Hazard assessment of hexagonal boron nitride and hexagonal boron nitride reinforced thermoplastic polyurethane composites using human skin and lung cells.

Hexagonal boron nitride (hBN) is an emerging two-dimensional material attracting considerable attention in the industrial sector given its innovative physicochemical properties. Potential risks are associated mainly with occupational exposure where inhalation and skin contact are the most relevant exposure routes for workers. Here we aimed at characterizing the effects induced by composites of thermoplastic polyurethane (TPU) and hBN, using immortalized HaCaT skin keratinocytes and BEAS-2B bronchial epithelial cells. The composite was abraded using a Taber® rotary abraser and abraded TPU and TPU-hBN were also subjected to photo-Fenton-mediated degradation mimicking potential weathering across the product life cycle. Cells were exposed to the materials for 24 h (acute exposure) or twice per week for 4 weeks (chronic exposure) and evaluated with respect to material internalization, cytotoxicity, and proinflammatory cytokine secretion. Additionally, comprehensive mass spectrometry-based proteomics and metabolomics (secretomics) analyses were performed. Overall, despite evidence of cellular uptake of the material, no significant cellular and/or protein expression profiles alterations were observed after acute or chronic exposure of HaCaT or BEAS-2B cells, identifying only few pro-inflammatory proteins. Similar results were obtained for the degraded materials. These results support the determination of hazard profiles associated with cutaneous and pulmonary hBN-reinforced polymer composites exposure.

A comparative investigation of the chemical reduction of graphene oxide for electrical engineering applications.

The presence of oxygen-containing functional groups on the basal plane and at the edges endows graphene oxide (GO) with an insulating nature, which makes it rather unsuitable for electronic applications. Fortunately, the reduction process makes it possible to restore the sp2 conjugation. Among various protocols, chemical reduction is appealing because of its compatibility with large-scale production. Nevertheless, despite the vast number of reported chemical protocols, their comparative assessment has not yet been the subject of an in-depth investigation, rendering the establishment of a structure-performance relationship impossible. We report a systematic study on the chemical reduction of GO by exploring different reducing agents (hydrazine hydrate, sodium borohydride, ascorbic acid (AA), and sodium dithionite) and reaction times (2 or 12 hours) in order to boost the performance of chemically reduced GO (CrGO) in electronics and in electrochemical applications. In this work, we provide evidence that the optimal reduction conditions should vary depending on the chosen application, whether it is for electrical or electrochemical purposes. CrGO exhibiting a good electrical conductivity (>1800 S m-1) can be obtained by using AA (12 hours of reaction), Na2S2O4 and N2H4 (independent of the reaction time). Conversely, CrGO displaying a superior electrochemical performance (specific capacitance of 211 F g-1, and capacitance retention >99.5% after 2000 cycles) can be obtained by using NaBH4 (12 hours of reaction). Finally, the compatibility of the different CrGOs with wearable and flexible electronics is also demonstrated using skin irritation tests. The strategy described represents a significant advancement towards the development of environmentally friendly CrGOs with ad hoc properties for advanced applications in electronics and energy storage.

Impact of physico-chemical properties on the toxicological potential of reduced graphene oxide in human bronchial epithelial cells.

The increasing use of graphene-based materials (GBM) requires their safety evaluation, especially in occupational settings. The same physico-chemical (PC) properties that confer GBM extraordinary functionalities may affect the potential toxic response. Most toxicity assessments mainly focus on graphene oxide and rarely investigate GBMs varying only by one property. As a novelty, the present study assessed the in vitro cytotoxicity and genotoxicity of six reduced graphene oxides (rGOs) with different PC properties in the human bronchial epithelial 16HBE14o - cell line. Of the six materials, rGO1-rGO4 only differed in the carbon-to-oxygen (C/O) content, whereas rGO5 and rGO6 were characterized by different lateral size and number of layers, respectively, but similar C/O content compared with rGO1. The materials were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, laser diffraction and dynamic light scattering, and Brunauer-Emmett-Teller analysis. Cytotoxicity (Luminescent Cell Viability and WST-8 assays), the induction of reactive oxygen species (ROS; 2',7'-dichlorofluorescin diacetate-based assay), the production of cytokines (enzyme-linked immunosorbent assays) and genotoxicity (comet and micronucleus assays) were evaluated. Furthermore, the internalization of the materials in the cells was confirmed by laser confocal microscopy. No relationships were found between the C/O ratio or the lateral size and any of the rGO-induced biological effects. However, rGO of higher oxygen content showed higher cytotoxic and early ROS-inducing potential, whereas genotoxic effects were observed with the rGO of the lowest density of oxygen groups. On the other hand, a higher number of layers seems to be associated with a decreased potential for inducing cytotoxicity and ROS production.

In vitro assessment of skin irritation and corrosion properties of graphene-related materials on a 3D epidermis.

