NANoREG Presentations and Publications brief review will summarize some of the most promising...

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No. Year Title Publication /Presentation

Author(s) Ben. Nr.

Beneficiary Summary Journal/Congres

TaskQ&Ns

Date Comment

01 2013 Measurement uncertainties of size, shape and surface 1 measurements using transmission electron microscopy of near-2 monodisperse, near-spherical nanoparticles

Publication Pieter-Jan De Temmermann et al

26 CODA-CERVA, Keywords Transmission electron microscopy Method validation Reference material Silica nanoparticles Measurement uncertainty Nanometrology

Research Paper Task 2.2aDel 2.10Q&Ns: 1, 2, 4

15-12-2013

02 2013 Semi-automatic size measurement of primary particles in aggregated nanomaterials by transmission electron microscopy

Publication Pieter-Jan De Temmerman et al

26 CODA-CERVA Keywords: transmission electron microscopy; image analysis; aggregate, nanomaterial , primary particles, fractal analysis

Powder technology Task 2.2aDel 2.10Q&Ns: 1, 2, 4

03 2013 Nano-(Q)SAR to Categorise Nanomaterial in the Assessment of Risk: What are the challenges?

Publication Ratna Tantra et al. 124660

NIA, NPL, UniLeeds, University of Gdansk

The paper identifies the challenges associated with the uptake of (Q)SAR in the assessment of hazard and risk. It reviews the use of (Q)SAR for chemical categorisation and summarises recent advances in (Q)SAR modelling of nanomaterial toxicity, which will subsequently be of use to allow categorisation and prediction of biological activities of untested or novel compounds. The authors identify a number of barriers: a) lack of guidelines in the development, validation and regulatory acceptance of the model and b) lack of good/reliable experimental data from which the model can be developed.

Nanotoxicology Q&N's: 5,8,10

04 2013 Physiologically based pharmacokinetic modeling of polyethylene glycol-coated 1 olyacrylamide nanoparticles in rats

Publication Yang Li et al 15 KI Keywords: nanoparticles, physiologically based pharmacokinetic modeling 13 biodistribution, phagocytosis Nanotoxicology Q&N's: 4,8

05 2014 Evaluation of genotoxic effects in peripheral blood cells of rats exposed to 6-month inhalation of CeO2 nanoparticles

Presentation Francesca Pacchierotti 18 ENEA In the framework of the European Project “NANoREG. A common European approach to the regulatory testing of manufactured nanomaterials”, a study is ongoing to assess potential toxicity and carcinogenicity of chronic CeO2 (OECD repository material, NM-212) inhalation in rats. At ENEA, genotoxic effects were evaluated in peripheral blood samples shipped from the BASF laboratories. Effects on DNA were analyzed in leukocytes by the comet assay. Gene mutation and chromosome damage were measured in erythrocytes, by the flow cytometric pig-a (MutaFlow®, Litron) and micronucleus (MicroFlow®, Litron) tests, respectively. The analyses were carried out after 6 months of exposure at the concentration levels of 0.1, 0.3, 1 or 3 mg/m3 CeO2, in groups of 5 female rats. An additional group of 7 unexposed animals (clean air only) served as negative controls. Positive controls were provided by blood samples withdrawn from rats exposed to 3 daily doses of 20 mg/kg of ethylnitrosourea (ENU) by gavage. As expected, ENU induced a highly significant increase of the comet, pig-a and micronucleus background values. Conversely, CeO2 inhalation exposure did not induce any significant effect on the analyzed genotoxicity endpoints. In particular, in any of the exposed groups, the mean tail intensity percentage was not significantly different from the 1.79 0

erythrocytes were recorded in all experimental groups, irrespectively of treatment. The percentages of micronucleated reticulocytes were very similar in all groups, ranging from 0.11 to 0.15. No evidence of toxicity was revealed by a decrease of the reticulocyte percentage. These overall results demonstrate that chronic inhalation exposure to low doses of CeO2 (NM-212) does not induce genotoxic effects on the hematopoietic system at the DNA, gene, chromosome or genome level.

NanoTOX20147th International Nanotoxicology Congress in Antalya, Turkey

23-4-2014

NANoREG Presentations and Publications

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06 2014 Genotoxic and immunotoxic effects of nanofibrillar cellulose in vitro

Presentation Hanna Lindberg et al 49 FIOH The presentation describes the backgrounds and the results of a in vitro study of the potential genotoxicity and immunotoxicity of four NFC materials. It summarises that none of the tested NFC materials or the bulk-sized cellulose were able to induce DNA damage in BEAS 2B cells. Preliminary results on the MN test in BEAS 2B cells neither indicated an increase in chromosomal damage by the materials. One of the NFCs produced cytotoxic and pro-inflammatory effects in THP-1 cell derived macrophages. Preliminary in vitro results suggest that the tested materials were not genotoxic in BEAS 2B cells as measured by the comet and micronucleus assays. Most of the tested materials neither showed any immunomodulatory properties except for one material and hence further studies are needed to clarify the reason behind this phenomenon

NanoTOX20147th International Nanotoxicology Congress in Antalya, Turkey

23-4-2014

07 2014 Influence of the dispersion methods in the study of the 1 ecotoxicity of CNTs

Publication Cristina Cerrilloa et al 51 IK4-TEKNIKER and the Departemento de Quimica Inorganica

The paper describes the effects of the dispersion methods on the calibration curves to measure MWCNTs concentrations by UV-Vis-NIR for ecotoxicity assessment. The presented work makes clear that different ultrasonic treatments does not result in the same absorbance results. Dispersions for calibration curves and those for toxicity assessment should therefore be prepared using the same dispersion methods. Moreover, the authors propose a procedure to select the most appropriate measurement wavelength for each type of MWCNT.

Environmental Sience & Technology

Q&Ns: 2,3

08 2014 Assessment of SiO2 nanoparticlegenotoxicity in blood cells of rats after sub -chronic exposure

Presentation Cordelli et al 18 ENEA In the framework of the European funded Project “NANoREG: A common European approach to the regulatory testing of nanomaterials”, a 90-day oral toxicity study of synthetic amorphous silica nanoparticles (NM-203) is being carried out at the Italian National Institute of Health. The study includes the assessment of genotoxicity in blood cells at the DNA, gene and chromosome level, by the comet, pig-a and micronucleus assay, respectively, carried out at ENEA laboratories.

EEMS2014 6-10 July 2014

09 2014 Toxicity Following Pulmonary Exposure to Carbon nanotubes

AbstractPresentation

Kristina Bram Knudsen et al

4 NRCWE Carbon nanotubes (CNTs) have numerous industrial applications. With increased production and widespread application of industrial and consumer products, the potential exposure will increase. CNTs are of great concern, due to their asbestos like properties. Previous in vivo studies suggest that multi-walled CNT can generate reactive oxygen species and induce inflammation in the exposed lungs and pleural cavity. The main concern about CNT exposure is whether these can over time pose cancer risk, similarly to asbestos.

