Predicting human neurotoxicity of propylene glycol methyl ether (PGME) by implementing in vitro neurotoxicity results into toxicokinetic modelling.

Although organic solvents have been associated with CNS toxicity, neurotoxicity testing is rarely a regulatory requirement. We propose a strategy to assess the potential neurotoxicity of organic solvents and predict solvent air concentrations that will not likely produce neurotoxicity in exposed individuals. The strategy integrated an in vitro neurotoxicity, an in vitro blood-brain barrier (BBB), and an in silico toxicokinetic (TK) model. We illustrated the concept with propylene glycol methyl ether (PGME), widely used in industrial and consumer products. The positive control was ethylene glycol methyl ether (EGME) and negative control propylene glycol butyl ether (PGBE), a supposedly non-neurotoxic glycol ether. PGME, PGBE, and EGME had high passive permeation across the BBB (permeability coefficients (Pe) 11.0 × 10-3, 9.0 × 10-3, and 6.0 × 10-3 cm/min, respectively). PGBE was the most potent in in vitro repeated neurotoxicity assays. EGME's main metabolite, methoxyacetic acid (MAA) may be responsible for the neurotoxic effects reported in humans. No-observed adverse effect concentrations (NOAECs) for the neuronal biomarker were for PGME, PGBE, and EGME 10.2, 0.07, and 79.2 mM, respectively. All tested substances elicited a concentration-dependent increase in pro-inflammatory cytokine expressions. The TK model was used for in vitro-to-in vivo extrapolation from PGME NOAEC to corresponding air concentrations (684 ppm). In conclusion, we were able to predict air concentrations that would not likely result in neurotoxicity using our strategy. We confirmed that the Swiss PGME occupational exposure limit (100 ppm) will not likely produce immediate adverse effects on brain cells. However, we cannot exclude possible long-term neurodegenerative effects because inflammation was observed in vitro. Our simple TK model can be parameterized for other glycol ethers and used in parallel with in vitro data for systematically screening for neurotoxicity. If further developed, this approach could be adapted to predict brain neurotoxicity from exposure to organic solvents.

Particle and metal exposure in Parisian subway: Relationship between exposure biomarkers in air, exhaled breath condensate, and urine.

Subway particulate toxicity results from in vitro and in vivo studies diverge and call for applied human research on outcomes from chronic exposures and potential exposure biomarkers. We aimed to (1) quantify airborne particulate matter (PM) concentrations (mass and number) and metal concentrations in exhaled breath condensate (EBC), urine, and PM; (2) investigate their associations (EBC vs. PM vs. urine); and (3) assess the relevance of EBC in biomonitoring. Nine subway workers in three jobs: station agents, locomotive operators and security guards were monitored during their 6-h shifts over two consecutive weeks. Six-hour weighed average mass concentrations expressed as PM10, PM2.5 and their metal concentrations were determined. Urine and EBC samples were collected pre- and post-shift. Ultrafine particle (UFP) number concentrations were quantified in PM and EBC samples. Metal concentrations in urine and EBC were standardized by creatinine and EBC volume, respectively, and log-transformed. Associations were investigated using Pearson correlation and linear mixed regression models, with participant's ID as random effect. PM concentrations were below occupational exposure limits (OEL) and varied significantly between jobs. Locomotive operators had the highest exposure (189 and 137 µg/m3 for PM10 and PM2.5, respectively), while station agents had the highest UFP exposure (1.97 × 104 particles/cm3). Five metals (Al, Fe, Zn, Cu, and Mn) in PM2.5 and three (Al, Fe, and Zn) in PM10 were above the limit of quantification (LOQ). Fe, Cu, Al and Zn were the most abundant by mass fraction in PM. In EBC, the metal concentrations in decreasing order were: Zn > Cu > Ni > Ba > Mn. Security guards had the highest EBC metal concentrations, and in particular Zn and Cu. Urinary metal concentrations in decreasing order were: Si > Zn > Mo > Ti > Cu > Ba ≈ Ni > Co. All urinary metal concentrations from the subway workers were similar to concentrations found in the general population. A statistically significant relationship was found for ultrafine particle number concentrations in PM and in EBC. Zn and Cu concentrations in post-shift EBC were associated with Zn and Cu concentrations in PM10 and with post-shift urinary Zn and Cu concentrations. Therefore, EBC appears a relevant matrix for assessing exposure to UFP in human biomonitoring when inhalation is a primary route of exposure. We found different temporal variation patterns between particle and metal exposures in three matrices (PM, urine, EBC) quantified daily over two full weeks in subway workers. These patterns might be related to metal oxidation, particulates' solubility and size as well as their lung absorption capabilities, which need to be further explored in toxicological research. Further research should also focus on understanding possible influences of low chronic exposures to subway particulates on health in larger cohorts.

