Hazard characterization of graphene-based nanomaterials in energy production and storage (GrapHazard)

The problem addressed

Growing demand for greener energy resources and energy devices with improved performance has greatly increased the development of materials and systems for energy production and storage. Unique electrochemical properties have made graphene-based materials (GBMs) one of the most promising tools in the development of batteries, supercapacitors and solar cells. The increasing market of GBMs calls for a thorough evaluation of their possible impact on human health. Previous studies have suggested that the hazard potential of various GBMs may vary considerably, depending on their physico-chemical properties, such as surface structure, functionalization, charge, impurities and aggregation/agglomeration state. The possible risk for human health in occupational settings is mainly associated with inhalation exposure to GBMs during their production, use, and waste disposal. The few available in vivo inhalation toxicity studies showed pulmonary inﬂammation, ﬁbrosis, and long GBMs persistence in rodents. Tentatively, some GBMs might have similar toxic properties as carbon nanotubes, some of which are known to be genotoxic and carcinogenic. Data on the genotoxicity of GBMs are contradictory, with no information on possible carcinogenicity. Thus, more studies on GBMs, based on speciﬁc guidelines adapted for nanomaterials, are required.

Keywords: toxicity nanomaterials

Research questions

To reach its overall aims, GrapHazard will: 1. Adapt toxicity test guidelines (TGs) for advanced materials and apply them for GBMs testing → By employing the latest principles and procedures developed within the OECD Manufactured Nanomaterials Working Party (MNMWP) programme, and comparing in vitro results with the human biomonitoring data on the same toxicological endpoints that will be obtained by FIOH within the EU Graphene Flagship programme. 2. Contribute to elucidating the mechanisms of action at the basis of human toxic responses after inhalation exposure to GBMs → By using in vitro approaches able to differentiate between primary (interaction with target cellular components) and secondary (mediated by an inflammatory response) mechanisms of actions. 3. Assess how the physico-chemical properties of GBMs can affect their toxicity → By evaluating GBMs with different physico-chemical properties for their in vitro effects on targeted cells, providing data that can be used in selecting safer materials in energy production and storage applications (Safe-by-Design approaches).

Expected outputs

Information on the toxicity of GBMs used in energy production and storage is presently scanty, despite the growing importance of this technology. Particularly, results obtained by utilizing test methods optimized for such advanced nanomaterials as GBMs are lacking. Up to now, very few in vitro studies have assessed the possible secondary effects of nanomaterials, although secondary toxic mechanisms are considered to be important in vivo. GrapHazard will fill these gaps, providing new scientific information on the primary and secondary toxicity of relevant GBMs in tests systems employing the latest adjustments required for regulatory testing of nanomaterials and our newly developed co-culture systems. GrapHazard also aims at correlating, for the first time to the best of our knowledge, the experimental in vitro toxicity results with human data on the same toxicity endpoints obtained from the Graphene Flagship studies of workers occupationally exposed to GBMs. This integrated approach will shed light on cutting-edge data related to both the identification and characterization of the hazard posed by GBMs as necessary steps for the risk assessment of GBMs in occupational settings. Furthermore, the GBMs to be studied will be thoroughly characterized, and the project will assess the possible influence of the physico-chemical properties of the GBMs features on toxicity, providing information that could be utilized in Safe-by-Design approaches to be applied in industrial applications for energy production and storage systems. The project is expected to be successful due to the great expertise of FIOH and UniTs on nanotoxicology, the commitment of the companies involved, and the complementary support provided by the Graphene Flagship.