7th International Nanotoxicology Congress in Antalya, Turkey

23-25 April 2014

10 2014 Toxicokinetics and toxicity of orally administered synthetic amorphous silica in rats: critical information for risk assessment of E551 in food

Presentation/Poster

Francesco Cubadda et al

17 ISS Synthetic amorphous silica (SAS) is a nanomaterial, consisting of aggregates and agglomerates of primary SiO2 particles in the nanorange. Besides other uses in consumer's products, SAS is a food additive (E551) widely employed as clearing agent, stabilizer, carrier, anticaking agent and flow aid. Notwithstanding its application in food for decades, the risk assessment of SAS has to be reconsidered in the light of up-to-date information on the toxicokinetics and potential toxicological hazards of nanomaterials. The distribution and retention in tissues are particularly relevant, e.g., a continuous oral intake might result in slow bioaccumulation, even if oral bioavailability is low.

7th International Nanotoxicology Congress in Antalya, Turkey

23-25 April 2014

11 2014 Perspectives in immunopharmacology: The future of immunosuppression

Publication Diana Boraschi,Giselle Penton-Rol

31 CNR Modulation of immune responses for therapeutic purposes is a particularly relevant area, given the central role of anomalous immunity in a wide variety of diseases, from the most typically immune-relatedsyndromes (autoimmune diseases, allergy and asthma, immunodeficiencies) to those in which alteredimmunity and inflammation define the pathological outcomes (chronic infections, tumors, chronicinflammatory and degenerative diseases, metabolic disorders, etc.). This brief review will summarize some of the most promising perspectives of immunopharmacology,in particular in the area of immunosuppression, by considering the following aspects:molecular pathways, personalized medicine, microbiome, natural products, nanomedicine: exploiting the intelligent design of engineered nanoparticles offered by nanotechnolo-gies in tailoring shape-, size-, surface-controlled carriers for targeted delivery of drugs.

Immunology Letters

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12 2014 Optimising the use of commercial LAL assays for the analysis of endotoxin contamination in metal colloids and metal oxide nanoparticles

Publication Yang Li et al 31 CNR Engineered nanoparticles (NP) are generally contaminated by bacterial endotoxin, an ubiquitous bacterial molecule with significant toxic and inflammatory effects. The presence of endotoxin, can be responsible for many of the in vitro and in vivo effects attributed to NPs. The Limulus Amoebocyte Lysate (LAL) assay, the test requested by regulatory authorities for assessing endotoxin contamination in products for human use, is not immediately applicable for testing endotoxin in NP. In this study, we have compared different commercially available LAL assays for detecting endotoxin in gold, silver and iron oxide NPs. The modified chromogenic LAL assay proved to be the most suitable assay for measuring endotoxin in NP samples.

Nanotoxicology

13 2014 The problem of nanosafety: how to assess the fate of nano-bio interaction and predict risk

Presentation/Abstract

Diana Boraschi, 31 CNR To assess whether NP may or not pose a risk for human health, reliable approaches should be used. The problem of devising assays that are representative of the human NP effects in vivo and that can be predictive of putative risk must be seriously addressed, since several of the current assays are non-specific, partially representative and eventually unreliable.Issues that should be considered when devising ways for testing nanosafety are therefore the following:1. toxicity of NP in real-life exposure scenarios;2. effects on population groups with frail or anomalous immunity (babies, elderly, patients with chronic diseases);3. NP changes due to interaction with surrounding agents (e.g., water, mucus, blood);4. co-challenge with other agents (e.g. bacteria, pollen, allergens).How these situations could be reproduced in simplified assays predictive of risk will be discussed.

Biodendrimer 2014 in Lugano

14 2014 A human primary monocytes based in vitro model for evaluating the effects of engineered nanoparticles on the course of an inflammatory defence reaction

Poster Presentation

Yang Li et al. 31 CNR The possible interference of nanoparticles with the normal development of the innate/inflammatory response was evaluated with an in vitro model based on human primary monocytes, which reliably represents the human inflammatory response. The course of the physiological inflammatory reaction, from initiation and development to eventual resolution, was reproduced by exposing CD14+ human blood monocytes to a sequence of different microenvironmental conditions (CCL2, LPS, TNF

the culture conditions, to mimic persistent pre-pathological inflammation

The effect of endotoxin-free Au nanoparticles was assessed on the course of the inflammatory reaction in the two in vitro models. Gene expression and protein production were analysed at different time points for several inflammatory cytokines and receptors and for anti-inflammatory factors.

Preliminary results show that Au nanoparticles are unable to affect in a significant fashion the course of either the physiological or the persistent inflammatory reaction in the in vitro monocyte-based models, suggesting that these nanoparticles do not alter the innate defensive ability of the human body.

We conclude that the use of a valid and representative in vitro model of human inflammation may allow us to realistically investigating the effects of nanoparticles on inflammatory/innate immune responses, thereby being useful to predicting the immunological risk posed by engineered nanomaterials.


15 2014 Nanoparticles and the Immune SystemSafety and Effects

Book Diana BoraschiAlbert Duschl

31 CNR Chapter 1 How Innate and Adaptive Immunity WorkChapter 2 Nanoparticles and Innate ImmunityChapter 3 Nanoparticles and Adaptive Immunity.Chapter 4 Nanoparticles and Allergy Chapter 5 Nanoparticles and Immunological FrailtyChapter 6 Nanoparticles in Medicine: Nanoparticle Engineering for Macrophage Targeting and Nanoparticles that Avoid Macrophage RecognitionChapter 7 The Invertebrate Immune System as a Model for Investigating the Environmental Impact of NanoparticlesChapter 8 Summary and Outlook

Boraschi, D. & A. Duschl (authors/editors). 2014. Nanoparticles and the immune system: safety and effects. Elsevier – Academic Press, Oxford, UK)

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16 2014 Bacterial endotoxin binds to the surface of gold nanoparticles and triggers inflammation

Presentation/Abstract

Yang Li et al 31 CNR The presence of bacterial endotoxin (lipopolysaccharide, LPS), a potent immune/inflammatory activator and toxic molecule, needs to be evaluated in engineered NP preparations, in order to be able to correctly attribute to NPs, the inflammatory/toxic effects that may be observed. In this study, the ability of LPS to associate with gold NPs was studied. The red-shift of UV-VIS spectra, the increase in NP size and the decrease in Z-potential all indicated a dose-dependent binding of LPS to the NP surface, which increased with time to reach maximal binding after about 60 min at room temperature. Preliminary results showed that LPS-treated NPs could induce the expression of the IL1B gene and the production of the mature IL-1 pro

illuminating that LPS-carrying NPs not only upregulated the IL1B gene but could also activate the inflammasome-dependent IL-1 maturat

production. These data suggest that unintentional adsorption of ubiquitous LPS contaminatin to engineered NP surface, if going undetected, could induce inflammatory/toxic effects that may be erroneously attributed to NPs. Distinguishing the intrinsic NP bio-effects from those caused by biologically active contaminants such as LPS is key to a correct and reliable nanosafety evaluation.