Respiratory Disease Occupational Biomonitoring Collaborative Project (ROBoCoP): A longitudinal pilot study and implementation research in the Parisian transport company.

The ROBoCoP project is launched within the EU COST Action CA16113 "CliniMARK" aiming to increase the number of clinically validated biomarkers and focused on chronic obstructive pulmonary disease (COPD) biomarker development and validation. ROBoCoP encompasses two consecutive studies consisting of a pilot study followed by a field study. The pilot study is a longitudinal exposure assessment and biomarker study aiming at: 1-understanding the suitability of the candidate biomarkers in surveying populations at risk such as workers exposed to COPD causing agents; 2-determining the best sampling plan with respect to the half-life of the candidate biomarkers; 3-implementing and validating the sampling procedures and analytical methods; 4-selecting the best suitable biomarkers to be measured in the field. Each study participant is surveyed every day during the 6-8 h work-shifts for two consecutive weeks. The field study has an implementation research designe that enabled us to demonstrate the applicability of the standardized protocol for biomarker measurements in occupational settings while also assessing the biomarkers' validity. ROBoCoP will focus on particulate matter (PM) exposure measurements, exposure biomarkers and a series of effect biomarkers, including markers of lipoperoxidation: 8-isoprostane, malondialdehyd in exhaled breath condensate (EBC) and urine, potential markers of nitrosative stress: NO2-, NO3- and formate anion in EBC; markers of DNA oxidation: 8-hydroxy-2'deoxyguanosine in EBC and urine, marker of genotoxicity: micronuclei in buccal cells, and oxidative potential in exhaled air (OPEA). OPEA appears particularly promising as a clinical biomarker for detecting COPD, and will be tested independently and as part of a biomarker panel. COPD diagnosis will be performed by an experienced occupational physician according to international diagnostic standards and confirmed by a pulmonologist.This research will include approximatively 300 underground subway workers randomly selected from the personnel registry of a large Parisian transport company. Underground subways are suggested as the most PM polluted urban transport environment. We believe this occupational exposure is relevant for biomonitoring of workers and early detection of respiratory diseases.

Influence of experimental parameters on in vitro human skin permeation of Bisphenol A.

Bisphenol A (BPA) in vitro skin permeation studies have shown inconsistent results, which could be due to experimental conditions. We studied the impact of in vitro parameters on BPA skin permeation using flow-through diffusion cells with ex-vivo human skin (12 donors, 3-12 replicates). We varied skin status (viable or frozen skin) and thickness (200, 400, 800 µm), BPA concentrations (18, 250 mg/l) and vehicle volumes (10, 100 and 1000 µl/cm2). These conditions led to a wide range of BPA absorption (2%-24% after 24 h exposure), peak permeation rates (J = 0.02-1.31 µg/cm2/h), and permeability coefficients (Kp = 1.6-5.2 × 10-3 cm/h). This is the first time steady state conditions were reached for BPA aqueous solutions in vitro (1000 µl/cm2 applied at concentration 250 mg/l). A reduction of the skin thickness from 800 and 400 µm to 200 µm led to a 3-fold increase of J (P < 0.05). A reduction of the vehicle volume from 1000 to 100 led to a 2-fold decrease in J (P > 0.05). Previously frozen skin led to a 3-fold increase in J compared to viable skin (P < 0.001). We found that results from published studies were consistent when adjusting J according to experimental parameters. We propose appropriate J values for different exposure scenarios to calculate BPA internal exposures for use in risk assessment.

Reflections on the OECD guidelines for in vitro skin absorption studies.

