEHP-d4 aerosols will be generated with a small, portable, aerosol-generating device. Participants will inhale DEHP-d4-containing aerosols themselves with this device at home. Air concentrations of the airborne phthalates will be less than or equal to their occupational exposure limit (OEL). DEHP-d4 and its metabolites will be quantified in urine and blood before, during, and after exposure. RESULTS: Our developed device can generate DEHP-d4 aerosols with diameters of 2.5 µm or smaller and a mean DEHP-d4 mass of 1.4 (SD 0.2) µg per puff (n=6). As of May 2023, we have enrolled 5 participants. CONCLUSIONS: The portable device can be used to generate phthalate aerosols for repeated exposure in human studies. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/51020.

Skin Absorption of Bisphenol A and Its Alternatives in Thermal Paper.

OBJECTIVES: Bisphenol A (BPA) is the most used colour developer in thermal paper for cashiers receipts, labels, and tickets. BPA can migrate onto the skin and be absorbed when handling these papers. BPA is a known endocrine disruptor and is therefore being replaced in thermal paper by some alternatives such as Bisphenol S (BPS), D-8, and Pergafast 201® (PF201). To our knowledge, no studies have characterized skin permeation of these BPA alternatives. METHODS: We measured/characterized skin absorption for BPA, BPS, D-8, and PF201 through ex vivo human skin using flow-through diffusion cells according to OECD guideline 428. Skin samples were 7-12 per test substance from three different skin donors. Skin metabolism was studied for BPA. Dermal absorption was expressed as the amount of the BPA alternatives in the receptor fluid over applied dose in percent (%). RESULTS: The absorbed dose after 24 h of exposure was 25% for BPA, 17% for D-8, 0.4% for BPS, and D-8 >> BPS > PF201. These results are in agreement with their log Kow and molecular weights. We provided here the necessary data to estimate the extent of skin absorption of BPA analogues, which is a necessary step in risk assessment, and ultimately evaluate public health risks posed by D-8, BPS, and PF201.

Simultaneous Quantification of Bisphenol A, Its Glucuronide Metabolite, and Commercial Alternatives by LC-MS/MS for In Vitro Skin Absorption Evaluation.

Bisphenol A (BPA) is the most used color developer in thermal paper products such as cashiers' receipts, followed by Bisphenol S (BPS), Wincon 8 (D-8), and Pergafast 201 (PF201). These chemicals can migrate from the paper onto the skin and possibly be absorbed and metabolized. Until now, D-8 and PF201 have not been analyzed in biological matrices, nor has a method been developed to simultaneously quantify them, even though they are often found as mixtures. Our aim was to develop and validate a method to quantify BPA, its glucuronide metabolite (BPA-G), BPS, D-8, and PF201 in in vitro skin absorption samples. After solid-phase extraction and reversed-phase chromatography, we quantified the substances in saline that had been in contact with human dermis for 24 h using a triple-quadrupole mass detector equipped with an electrospray ionization source. We assessed the method in three in vitro skin absorption assays using ex vivo human skin from one skin donor per test substance. The quantification ranges of our method were 0.2-200 µg/L for BPA and 0.2-20 µg/L for BPA-G, BPS, D-8, and PF201. Accuracies were within ±8% of nominal concentrations. Intra-day and total precisions (%RSD) were <10% for all analytes, except for BPA in low-concentration quality control solutions (low QCs) (12.2% and 15.5%, respectively). Overall, the process efficiency was 100-113% for all analytes, except BPS low and high QCs (80% and 71%, respectively) and BPA low QCs (134%). The absorbed dose ranged from 0.02% to 49% depending on the test substance, and was not determinable for PF201. This is the first analytical method to quantify simultaneously BPA, BPA-G, and BPA alternatives in saline from in vitro skin absorption samples.