17 2014 Principal Component Analysis, a useful tool in nanotoxicological studies

Presentation Ratna Tantra et al 4660

NPLUnivLeeds

The presentation adresses the need for better measurement regarding the EHS aspects of NMs. Keywords and key elements: dispersion, sonication protocolRepeatibility of four different (dispersion) protocols as asssesed by PCA and sources of irreproducibility. It is concluded that for the purpose of harmonisation, experimental protocols reported by researchers must be described in sufficient detail. For the purpose of data comparability between labs, detailed dispersion protocol must be followed. Dispersion protocol followed will be nano-specific and hence we cannot generalise. For this case study the largest source of variability is associated with particle concentration, with pre-wetting resulting in least significant source of variability. Why use PCA? Able to handle large datasets to compress, cluster and visualise particle size distribution data, with minimal loss of information

IUMRS-ICA 2014 24–30 August 2014, Fukuoka, Japan

18 2014 Is there a link between the physicochemical characteristics of multiwalled carbon nanotubes and their genotoxicity in cells

Presentation/abstract

Henriqueta Louro et al 53 ISQ The human exposure to nanomaterials (NM) has been increasing worldwide. In particular, multi-walled carbon nanotubes (MWCNTs) have been developed for industrial purposes, but the same properties that render these materials so attractive may also cause higher toxicity. However, contradictory results concerning their genotoxicity and carcinogenicity have been reported suggesting that minor changes in physicochemical properties may determine their toxicity. The objective of this work was to characterize the cyto- and genotoxic effects of MWCNTs that differ in thickness, length and aspect ratio (NM-400, NM-401, NM-402 and NM-403, JRC repository) in human-derived bronchial epithelial cells (BEAS-2B), in order to ascertain which characteristic may imply higher genotoxicity.

43rd Annual Meeting of the EEMS hosted by the UKEMS

6-10th July, 2014

19 2014 Toxicity of nanofibrillar cellulose in BEAS 2B cells and THP-1 cell derived macrophages in vitro

Conference abstract

Saila Pesonen et al 495758

FIOHStora Enso UPM

After a NM dispersion protocol using 0.05% BSA-water with ethanol pre-wetting, cell cultures were exposed to several NM concentrations (0-256 μg/ml). The cell viability was evaluated with the Trypan blue dye exclusion assay. For genotoxicty testing, the alkaline comet assay was applied, after 3 or 24h- exposure and the cytokinesis-blocked micronucleus assay was carried out at 48h-exposure.

2014 TAPPI International Conference on Nanotechnology for Renewable Materials

20 2014 Hazard identification and characterization of synthetic amorphous silica (SAS), food additive E551, through a 90-day oral toxicity study in rat

Conference abstract

R. Tassinari et al 17 ISS The results show a decrease in cell viability, seen 3h after exposure above 64 μg/ml of NM-402, while other MWCNTs were not cytotoxic in this timepoint. A decrease in viability was observed after 24h-exposure to NM-401 and NM-403. Considering the genotoxicity assays, both the thick (diameter 62.8 nm) and long (3366.4 nm) NM-401, but also the thin (diameter 10.7 nm) but not so long (1141.3 nm) NM-402 were negative. Furthermore, the thin (diameter 11 nm) and short (394.3 nm) NM-403 was also negative and data on NM-400 is underway.

First national Congress of the Italian Society of Nanotoxicology

WP4Task 4.5.5Q&Ns: 6,7, 8, 9 10

Lancaster University, United Kingdom, from 6-10th July, 2014.

21 2014Evaluation of particle tracking analysis as an alternative for transmission electron microscopy.

Publication Pieter-Jan De Temmerman et al

26 Coda Cerva Overall, we show that standard genotoxicity tests can be applied for the safety evaluation of nanomaterials, although further investigation is necessary to understand how to predict which type of MWCNT is actually harmful.

Journal of Colloidal and Interface Science.

WP2Task 2.2aDeliverable 2.10Q&Ns: 1, 2, 4

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22 2014

Classification and Reporting of Nanostructured Silica Materials

Publication Rambabu Atluri, Keld Alstrup Jensen

4 NRCWE Nanostructured silica materials (NSMs) as an example, we highlight the importance of not only composition but also the structural complexity of NSMs for their identification and registration. We reviewed various forms of NSMs and for the first time, recommend a classification system by their composition, extent and location of surface treating agents. Bare silica nanoparticles are grouped to 1st generation NSMs (1G-NSMs), nanocomposites of silica with organic or inorganic as their secondary phase are in the 2nd Generation NSMs (2G-NSMs) and finally, nanocomposites of silica comprising both organic and inorganic as their counterparts as 3rd Generation NSMs (3G-NSMs). In addition, we have identified volume specific surface area (VSSA) as an additional identifier, and proposed a scheme for identification and reporting of NSMs. Our methodology of NSMs will enable scientists, industrialists and regulators to identify the nanomaterials systematically and guide them to identify their unique properties towards risk and hazard assessment.

Chemical Reviews WP2, Task 2.1Q&Ns: 1, 2

23 2014 Assessing Suitability of Analytical Methods to Measure Solubility for the Purpose of Nano-Regulation

Publication Ratna Tantra et al. 46, 22, 30, 43

NPL, UNAMUR, DLO RIKILT, UdL

Solubility is an important physicochemical parameter in nano-regulation. This review assesses potential techniques for the measurement of nanomaterial solubility and evaluates the performance against a set of analytical criteria (based on satisfying the requirements as governed by the cosmetic regulation as well as the need to quantify the concentration of free (hydrated) ions). Our findings show that no universal method exists. A complementary approach is thus recommended, to comprise of an atomic spectrometry based method in conjunction with an electrochemical (or colorimetric) method. This article shows that although some techniques are more commonly used than others, a huge research gap remains, related with the need to ensure data reliability. The importance of conducting appropriate validation studies is thus highlighted here.