At the 8th conference of Occupational and Environmental Exposure of the Skin to Chemicals (OEESC) (16-18 September 2019) in Dublin, Ireland, several researchers performing skin permeation assays convened to discuss in vitro skin permeability experiments. We, along with other colleagues, all of us hands-on skin permeation researchers, present here the results from our discussions on the available OECD guidelines. The discussions were especially focused on three OECD skin absorption documents, including a recent revision of one: i) OECD Guidance Document 28 (GD28) for the conduct of skin absorption studies (OECD, 2004), ii) Test Guideline 428 (TGD428) for measuring skin absorption of chemical in vitro (OECD, 2004), and iii) OECD Guidance Notes 156 (GN156) on dermal absorption issued in 2011 (OECD, 2011). GN156 (OECD, 2019) is currently under review but not finalized. A mutual concern was that these guidance documents do not comprehensively address methodological issues or the performance of the test, which might be partially due to the years needed to finalize and update OECD documents with new skin research evidence. Here, we summarize the numerous factors that can influence skin permeation and its measurement, and where guidance on several of these are omitted and often not discussed in published articles. We propose several improvements of these guidelines, which would contribute in harmonizing future in vitro skin permeation experiments.

Urinary 8-isoprostane as a biomarker for oxidative stress. A systematic review and meta-analysis.

Oxidative stress is defined as an imbalance between the production and elimination of reactive oxygen species (ROS) are associated with various inflammation-related human disease. ROS can oxidize lipids, which subsequently undergo fragmentation to produce F2-isoprostanes (F2-IsoPs). Eight-isoprostane is one of the most extensively studied F2-IsoPs and the most commonly used biomarker for the assessment of oxidative stress in human studies. This urinary biomarker is quantified using either chemical or immunological techniques. A "physiological" range for 8-isoprostanes is needed to use this biomarker as a measure of excess oxidative stress originating from occupational exposures. However, ranges reported in the literature are inconsistent. We designed a standardized protocol of a systematic review and meta-analysis to assess baseline values for 8-isoprostane concentrations in urine of healthy adults and identify determinants of their inter- and intra-individual variability. We searched PubMed from journal inception and up to April 2019, and screened articles for studies containing F2-IsoPs concentrations in urine for healthy adult participants. We grouped studies in three biomarker groups: "8-isoprostane", "Isoprostanes" "15- F2t-Isoprostane". We computed geometric mean (GM) and geometric standard deviation (GSD) as the basis for the meta-analysis. Of the initial 1849 articles retrieved, 63 studies were included and 107 subgroups within these study populations were identified. We stratified the subgroups analyzed with the chemical methods by body mass index (BMI) reported. We provide pooled GM values for urinary 8-isoprostane concentrations in healthy adults, separately for chemical and immunological analysis in this review. The interquartile range (IQR) in subgroups with a mean BMI below 25 measured using chemical methods was 0.18 to 0.40 µg/g creatinine. We show that there is a significant positive association between BMI and urinary 8-isoprostane concentrations. We recommend adjusting urinary 8-isoprostane concentrations in spot urine with creatinine, quantifying 8-isoprostane with chemical analytical methods, and reporting results as median and quartiles. This will help in comparing results across studies.

State of knowledge on the occupational exposure to carbon nanotubes.

Carbon nanotubes (CNT) trigger fascination as well as anxiety, given their unique physical and chemical properties, and continuing concerns around their possible health effects. CNT exposure assessment is an integral component of occupational and environmental epidemiology, risk assessment, and management. We conducted a systematic review to analyze the quality of CNT occupational exposure assessments in field studies and to assess the relevance of available quantitative data from occupational hygiene and epidemiological perspectives. PubMed and Scopus databases were searched for the period 2000-2018. To grade the quality of each study, we used a standardized grid of seven criteria. The first criterion addressed 12 items deemed most relevant CNT physical-chemical properties with respect to their in vitro and in vivo toxicity. We included 27 studies from 11 countries in the review and graded them high (n = 2), moderate (n = 15) and low quality (n = 10). Half of the studies measured elemental carbon mass concentration (EC) using different methods and aerosol fractions. In 85% of studies, the observed values exceed the US National Institute for Occupational Safety and Health Recommended Exposure Limit. The quantification of CNT agglomerates and/or CNT contained fibers becomes increasingly common although lacking methodological standardization. Work activities with the greatest mean CNT mass concentrations were non-enclosed and included sieving, harvesting, packaging, reactor cleaning, extrusion and pelletizing. Some of the large studies defined standardized job titles according to exposure estimates at corresponding workstations and classified them by decreasing CNT exposure level: technicians > engineers > chemists. The already initiated harmonization of CNT exposure assessment and result reporting need to continue to favor not only studies in the field, but also to identify companies and workers using CNTs to characterize their exposures as well as monitor their health. This will enable an objective and realistic evaluation of risks associated with CNT applications and an appropriate risk management.

