Grouping of nanomaterials to read-across hazard endpoints: from data collection to assessment of the grouping hypothesis by application of chemoinformatic techniques

An increasing number of manufactured nanomaterials (NMs) are being used in industrial products and need to be registered under the REACH legislation. The hazard characterisation of all these forms is not only technically challenging but resource and time demanding. The use of non-testing strategies like read-across is deemed essential to assure the assessment of all NMs in due time and at lower cost. The fact that read-across is based on the structural similarity of substances represents an additional difficulty for NMs as in general their structure is not unequivocally defined. In such a scenario, the identification of physicochemical properties affecting the hazard potential of NMs is crucial to define a grouping hypothesis and predict the toxicological hazards of similar NMs. In order to promote the read-across of NMs, ECHA has recently published “Recommendations for nanomaterials applicable to the guidance on QSARs and Grouping”, but no practical examples were provided in the document. Due to the lack of publicly available data and the inherent difficulties of reading-across NMs, only a few examples of read-across of NMs can be found in the literature. This manuscript presents the first case study of the practical process of grouping and read-across of NMs following the workflow proposed by ECHA. The workflow proposed by ECHA was used and slightly modified to present the read-across case study. The Read-Across Assessment Framework (RAAF) was used to evaluate the uncertainties of a read-across within NMs. Chemoinformatic techniques were used to support the grouping hypothesis and identify key physicochemical properties. A dataset of 6 nanoforms of TiO2 with more than 100 physicochemical properties each was collected. In vitro comet assay result was selected as the endpoint to read-across due to data availability. A correlation between the presence of coating or large amounts of impurities and negative comet assay results was observed. The workflow proposed by ECHA to read-across NMs was applied successfully. Chemoinformatic techniques were shown to provide key evidence for the assessment of the grouping hypothesis and the definition of similar NMs. The RAAF was found to be applicable to NMs

Towards an alternative testing strategy for nanomaterials used in nanomedicine: lessons from NanoTEST.

In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project ( www.nanotest-fp7.eu ) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed

Impact of agglomeration and different dispersions of titanium dioxidenanoparticles on the human related in vitro cytotoxicity and genotoxicity

The published results on nanoparticles cytotoxicity and genotoxicity such as titanium dioxide nanoparticles (TiO2 NPs) are inconsistent, and often conflicting and insufficient. Since different parameters may have impact on the toxicity results, there is need to lay stress on detailed characterization of NPs and the use of different testing conditions for assessment of NPs toxicity. In order to investigate whether dispersion procedures influence NP cytotoxicity and genotoxicity, we compared two protocols giving TiO2 NP dispersions with different stability and agglomeration states. Detailed primary and secondary characteristics of both TiO2 NP dispersions in culture media were carried out before toxicological testing; TK6 human lymphoblast cells, EUE human embryonic epithelial cells and Cos-1 monkey kidney fibroblasts were used to assess cytotoxicity (by trypan blue exclusion, proliferation activity and plating efficiency assays) and genotoxicity (by the comet assay). DNA strand breaks were detected by the alkaline comet assay. DNA oxidation lesions (especially 8-oxo-7,8-dihydroguanine, 8-oxoG) were measured with a modified comet assay including incubation with specific repair enzyme formamidopyrimidine DNA glycosylase (FPG). The TiO2 NPs dispersion with large agglomerates (3 min sonication and no serum in stock solution) induced DNA damage in all three cell lines, while the TiO2 NPs dispersed with agglomerates less than 200 nm (foetal serum in stock solution and sonication 15 min) had no effect on genotoxicity. An increased level of DNA oxidation lesions detected in Cos-1 and TK6 cells indicates that the leading mechanism by which TiO2 NPs trigger genotoxicity is most likely oxidative stress. Our results show that the dispersion method used can influence the results of toxicity studies. Therefore at least two different dispersion procedures should be incorporated into assessment of cyto- and genotoxic effects of NPs. It is important, when assessing the hazard associated with NPs, to establish standard testing procedures and thorough strategies to consider the diverse conditions relevant to possible exposures

Toxicity screenings of nanomaterials: Challenges due to interference with assay processes and components of classic in vitro tests

Given the multiplicity of nanoparticles (NPs), there is a requirement to develop screening strategies to evaluate their toxicity. Within the EU-funded FP7 NanoTEST project, a panel of medically relevant NPs has been used to develop alternative testing strategies of NPs used in medical diagnostics. As conventional toxicity tests cannot necessarily be directly applied to NPs in the same manner as for soluble chemicals and drugs, we determined the extent of interference of NPs with each assay process and components. In this study, we fully characterized the panel of NP suspensions used in this project (poly(lactic-co-glycolic acid)-polyethylene oxide [PLGA-PEO], TiO2, SiO2, and uncoated and oleic-acid coated Fe3O4) and showed that many NP characteristics (composition, size, coatings, and agglomeration) interfere with a range of in vitro cytotoxicity assays (WST-1, MTT, lactate dehydrogenase, neutral red, propidium iodide, (3)H-thymidine incorporation, and cell counting), pro-inflammatory response evaluation (ELISA for GM-CSF, IL-6, and IL-8), and oxidative stress detection (monoBromoBimane, dichlorofluorescein, and NO assays). Interferences were assay specific as well as NP specific. We propose how to integrate and avoid interference with testing systems as a first step of a screening strategy for biomedical NPs

Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles.

Nanogenotoxicity is a crucial endpoint in safety testing of nanomaterials as it addresses potential mutagenicity, which has implications for risks of both genetic disease and carcinogenesis. Within the NanoTEST project, we investigated the genotoxic potential of well-characterised nanoparticles (NPs): titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled amorphous silica 25 (Fl-25 SiO2) and 50 nm (Fl-50 SiO) and polylactic glycolic acid polyethylene oxide polymeric NPs - as well as Endorem® as a negative control for detection of strand breaks and oxidised DNA lesions with the alkaline comet assay. Using primary cells and cell lines derived from blood (human lymphocytes and lymphoblastoid TK6 cells), vascular/central nervous system (human endothelial human cerebral endothelial cells), liver (rat hepatocytes and Kupffer cells), kidney (monkey Cos-1 and human HEK293 cells), lung (human bronchial 16HBE14o cells) and placenta (human BeWo b30), we were interested in which in vitro cell model is sufficient to detect positive (genotoxic) and negative (non-genotoxic) responses. All in vitro studies were harmonized, i.e. NPs from the same batch, and identical dispersion protocols (for TiO2 NPs, two dispersions were used), exposure time, concentration range, culture conditions and time-courses were used. The results from the statistical evaluation show that OC-Fe3O4 and TiO2 NPs are genotoxic in the experimental conditions used. When all NPs were included in the analysis, no differences were seen among cell lines - demonstrating the usefulness of the assay in all cells to identify genotoxic and non-genotoxic NPs. The TK6 cells, human lymphocytes, BeWo b30 and kidney cells seem to be the most reliable for detecting a dose-response