Nanotoxicology WP5,Task 5.2

24 2014 Deposition of 30 to 150 nm Au particles in the airways of C57BL/6 mice

Presentation Kirsten Inga Kling et al 4 NRCWE The aim of this study is to quantify the regional air-way deposition efficiencies of nm- to μm-size particles in the often used C57BL/6 mouse model as using modern aerosol measurement devices for exposure measurement. Here we present the initial results from inhalation study using highly controlled 30 to 150 nm-size spherical and aggregate Au nanoparticles.We investigated the deposition efficiencies of high density spherical (30, 50, 70 nm) and lower density aggregated (30, 50, 70, 100 and 150 nm) gold particles. Groups of 9 mice were exposed head-only in body plethysmographs. Their respiration was monitored for 15 min prior to exposure (baseline) and during the 1.5 hr exposure. After exposure, all mice were anaesthetized. The mean inhaled volume was calculated based on respiratory frequency and tidal volume, and averaged 3.6 ± 1 L (SD), range 2.1-6.3 L. The large variation in respiratory volume suggests that respiration should be measured in inhalation exposure studies to estimate the deposited dose from experimental data.The detailed exposure characteristics will be presented including supporting studies on particle morphologies and effective densities and discussed with respect to the observed upper airway irritation. The regional deposition data will be discussed as function of particle size and morphology based on the results from over-view elemental mapping and the Au-concentrations in dissected organ fractions. Electron microscopy studies will reveal whether local differences in deposition occur in local biological structures such as the carina and different lung lobes.

Presentation during the 2014 Intl Aerosol Conference in Busan

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25 2014 Systematic Investigation of the Physicochemical Factors ThatContribute to the Toxicity of ZnO Nanoparticles

Publication Qingshan Mu et al 43 UdL ZnO nanoparticles (NPs) are prone to dissolution, and uncertainty remains whether biological/cellular responses to ZnO NPs are solely due to the release of Zn2+ or whether the NPs themselves have additional toxic effects. We address this by establishing ZnO NP solubility in dispersion media (Dulbecco’s modified Eagle’s medium, DMEM) held under conditions identical to those employed for cellculture (37 °C, 5% CO2, and pH 7.68) and by systematic comparison of cell−NP interaction for three different ZnO NP preparations. For NPs at concentrations up to 5.5 μg ZnO/mL, dissolution is complete (with the majority of the soluble zinc complexed to dissolved ligands in the medium), taking ca. 1 h for uncoated and ca. 6 h for polymer coated ones. Above 5.5 μg/mL, the results are consistent with theformation of zinc carbonate, keeping the solubilized zinc fixed to 67 μM of which only 0.45 μM is as free Zn2+, i.e., not complexed to dissolved ligands. At these relatively high concentrations, NPs with an aliphatic polyether-coating show slower dissolution (i.e., slower free Zn2+ release) and reprecipitation kinetics compared to those of uncoated NPs, requiring more than 48 h to reach thermodynamic equilibrium. Cytotoxicity (MTT) and DNA damage (Comet) assay dose−response curves for three epithelial cell lines suggest that dissolution and reprecipitation dominate for uncoated ZnO NPs. Transmission electronmicroscopy combined with the monitoring of intracellular Zn2+ concentrations and ZnO−NP interactions with model lipid membranes indicate that an aliphatic polyether coat on ZnO NPs increases cellular uptake, enhancing toxicity by enabling intracellular dissolution and release of Zn2+. Similarly, we demonstrate that needle-like NP morphologies enhance toxicity by apparently frustrating cellular uptake. To limit toxicity, ZnO NPs with nonacicular morphologies and coatings that only weakly interact with cellular membranes are recommended.

Chemical Research in Toxicology

http://pubs.acs.org/doi/abs/10.1021/tx4004243

WP2, 5TASKS 2.2, 2.4, 5.2 Q&Ns 2, 3, 6, 8, 9, 12

feb-14

27 2015 Selection of reference organic matter to analyze the colloidal 1 stability and ecotoxicity of multiwalled carbon nanotubes

Publication Cristina Cerrillo et al 51 TEKNIKER In the last few years the release of multiwalled carbon nanotubes (MWCNTs) into the environment has raised serious concerns regarding the induction of ecotoxic effects. The divergent results published on their experimental toxicity data pose a threat to their potential applications. Aquatic organisms represent one of the most important pathways for the entrance and transfer throughout the food webs in ecosystems. However, the strong hydrophobicity of carbon nanotubes (CNTs) usually results in agglomeration and settlement behaviors. On the other hand, the solution chemistries of aquatic environments influence greatly their stability and bioavailability. Several types of organic matter have been analyzed in the literature, demonstrating the promotion of the stabilization of CNTs, but influencing otherwise their toxicity results. The current demand for standardization of materials and procedures to assess their ecotoxicity requires the equilibrium between methodologies similar to realistic environmental conditions and also accessible for laboratories. An important aspect of these 2 requirements, which remain still unsatisfied, is the preparation of CNTs dispersions by means of synthetic or natural organic matter. The present study analyzes their influence in the agglomeration kinetics and ecotoxicity of MWCNTs. Inhibitory effects were studied using the key invertebrate organism in regulatory testing Daphnia magna.

Environmental Sience & Technology journal

WP 4.6

28 20142015

Genotoxic effects of nanofibrillar cellulose

presentation Kukka Aimonen et al 49 FIOH Nanofibrillar cellulose (NFC) is among the most promising innovations in the forestindustry. NFC possesses unique properties compared with similar materials of larger size, but it is unclear whether this also results in differential toxic properties. Toxicity studies on nanocellulose materials are still scarce, and it is important to investigate the possible toxicity of NFC at an early stage of product development. The objective of the present study was to examine in vitro and in vivo the potential genotoxicity of four NFC materials (fibril diameter: 2-15 nm; length: several micrometers) and a bulksized cellulose material.

Nanosafety Forum for Young Scientist

Q&N 15, 17 8th – 9th of October in Syracuse, Italy

SENN201512-15 April 2015 Helsinki, Finland

29 2014 Towards a harmonized Safety Assessment of Nanomaterials

Presentation Carol Aristimuño 11 GAIKER Traditional toxicology´s main concern is to study the adverse effects of chemicals on a given set of known cytological, physiological and morphological parameters. However, these set of well defined studies do not take into account the special nature of nanomaterials, such as their small size, aggregation capacity and reactivity. These new properties may be able to alter the absorption and transport capacity of nanomaterials across membranes. There is also a potential for nanomaterials to accumulate in organs, enter into blood circulation or even cross the placental-foetal barrier.

Opentox conference

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30 2014 How horizon scanning can contribute to the safe(r)-by-design concept

Presentationposter

Christian Micheletti et al

41 VN There is a growing regulatory body addressing the manufactured nanomaterial (MNMs) Environmental, Health, and Safety (EHS) issues, which is promising to support a sustainable innovation driven by nanotechnology. However, the lack of proper understanding of the relevance of the available EHS data for regulatory purposes, is limiting the nanotechnology innovative potential, with impacts on the economy. Besides these short term issues, there is the need to tackle the necessity of being prepared for the foreseen multifaceted nanotech uses [1]. The case-by-case approach that is currently applied to nanosafety is not sufficient to balance safe use and economic development. This approach is also not well suited to address the needs of the different steps in the innovation chain.