A new alternative method for testing skin irritation using a human skin model: a pilot study.

BACKGROUND: Studies assessing skin irritation to chemicals have traditionally used laboratory animals; however, such methods are questionable regarding their relevance for humans. New in vitro methods have been validated, such as the reconstructed human epidermis (RHE) model (Episkin®, Epiderm®). The comparison (accuracy) with in vivo results such as the 4-h human patch test (HPT) is 76% at best (Epiderm®). There is a need to develop an in vitro method that better simulates the anatomo-pathological changes encountered in vivo. OBJECTIVES: To develop an in vitro method to determine skin irritation using human viable skin through histopathology, and compare the results of 4 tested substances to the main in vitro methods and in vivo animal method (Draize test). METHODOLOGY: Human skin removed during surgery was dermatomed and mounted on an in vitro flow-through diffusion cell system. Ten chemicals with known non-irritant (heptylbutyrate, hexylsalicylate, butylmethacrylate, isoproturon, bentazon, DEHP and methylisothiazolinone (MI)) and irritant properties (folpet, 1-bromohexane and methylchloroisothiazolinone (MCI/MI)), a negative control (sodiumchloride) and a positive control (sodiumlaurylsulphate) were applied. The skin was exposed at least for 4h. Histopathology was performed to investigate irritation signs (spongiosis, necrosis, vacuolization). RESULTS: We obtained 100% accuracy with the HPT model; 75% with the RHE models and 50% with the Draize test for 4 tested substances. The coefficients of variation (CV) between our three test batches were <0.1, showing good reproducibility. Furthermore, we reported objectively histopathological irritation signs (irritation scale): strong (folpet), significant (1-bromohexane), slight (MCI/MI at 750/250ppm) and none (isoproturon, bentazon, DEHP and MI). CONCLUSIONS: This new in vitro test method presented effective results for the tested chemicals. It should be further validated using a greater number of substances; and tested in different laboratories in order to suitably evaluate reproducibility.

Skin permeation and metabolism of di(2-ethylhexyl) phthalate (DEHP).

Phthalates are suspected to be endocrine disruptors. Di(2-ethylhexyl) phthalate (DEHP) is assumed to have low dermal absorption; however, previous in vitro skin permeation studies have shown large permeation differences. Our aims were to determine DEHP permeation parameters and assess extent of skin DEHP metabolism among workers highly exposed to these lipophilic, low volatile substances. Surgically removed skin from patients undergoing abdominoplasty was immediately dermatomed (800 µm) and mounted on flow-through diffusion cells (1.77 cm(2)) operating at 32°C with cell culture media (aqueous solution) as the reservoir liquid. The cells were dosed either with neat DEHP or emulsified in aqueous solution (166 µg/ml). Samples were analysed by HPLC-MS/MS. DEHP permeated human viable skin only as the metabolite MEHP (100%) after 8h of exposure. Human skin was able to further oxidize MEHP to 5-oxo-MEHP. Neat DEHP applied to the skin hardly permeated skin while the aqueous solution readily permeated skin measured in both cases as concentration of MEHP in the receptor liquid. DEHP pass through human skin, detected as MEHP only when emulsified in aqueous solution, and to a far lesser degree when applied neat to the skin. Using results from older in vitro skin permeation studies with non-viable skin may underestimate skin exposures. Our results are in overall agreement with newer phthalate skin permeation studies.