The increasing use of graphene-related materials (GRMs) in many technological applications, ranging from electronics to biomedicine, needs a careful evaluation of their impact on human health. Skin contact can be considered one of the most relevant exposure routes to GRMs. Hence, this study is focused on two main adverse outcomes at the skin level, irritation and corrosion, assessed following two specific Test Guidelines (TGs) defined by the Organization for Economic Co-operation and Development (OECD) (439 and 431, respectively) that use an in vitro 3D reconstructed human epidermis (RhE) model. After the evaluation of their suitability to test a large panel of powdered GRMs, it was found that the latter were not irritants or corrosive. Only GRMs prepared with irritant surfactants, not sufficiently removed, reduced RhE viability at levels lower than those predicting skin irritation (≤50%, after 42 min exposure followed by 42 h recovery), but not at levels lower than those predicting corrosion (<50%, after 3 min exposure or <15% after 1 h exposure). As an additional readout, a hierarchical clustering analysis on a panel of inflammatory mediators (interleukins: IL-1α, IL-1ß, IL-6, and IL-18; tumor necrosis factor-α and prostaglandin E2) released by RhE exposed to these materials supported the lack of irritant and pro-inflammatory properties. Overall, these results demonstrate that both TGs are useful in assessing GRMs for their irritant or corrosion potential, and that the tested materials did not cause these adverse effects at the skin level. Only GRMs prepared using toxic surfactants, not adequately removed, turned out to be skin irritants.

Role of Chemical Reduction and Formulation of Graphene Oxide on Its Cytotoxicity towards Human Epithelial Bronchial Cells.

Graphene-based materials may pose a potential risk for human health due to occupational exposure, mainly by inhalation. This study was carried out on bronchial epithelial 16HBE14o- cells to evaluate the role of chemical reduction and formulation of graphene oxide (GO) on its cytotoxic potential. To this end, the effects of GO were compared to its chemically reduced form (rGO) and its stable water dispersion (wdGO), by means of cell viability reduction, reactive oxygen species (ROS) generation, pro-inflammatory mediators release and genotoxicity. These materials induced a concentration-dependent cell viability reduction with the following potency rank: rGO > GO >> wdGO. After 24 h exposure, rGO reduced cell viability with an EC50 of 4.8 µg/mL (eight-fold lower than that of GO) and was the most potent material in inducing ROS generation, in contrast to wdGO. Cytokines release and genotoxicity (DNA damage and micronucleus induction) appeared low for all the materials, with wdGO showing the lowest effect, especially for the former. These results suggest a key role for GO reduction in increasing GO cytotoxic potential, probably due to material structure alterations resulting from the reduction process. In contrast, GO formulated in a stable dispersion seems to be the lowest cytotoxic material, presumably due to its lower cellular internalization and damaging capacity.

Assessment of skin sensitization properties of few-layer graphene and graphene oxide through the Local Lymph Node Assay (OECD TG 442B).

Skin contact is one of the most common exposure routes to graphene-based materials (GBMs) during their small-scale and industrial production or their use in technological applications. Nevertheless, toxic effects in humans by cutaneous exposure to GBMs remain largely unexplored, despite skin contact to other related materials has been associated with adverse effects. Hence, this in vivo study was carried out to evaluate the cutaneous effects of two GBMs, focusing on skin sensitization as a possible adverse outcome. Skin sensitization by few-layer graphene (FLG) and graphene oxide (GO) was evaluated following the Organization for Economic Cooperation and Development (OECD) guideline 442B (Local Lymph Node Assay; LLNA) measuring the proliferation of auricular lymph node cells during the induction phase of skin sensitization. Groups of four female CBA/JN mice (8-12 weeks) were daily exposed to FLG or GO through the dorsal skin of each ear (0.4-40 mg/mL, equal to 0.01-1.00 mg/ear) for 3 consecutive days, and proliferation of auricular lymph node cells was evaluated 3 days after the last treatment. During this period, no clinical signs of toxicity and no alterations in body weight and food or water consumptions were observed. In addition, no ear erythema or edema were recorded as signs of irritation or inflammation. Bromo-deoxyuridine (BrdU) incorporation in proliferating lymphocytes from ear lymph nodes (stimulation indexes <1.6) and the histological analysis of ear tissues excluded sensitizing or irritant properties of these materials, while myeloperoxidase activity in ear biopsies confirmed no inflammatory cells infiltrate. On the whole, this study indicates the absence of sensitization and irritant potential of FLG and GO.

Acute Toxicity by Oral Co-Exposure to Palytoxin and Okadaic Acid in Mice.

The frequent occurrence of marine dinoflagellates producing palytoxin (PLTX) or okadaic acid (OA) raises concern for the possible co-presence of these toxins in seafood, leading to additive or synergistic adverse effects in consumers. Thus, the acute oral toxicity of PLTX and OA association was evaluated in mice: groups of eight female CD-1 mice were administered by gavage with combined doses of PLTX (30, 90 or 270 µg/kg) and OA (370 µg/kg), or with each individual toxin, recording signs up to 24 h (five mice) and 14 days (three mice). Lethal effects occurred only after PLTX (90 or 270 µg/kg) exposure, alone or combined with OA, also during the 14-day recovery. PLTX induced scratching, piloerection, abdominal swelling, muscle spasms, paralysis and dyspnea, which increased in frequency or duration when co-administered with OA. The latter induced only diarrhea. At 24 h, PLTX (90 or 270 µg/kg) and OA caused wall redness in the small intestine or pale fluid accumulation in its lumen, respectively. These effects co-occurred in mice co-exposed to PLTX (90 or 270 µg/kg) and OA, and were associated with slight ulcers and inflammation at forestomach. PLTX (270 µg/kg alone or 90 µg/kg associated with OA) also decreased the liver/body weight ratio, reducing hepatocyte glycogen (270 µg/kg, alone or combined with OA). No alterations were recorded in surviving mice after 14 days. Overall, the study suggests additive effects of PLTX and OA that should be considered for their risk assessment as seafood contaminants.