NanotechItaly 2014 WP6 – Task 6.1 Q&N:15, 16

31 2014 IT on 90 day oral toxicity study on SAS within the NANoREG project

Presentation Francesco Cubadda 17 ISS A repeated-dose 90-day oral toxicity study in rat on synthetic amorphous silica (SAS) has been carried out as Task 4.5.5 within WP4 of the FP7 project NANoREG on the basis of the OECD TG 408. This protocol has been considered by EFSA as the minimum requirement to identify hazards and obtain dose-response data to characterize the hazards of nanomaterials relevant for food safety (EFSA, 2011).

4th meeting of the EFSA Scientific Network for Risk Assessment of Nanotechnologies in Food and Feed EFSA

21-21 October 2014, Parma

32 2014 Speciation with AGNES: the case of ZnO nanoparticles"

Presentation J. Galceran et al 43 UdL AGNES is an emerging electroanalytical technique specially designed to determine free metal concentrations. It has proved to be specially indicated to contribute to the current debate on whether the toxicity of ZnO nanoparticles stems from its nanoparticulate nature or from the Zn ions generated in their dissolution. One key aspect is that AGNES can measure in a dispersion without any previous separation. AGNES measurements confirm the expected impact of nanoparticle radius on the solubility and allow to establish their surface energy. The co-existence of humic acid and nanoparticles does not alter the free Zn equilibrium concentration, but the solubility is affected. In media used for toxicological tests with mammal cells, AGNES mesurements pin-point transformation products. AGNES can also access to kinetic information, because the measurements along ZnO dissolution can be made in a much shorter time (say every 5 minutes) than competing techniques such as dialysis (24 h) or centrifugation + ICP-MS (say 1 hour) and avoid artifacts derived from very small nanoparticles not being totally excluded in some separations.

BNASS / TraceSpec Tandem Conference Aberdeen, Scotland

WP2, WP5; Task 2.3, 2.4 and 5.2, Q&Ns: 2, 3, 6, 8, 9 and 12

31st August - 4th September 2014

33 2014 Physical characterization of nanomaterials in dispersion by transmission electron microsco-py in a regulatory framework

Publication Jan Mast et al 26 CODA-CERVA TEM is one of the few techniques that can identify nanoparticles ac-cording to the current definitions. This chapter focuses on the different steps re-quired to analyze dispersed nanomaterials by TEM. Methodologies to obtain ho-mogeneous and stable dispersions of colloidal nanomaterials and powders are presented. The preparation of TEM specimens to obtain a representative distribu-tion of particles on the grid is discussed. The application of TEM imaging meth-ods, electron diffraction and analytical TEM to obtain complementary information on the size, morphology, crystallographic structure, electronic structure and com-position of nanomaterials is reviewed. In a qualitative TEM analysis the key properties of the physical form of the na-nomaterial under which it is exposed to in vitro and in vivo test systems are de-scribed based on TEM micrographs. Subsequently, a quantitative analysis which includes detection, classification and measurement of primary particle properties, and validation of the measurement results can be performed. The possibility to ex-tract 3D information by fractal analysis of electron micrographs of aggregated na-nomaterials with a fractal-like structure is explored.

Electron Microscopy of Materials

34 2014 Nanomaterial and Food Safety: toxicological evaluation of food additive E551 in NANoREG project

News letterPublication

Francesca Maranghi - Roberta Tassinari

17 ISS Synthetic amorphous silica (SAS), the silica dioxide (SiO2 NM-203, JRC repository) – is the food additive E551 which has been selected as nanomaterial model in the Nanogenotox Joint Action - (http://www.nanogenotox.eu/) for oral testing due to its relevance in food safety and successfully tested in short-term oral studies in rodents. E551 is widely employed as clearing agent, stabilizer, carrier, anticaking agent and flow aid.

EFSA Italian Focal Point http://www.iss.it/efsa/index.php?lang=1&anno=2014&tipo=28)

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35 2014 NANoREG first highlights and challenges

Presentation by poster

Consiglio Nazionale delle Ricerche et al

31 CNR NANoREG is the first FP7 project aiming to deliver the answers needed by industry, regulators and legislators to questions related to nanomaterials’ (NM) environmental, health and safety by linking them to a scientific evaluation of data and test methods. The Italian partnership within the NANoREG project consortium encompasses 5 partners (CNR, ENEA, IIT, ISS, VN) and 3 third parties (UniTO, UniTorVergata, INAIL), committed in different activities that together cover all the project pillars. We present here the preliminary results obtained and the challenges encountered during the first 18 months. The Ministero della Salute, acting as National Coordinator (NC) supported this process both at the national and at the European level.

NanotechItaly 2014, Venice

WP1, WP2, WP4, WP5, WP6, WP7

26-28 November 2014

36 2014 Unmasking nanoparticle subcellular localization artifacts by electron tomography

Publication Ivan Mičetić et al 41 VN Biological effects of engineered nanoparticles (NPs) are under constant investigation due to their peculiar physico-chemical properties and promising roles in several applications. NP cellular uptake and intracellular distribution are pre-requirements for understanding their mechanisms of action. Much effort has been made trying to elucidate intracellular pathways and fate of NPs in exposed biological samples. Due to their small size, the method of choice for NPs determination and localization is given by transmission electron microscopy (TEM). However, high resolution localization of NPs at subcellular level can be impaired by artifacts due to the complex sample preparation procedures. NPs may detach from ultrathin sections and be dragged by the microtome knife to other locations. Another source of possible contamination with ex situ NPs is the water on which freshly cut sections float waiting to be collected on TEM grids. The location of displaced NPs can be erroneously assumed as correct during sample observation. Consequently, a method for identifying possible erroneous NP localization in thin sections is mandatory. We show that depth-sectioning samples by electron tomography allows to successfully discriminate NPs belonging to the biological sample from those accidentally deposited on the sections. Electron tomography of resin embedded sections at low resolution is a relatively low cost, medium effort method that should complement TEM every time an uncommon subcellular NP localization is observed or claimed.

37 2014 Interaction of Nanoparticles with the immune system

Publication Diana Boraschi, Paola Italiani

31 CNR Interaction with the immune system is among the first events that take place upon human exposure to nanoparticles. The immune system has the role of recognising possible dangers and eliminating them, so as to preserve the body integrity. Thus, the nanoparticle--–immune system interaction often results in a defensive reaction aiming at eliminating the particle. Phagocytic cells, such as neutrophils and, most importantly, macrophages, are the immune cells that are mainly involved in nanoparticle recognition and elimination, via an array of mechanisms such as opsonisation, extracellular trapping, enzymatic degradation, phagocyotosis and endocytosis. A major issue in the interaction of nanoparticles with the immune system is their immunosafety, i.e. inability to trigger an excessive immune response that may derange into a pathological situation. In addition, in the case of nanomedicines, a significant challenge is the design of nanoparticles able to escape immune recognition, thereby successfully reaching their tissue target. In this perspective, targeting the phagocytic immune cells can be achieved very efficiently by nanoparticles, a strategy that has worked excellently for many year in vaccination and that is also becoming an effective diagnostic and therapeutic and diagnostic strategy in inflammation--–based diseases such as chronic inflammatory diseases, autoimmunity, and degenerative diseases.