Workplan

Task 1: Characterization of GBMs [UniTs (leader), FIOH] (M1-M12) Each material will be fully physico-chemically characterized by transmission electron microscopy (TEM), Raman analysis, elemental analysis, thermogravimetric analysis, dynamic light scattering, and z-potential analysis, both as such and dispersed in culture media. In this manner, for each GBM, specific information on their dimension, shape, number of layers, defects, elemental composition, oxidation state, charge and agglomeration/aggregation state will be available. In addition, each material will be characterized for its endotoxin content, frequently present in nanomaterials and potentially leading to false-positive results, especially when evaluating inflammatory parameters. Expected results: complete set of characterization data for the selected GBMs.
Task 2: Adjustment of test protocols for assessing GBMs [FIOH (leader), UniTs] (M1-M10) Only few OECD test guidelines (TGs), originally validated for chemicals, are currently adapted and validated also for nanomaterials. Testing of advanced nanomaterials, such as GBMs, may require additional adjustments due to the complexity of these substances. The OECD MNWP is leading international efforts on such adaptations. Many European institutions are contributing to the so-called Malta initiative through National and European projects - FIOH is involved in the adaptation of in vitro genotoxicity assays for nanomaterials. In the Graphene Flagship, UniTs participates in the adaptation of OECD TGs for skin toxicity. In GrapHazard, FIOH and UniTs will modify the in vitro toxicity assays for GBMs, based on the ongoing efforts in the Malta initiative. FIOH will contribute to adjusting in vitro genotoxicity tests, to be used for testing the GBMs selected for the project. UniTs will contribute to adjusting the test protocols developed in the Graphene Flagship to the cellular systems (bronchial epithelial and macrophage cell lines) used in the present project (optimal dispersion, exposure time, dose range, etc.), as concerns assessing the cytotoxicity, production of reactive oxygen species (ROS), inflammation and immunotoxicity of the selected GBMs. Expected results: test protocols adjusted for assessing in vitro toxicity of inhaled GBMs.
Task 3: In vitro toxicity assessment of primary effects of GBMs [FIOH (leader), UniTs] (M8-M24) It is still unclear whether carbon-based nanomaterials showing a genotoxic effect (e.g. carbon black) act via a primary mechanism (interaction with DNA, directly or indirectly through reactive oxygen species, ROS) or a secondary mechanism (mediated by an inflammatory response). Similarly, ROS production could be induced by primary exposure to GBMs or through their pro-inflammatory effects. In vitro approaches able to differentiate between primary and secondary mechanisms of actions (i.e., co-cultures of bronchial epithelial cells and inflammatory cells) will be used in GrapHazard. To distinguish between direct and indirect primary mechanisms, parallel experiments on the capacity of GBMs to induce direct genotoxic effects or oxidative stress/pro-inflammatory responses will be carried out. The in vitro toxic effects induced by exposing cell monocultures to the selected GBMs will be evaluated using two human bronchial epithelial cell lines (BEAS-2B and 16HBE14o−), by means of cytotoxicity (FIOH, UniTs), ROS production (UniTs), release of pro-inflammatory cytokines (UniTs) and genotoxicity (FIOH). Expected results: knowledge on the primary mechanisms of action in toxic response of pulmonary cells to GBMs.
Task 4: In vitro toxicity assessment of secondary effects of GBMs [FIOH (leader), UniTs] (M11-M24) Secondary pro-inflammatory effects at the pulmonary level will be assessed in vitro by co-cultures of human THP-1 monocytes with human bronchial epithelial cells: the levels of selected differentiation markers and the release of pro-inflammatory cytokines by THP-1 cells exposed to GBMs (UniTs) will at first be evaluated to select the most inflammogenic conditions to be studied later on in the co-culture system. Secondary toxic effects mediated by THP-1-released cytokines and pro-inflammatory factors will be evaluated in the human bronchial epithelial cells co-cultured with GBM-exposed THP-1 cells by means of genotoxicity (FIOH) and ROS production (UniTs). Depending on the results, a second approach could investigate the ability of GBM-exposed bronchial epithelial cells to activate THP-1 monocytes as a pro-inflammatory response. To this aim, the highest inflammogenic conditions, as evaluated in Task 3, will be chosen, and GBM-exposed bronchial cells will be co-cultured in a Transwell® system with THP-1 undifferentiated monocytes. Activation of THP-1 monocytes will then be evaluated measuring selected differentiation markers, the release of pro-inflammatory cytokines and monocytes chemotaxis (UniTS). Expected results: knowledge on the secondary mechanisms of action involved in toxicological response of pulmonary cells to GBMs.
Task 5: Establishing correlations between physico-chemical properties of GBMs and their in vitro toxicity [UniTs (leader), FIOH] (M12-M24) GBMs with different physico-chemical properties will be evaluated for their in vitro effects on targeted cells, to define structural features strictly correlated with the toxic potential of these materials. In particular, shape, number of layers, defects, elemental composition, oxidation state, charge and agglomeration/aggregation state will be considered. This information will be relevant in choosing safer materials to be used in energy production and storage applications (Safe-by-Design approaches). In addition, knowledge on similarities and dissimilarities in toxic responses to different GBMs can help GBMs grouping when registering them under European regulations. Data on physico-chemical characterization of GBMs obtained in Task 1 will be correlated to the toxicity data obtained in Tasks 3 and 4, to identify which physico-chemical properties could mostly affect the toxic response to inhaled GBMs. Expected results: knowledge on parameters relevant for Safe-by-Design and grouping approaches concerning GBMs used in energy production and storage applications.
Task 6: Correlations between in vitro toxicity data and human data from workers exposed to GBMs [FIOH (leader), UniTs] (M18-M30) Data from human biomonitoring studies performed by FIOH within the Graphene Flagship will be correlated with the toxicity data obtained in Tasks 3 and 4, to assess i) whether the in vitro settings are predictive of human in vivo response, and ii) the mechanisms of action operating in possible toxic responses observed in humans exposed to GBMs. This information will be used by FIOH during the last 6 months of the project, to support the derivation of suggestive occupational exposure limits for the risk assessment of GBMs in occupational settings performed at the Graphene Flagship. Expected results: improved knowledge on the mechanism of action in toxic response to inhaled GBMs and derivation of occupational exposure limits (OELs).
Task 7: Dissemination of the results [FIOH (leader), UniTs] (M1-M30) A GrapHazard website will be built up and maintained. It will store the GrapHazard database, a list of produced communications and publications, and a record of all the dissemination activities (e.g. attendance to conferences and workshops) performed during the project. FIOH will be responsible for creating the website and both partners will contribute to keeping it updated. Both FIOH and UniTs will guarantee the dissemination of the most important results related to safety issues of GBMs in occupational settings by presenting them in international conferences, educational events, and by direct interaction with the other relevant stakeholders (who are described in the next section). Expected results: increased impact of the project results on relevant stakeholders, expanding the knowledge on GBMs effects after inhalation exposure and awareness to improve occupational health and safety measures.