Use of a common European approach for nanomaterials’ testing to support regulation: a case study on titanium and silicon dioxide representative nanomaterials

Abstract

The European Union (EU) continuously takes ensuring the safe use of manufactured nanomaterials (MNMs) in consumer products into consideration. The application of a common approach for testing MNMs, including the use of optimized protocols and methods' selection, becomes increasingly important to obtain reliable and comparable results supporting the regulatory framework. In the present study, we tested four representative MNMs, two titanium dioxides (NM100 and NM101) and two silicon dioxides (NM200 and NM203), using the EU FP7-NANoREG approach, starting from suspension and dispersion preparations, through to their characterization and final evaluation of biological effects. MNM dispersions were prepared following a refined NANOGENOTOX protocol and characterized by dynamic light scattering (DLS) in water/bovine serum albumin and in media used for in vitro testing. Potential genotoxic effects were evaluated on human bronchial BEAS-2B cells using micronucleus and Comet assays, and pro-inflammatory effects by cytokines release. Murine macrophages RAW 264.7 were used to detect potential innate immune responses using two functional endpoints (pro-inflammatory cytokines and nitric oxide [NO] production). The interaction of MNMs with RAW 264.7 cells was studied by electron microscopy. No chromosomal damage and slight DNA damage and an oxidative effect, depending on MNMs, were observed in bronchial cells. In murine macrophages, the four MNMs directly induced tumor necrosis factor α or interleukin 6 secretion, although at very low levels; lipopolysaccharide-induced NO production was significantly decreased by the titania and one silica MNM. The application of this approach for the evaluation of MNM biological effects could be useful for both regulators and industries.

Safety assessment of titanium dioxide (E171) as a food additive

“…Thirteen studies performed with lung cells were classified as relevant. Four out of five studies available in human lung epithelial cells (BEAS‐2B) were negative with MN tests after exposure at different concentrations of TiO 2 NPs and for different times ((NANOGENOTOX Project, 2013 Documentation provided to EFSA No 7 and 8); Vales et al., 2015; Di Bucchianico et al., 2017; Zijno et al., 2020). In Falck et al.…”

FAIRification of nanosafety data to improve applicability of (Q)SAR approaches: A case study on in vitro Comet assay genotoxicity data

“…For the interpretation of the toxicological experiments of NMs in general, as well as for the in vitro Comet assays, monitoring of critical physical–chemical parameters during the experiment is crucial. For instance, knowledge of dispersion state [70] , [61] is an important factor in determining the specific entity interacting with the biological environment [4] . Furthermore, cells healthiness is an essential requisite of in vitro experiments and associated cytotoxicity data should be available for control to be able to evaluate the outcome of a genotoxicity assay [71] , [72] .…”

“…The in vitro Comet assays were performed in three different cell lines (Caco-2, A549, BEAS 2B) following the international validated protocols and literature for standard [47] and enzyme-modified protocol [59] to study DNA strand breaks and oxidized DNA lesions induced by different TiO2, SiO2, and ZnO nanoforms (from JRC Repository [60] ). The treatment at 3 and 24 h was performed with 5 different concentrations of NMs ( e.g., 0, 1, 10, 50 and 100 µg/ml), following the protocol for NM suspension preparation used in NanoReg project [61] . Chemicals, such as H 2 O 2 , were used for the positive control.…”

A harmonized and standardized in vitro approach produces reliable results on silver nanoparticles toxicity in different cell lines

“…The use of standardized methods and standard operating procedures (SOPs) for both the physico-chemical characterization of MNMs under study, in water and in biological media, and the measurement of different endpoints in a number of cell lines, is recommended to understand their behavior in biological experiments and to evaluate their toxicological potential (Zijno et al, 2020). In their review, Drasler et al (2017) suggested some recommendations for in vitro testing of MNMs in order to increase the production of reliable and relevant data.…”

“…After exposure to AgNPs, cytotoxicity was measured by 3-( 4 toxicities in response to 20-nm AgNPs; endpoints evaluated in the cell lines included viability, AgNPs uptake, generation, and modulation of inflammatory responses in terms of secretion of the inflammatory cytokines IL-6 and TNF-α and production of the effector molecule nitric oxide (NO). The same approach was used in NANoREG project to test immunotoxicity of different MNMs (Zijno et al, 2020).…”