Nanoparticles for the Delivery of Nanothera-peutics. J. Ramey, and L. Forrest (Eds.). Future Science e-book series

WP5,tasks 5.3-5Q&M: 9

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38 2014 Human primary monocytes as model for evaluating the effects and safety of engineered nanoparticles

Presentation Boraschi, D. 31 CNR Monocytes/macrophages have a central role in initiating defensive responses to external or endogenous challenges. Thus, monocyte reactivity is paradigmatic of the immediate host reaction to engineered nanoparticles (NPs), and exploitable both for evaluating their safety and for identifying possible use in nanomedicine, in particular vaccination. The interaction of NPs with human monocytes/macrophages can be modelled in vitro in a series of relevant and representative systems.We conclude that the use of a valid and representative in vitro model of human inflammation may allow us to realistically investigating the effects of NPs on inflammatory/innate immune responses, thereby being useful to predicting the immunological risk posed by engineered nanomaterials as well as for testing the possible immuno-stimulating effects of NP-based adjuvants.

2014 CFB Agricultural Biotechnology Symposium on “Biomaterials and Immune Modulation”, Seoul (Korea),

WP5,tasks 5.3-5Q&M: 9

November 5, 2014

39 2015 Characteristics of airborne gold aggregates generated by spark 1 discharge and high temperature evaporation furnace: mass-mobility 2 relationship and surface area

Publication Svensson, C.R. et al 37 LTH A common particle type in the air, both in indoor and outdoor environments, is that of aggregates – irregular particles built up by smaller primaries/monomers, kept together by weak or strong forces. Due to their size and physical characteristics, they are of special interest in the fields of toxicology and environmental health, but also in the field of nanotechnology. However, characterizing aggregates is not entirely straightforward. The performance of a new custom-built spark discharge generator (SDGc) was investigated and compared to a commercially available spark discharge generator (SDGp) and a high temperature evaporation furnace (HT) regarding the output and characteristics of gold aggregates.The SDGc has a greater output regarding mass and estimated total surface area compared to the commercial SDGp. The characteristics of Au aggregates generated using the SDPp differ from the HT furnace with respect to the effective density, mass-mobility relationship, primary particle size, shape factor and size distribution.

Journal of Aerosol Science and technology

WP2

40 2014 Inflammatory effects of nanofibrillar cellulose

Presentation Sara Vilske et al 49 FIOH Nanofibrillar cellulose (NFC) is among the most promising innovations in the forest industry. NFC possess unique properties compared with similar materials of larger size, but it is unclear whether these properties could cause adverse health effects. Thus, it is important to investigate the safety of NFC. We examined the potential inflammatory effects of four NFC materials (fibril diameter: 2-15 nm; length: several micrometers). Immunotoxic effects in vitro were investigated in THP-1 derived macrophages at 1, 10 and 100 μg/ml. Cytotoxicity, mRNA expression and protein secretion of pro-inflammatory cytokines IL-1β and TNF-α were assessed after 3, 6, and 24 h. Inflammatory effects in vivo were studied in C57BL/6 mice after a single pharyngeal aspiration of 10 and 40 μg/mouse NFC. Influx of inflammatory cells, mRNA expression levels of relevant cytokines and histopathological changes in lungs were investigated 24 h and 28 days after the administration. Two NFCs showed activation of inflammatory response. One of these induced a decrease in cell viability and up-regulated mRNA expression and protein secretion of pro-inflammatory cytokines in vitro. Furthermore, the recruitment of neutrophils and eosinophils in BAL and lung tissue, and expression of several cytokines was found in response to the NFC after 24 h in vivo. The other material did not activate macrophages in vitro, however, the effects in vivo were somewhat comparable to the ones caused by the first material. None of the materials caused long-term toxic effects. Our study provides new information about the immunotoxicity of NFC and thereby contributes to risk assessment of these materials.

SENN2015 Helsinki - Finland 12-15 April 2015

WP 4 task 4.) WP 5 task 5.5.Q&Ns: 15, 17.

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41 Genotoxicity of nanofibrillar cellulose

Presentation Kukka Aimonen et al 49 FIOH Nanofibrillar cellulose (NFC) is among the most promising innovations in the forest industry. Due to its unique properties, NFC has wide-variety of application possibilities. Toxicity studies on nanocellulose materials are still scarce, and it is important to investigate the safety of NFC at an early stage of product development. The objective of the present study was to examine the potential genotoxicity of four NFC materials (fibril diameter: 2-15 nm; length: several micrometers) and a bulk-sized cellulose material.In vitro genotoxicity was assessed in human bronchial epithelial (BEAS 2B) cells by the single cell gel electrophoresis (comet) assay (24-h exposure, doses 9.5-950 µg/ml) to detect DNA strand breakage and by the cytokinesis-block micronucleus (MN) assay (48-h exposure, doses 25-1250 µg/ml) to show possible chromosomal damage.For genotoxicity assessment in vivo, the comet assay was performed on lung cells and bronchoalveolar lavage (BAL) fluid cells and the MN assay on bone marrow polychromatic erythrocytes, after single pharyngeal aspiration to female C57BL/6 mice (24-h and 28-d follow up; doses 10, 40, 80 and 200 µg/mouse).The tested NFC materials did not induce significant DNA strand breakage or chromosomal damage in vitro. One of the tested nanocelluloses induced significant genotoxic effects in vivo, as determined by the comet assay 24 h after the exposure. None of the NFCs was shown to possess systemic genotoxic properties as measured by the micronucleus assay.Our study contributes to the risk assessment of nanocellulose by providing new information about the genotoxicity of the NFC materials.

SENN2015 Helsinki - Finland 12-15 April 2015

WP 4, task 4.7,WP 5 task 5.5Q&Ns:6, 8,10.

42 2015 Metrological characterisation of the size distribution of airborne nanoparticles generated from nanomaterials in the form of colloidal suspension

Presentation Charles Motzkus 47 LNE Key words: Number size distribution, Airborne nanoparticles, Nanomaterials, Scanning Mobility Particle Sizer http://www.metrologie2015.com/

Deliverable D2.10 of the WP2 international

congress of metrology 2015

43 2015 From scientific evidence to regulation: the NANOREG EU project

Presentation Maria Letizia Polci NC/IT A serious threat to the capitalization of the innovative and economic potential of nanotechnology is the limited understanding of the Environmental, Health and Safety (EHS) aspects of nanomaterials (NMs). This lack of knowledge leads to uncertainty on how to judge the EHS aspects of these materials in a regulatory context. This has a negative impact on the investment climate and on societal appreciation of products containing NMs. NANoREG is the first FP7 project aiming to deliver the answers needed by industry, regulators and legislators to questions related to nanomaterials’ environmental, health and safety by linking them to a scientific evaluation of data and test methods. The NANoREG unique approach includes four stakeholder groups involved in the same project (regulators; industry; researchers; NGOs, the public, and others). There is a special focus on the needs and challenges of regulators, based on the identified knowledge gaps and with the overall objective being the elimination of these uncertainties. On one hand research efforts are devoted to fill in those identified gaps and on the other a framework is developed for testing the EHS aspects and for the assessment and management of the risks.To deal with the uncertainties that innovations give rise to, a safe-by-design approach is considered, aimed at integrated and iterative process where safety and functionality are weighed, and eventually reach a more efficient process from design to application.

Symposium on the health protection of the Nanomaterial Workers. International Commission on Occupational Health. Rome

WP7 26 February 2015

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44 2015 Visualization of nanocellulose in biological tissues using a biotinylated cellulase CBM domain, EXG:CBMAuthors: Kristina Bram Knudsen et al

Publication Kristina Bram Knudsen et al

4 NRCWE Nanocellulose is regarded as a very promising innovation with a wide variety of applications. Production of nanocellulose on an industrial scale may possibly impact people in the working environment as well as the environment. Methods Nanocellulose consists primarily of glucose moieties and is difficult to detect in biological tissues. We have developed a simple and effective method for specific and sensitive detection of celluloses fibers, including nanocellulose in biological tissue, using a biotinylated, modified carbohydrate binding module of beta-1,4-glycanase from the bacterium Cellulomonas fimi (EXG:CBM) synthesized in Eschericia coli. EXG:CBM was purified and biotinylated at two sites.

The publication shows that the biotinylated EXG:CBM can be used to visualize different kinds of cellulose including several different nanocelluloses both by fluorescence- and by horse radish peroxidase (HRP) -tagged Avidin labeling. HRP-EXG:CBM enzyme was used to visualize cellulose fibers in cryopreserved and paraffin embedded lung tissue from mice dosed with 10-200 µg/mouse of 5 different cellulose fibers by aspiration.

The current method represents a new concept for the design of simple, robust and specific detection methods for nanomaterials which are otherwise difficult to detect. Protein-based detection of nanomaterials may enable development of quantitative assays for detection of specific nanomaterials in the future.

P&FT WP 4 task 4.5.1Q 10 and 14

45 2015 Freshwater dispersion stability of PAA-stabilised cerium oxide nanoparticles and toxicity towards Pseudokirchneriella subcapitata

Publication Andy Booth 44596

SINTEF, SPBfr

An aqueous dispersion of poly (acrylic acid)-stabilised cerium oxide (CeO2) nanoparticles (PAA-CeO2) was evaluated for its stability in a range of freshwater ecotoxicity media (MHRW, TG 201 and M7), with and without natural organic matter (NOM). In a 15 day dispersion stability study, PAA-CeO2 did not undergo significant aggregation in any media type. Zeta potential varied between media types and was influenced by PAA-CeO2 concentration, but remained constant over 15 days. NOM had no influence on PAA-CeO2 aggregation or zeta potential. The ecotoxicity of the PAA-CeO2 dispersion was investigated in 72 h algal growth inhibition tests using the freshwater microalgae Pseudokirchneriella subcapitata. PAA-CeO2 EC50 values for growth inhibition (GI; 0.024 mg/L) were 2–3 orders of magnitude lower than pristine CeO2 EC50 values reported in the literature. TOF-SIMS analysis of algal cell wall compounds indicated three different modes of action, including a significant oxidative stress response to PAA-CeO2 exposure.

Science of the Total Environment

Task 2.4 and 2.6

46 2015 Hazard identification of the food additive E551 - synthetic amorphous SiO2: the EFSA approach in NANoREG project

Poster presentation

Maranghi Fet al.

17 ISS NANoREG Task 4.5.5 performed a 90-day oral toxicity study, on the basis of the OECD TG 408, for hazard identification of SAS nanoparticles. The test material is a pyrogenic amorphous SiO2 (NM-203, JRC repository); biodistribution and toxicokinetics profile were characterized in the Nanogenotox Joint Action (http://www.nanogenotox.eu/). Sprague Dawley rats of both sexes were used. The dose levels selected were 0 (vehicle only – distilled water), 2, 5, 10, 20 and 50 mg/kg body weight a day, administered orally by gavage, 5 days/week for 90 days. Silicic acid, the soluble counterpart of SAS, was administered at 50 mg/kg body weight a day for comparative tissue biodistribution. Rats were fed a standard diet showing the lowest silica concentration.The results of the study will be used to identify a NOAEL/LOAEL for regulatory purposes. The Benchmark Dose lower confidence limit (BMDL) will be also calculated. The preliminary data showed no marked general toxicity in both sexes at the selected dose levels, although gender-specific differences both in immune and biochemical endpoints are observed. As commonly reported with nanomaterials, this study highlights again the difficulty in identifying a clear dose-response relationship for key endpoints, which would help for risk assessment of SAS.

EFSA Meeting “Shaping the future of food safety, together”

Task 4.5.5 14 October 2015

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47 2015 Techniques and protocols for dispersing nanoparticle powders in aqueous media – (what) are we ready to harmonise?

Publication Nanna B. Hartmann, et al.

4 NRCWE Appropriate ways of bringing engineered nanoparticles (ENPs) into aqueous dispersion is a main obstacle for testing and thus for understanding and evaluating their potential adverse effects to the environment and human health. Harmonisation and standardisation of dispersion methods applied in mammalian and ecotoxicity testing are needed to ensure a comparable data quality and to minimise test artefacts caused by modifications of ENPs during the dispersion preparation process. This paper discusses the essential parameters in dispersion protocols for ENPs. The parameters are identified from individual scientific studies and from consensus reached in larger-scale research projects and international organisations. We propose a step-wise approach to develop tailored dispersion protocols for ecotoxicological and mammalian toxicological testing of ENPs. The recommendations of this analysis may serve as a guide to researchers, companies, and regulators when choosing and evaluating the appropriateness of dispersion methods applied in mammalian and ecotoxicity testing. However, additional research is needed to further document the protocol parameters and investigate to what extent different stock dispersion methods do indeed affect the ecotoxicological and mammalian toxicological responses of ENPs.

Journal of Toxicology and Environmental Health, Part B: Critical Reviews

WP2, T2.4 Q&N 10

21 March 2015

48 2015 Assessment of SiO2 nanoparticle systemic genotoxicity in rats after sub-chronic oral exposure

Poster presentation

Cordelli E, et al. 18 ENEA A 90-day oral toxicity study for hazard identification of SAS (NM-203, the pyrogenic form of the food additive E551) has been carried out at the Italian National Institute of Health on the basis of the OECD TG 408. The study includes the assessment of genotoxicity in different organs. Male and female Sprague Dawley rats were exposed daily by gavage to 0, 2, 5, 10, 20, or 50 mg/kg b.w. SiO2 nanoparticles for a total of 90 days. At the end of treatment, comet assay was applied to assess DNA damage in bone marrow, intestine, colon, kidney, liver, spleen, ovary of female rats and in blood, bone marrow, spleen and testis of male rats. Gene mutation and chromosome damage were measured in erythrocytes of male rats at an intermediate exposure timepoint and at the end of exposure to 2, 20 and 50 mg/kg, by the flow cytometric Pig-a assay and by the micronucleus test, respectively. Finally, micronucleus test was applied to detect chromosomal damage in colon cells of female rats. General toxicity showed no differences among groups in body weight gain and feed consumption in both sexes. Analyses of genotoxicity are still in progress; preliminary data show spotted increases of DNA damage in bone marrow and a slight but significant dose related increase of DNA damage in spleen cells of male rats. In the same rats, no increase of DNA damage was observed in blood, liver and testis cells. In females, spotted, dose unrelated, increase of DNA damage was observed in intestine, kidney, bone marrow, spleen and ovary cells. No increase of Pig-a mutant cell frequencies and of micronucleated reticulocyte percentages were observed at any tested timepoint.

2015 Annual Congress of the European Environmental Mutagenesis and Genomics Society

WP4, T4.5.5Q&N6,7,9,10,14

23-27 August 2015 Praque - Czech Republique

49 2015 Role of electrostatics in silica nanoparticle effect studies

Presentation Mikael Lilja, et al. 52 Chalmers The present study deals with physicochemical characterization. We aim to extend this work, in a long perspective, to structure-activity relationships which can perhaps help answer questions related to driving forces for persistence and long-term effects on organisms. A nanosized material can be transported in very different ways from bulk materials, in the body and in the environment. #7 It is likely possible to extrapolate some results from relevant model surfaces (of bulk or nanostructured materials) to predictions on kinetics and fate. Examples of characteristics which should be avoided for any MNM emitted to the environment:

?? ?? ??

50 2015 LCA of alumina nanofluid used as coolant in Power ElectronicTraction system

Presentation Simona Scalbi, et al. 18 ENEA The abstract deals with a case study highlighting the importance for comparing the environmental performance of emerging nanotechnologies and products with conventional technologies.

SETAC Europe 25th Annual Meeting in Barcelona, Spain.

Task 1.4 3-7 May, 2015

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51 2015 Towards the identification of suitable models for nanoparticle-mediated inhalation cytotoxicity

Poster presentation

Karen McQuillan et al TCD Variance in experimental conditions, nanoparticle concentration and type often precludes their direct comparison, and can sometimes result in conflicting evidence. This latter issue combined with the lack of standardization and appropriate in vitro inhalation toxicity models available are contributing to the current scientific discussion and disagreement on the determination of NP-mediated cytotoxicity.Inhaled or aerosolised NPs have been associated with a variety of respiratory disorders including: pulmonary fibrosis and granulomas, silicosis, chronic obstructive pulmonary disease and lung cancer1.In order to adequately assess the biological effects of NPs on the alveolar respiratory epithelium, in vitro models must be employed that closely mimic the in vivo physiological interactions. These models must reflect the natural structure of the alveolar epithelium and its function as a barrier between the bloodstream and the external environment.This study is focused around Air Liquid Interface (ALI) tissue culture conditions as a representative model to mimic the alveolar epithelial barrier. Cells grown in ALI conditions are in direct contact with the gas phase, allowing a more natural model of NPs interaction, compared to traditional submerged culture.The outcome of this work aims to provide some qualitative and quantitative evidence in support to the definition of future inhalation cytotoxicty models.

EVOLVE Biomed 2015 (http://www.evolvebiomed2015.com

Task 5.3 Q&Ns 6, 7

52a 2015 Visualization of nanocellulose in biological tissues using a biotinylated cellulase CBM domain, EXG:CBM

Presentation Kristina Bram Knudsen et al

NRCWE Nanocellulose consists primarily of glucose moieties and is difficult to detect in biological tissues. We have developed a simple and effective method for specific and sensitive detection of celluloses fibers, including nanocellulose in biological tissue, using a biotinylated, modified carbohydrate binding module of beta-1,4-glycanase from the bacterium Cellulomonas fimi (EXG:CBM) synthesized in Eschericia coli.We demonstrated that the biotinylated EXG:CBM can be used to visualize different kinds of cellulose including several different nanocelluloses both by fluorescence- and by horse radish peroxidase (HRP) -tagged Avidin labeling. HRP-EXG:CBM enzyme was used to visualize cellulose fibers incryo preserved and paraffin embedded lung tissue from mice dosed with 10-200µg/mouse of 5 different cellulose fibers by aspiration. The current method represents a new concept for the design of simple, robust and specific detection methods for nanomaterials, which are otherwise difficult to detect. Protein-based detection of nanomaterials may enable development of quantitative assays for detection of specific nanomaterials in the future.

7th International Symposium on Nanotechnology, Occupational and Environmental health in South Africa

Task 4.5.1Q&Ns: 10 and 14

18th-22nd of October

52b 2015 A One Year Toxicity Study Following Pulmonary Exposure to Twelve Different Carbon Nanotubes

Presentation Kristina Bram Knudsen et al

4 NRCWE Objective:Pulmonary exposure to 12 different multi-walled CNTs was tested in a one year in vivo toxicity study in mice. Single doses of 54 µg CNTs were instilled into lungs of C57BL/6J mice by intra tracheal instillation. We assessed genotoxicity (DNA strand breaks) in liver tissue and lung histopathology in tissue from mice after one year. Fifty-four µg correspond to a 3 x ‘safe’ lifetime exposure in mice and therefore considered are levant max dose. OECD reference materials NM401, 402, and 403 were included along with CNTs with different surface functionalizations(pristine, -OH or -COOH).Results and

The CNTs were well distributed throughout the lung one year after instillation and the CNTs were still visible after one year. Based on the histopathological evaluations, we found no consistent lung fibrosis, pleural fibrosis, nor lung tumors or any mesotheliomas. Instead, great lymphocyte and macrophage infiltration, cells with CNT material and granuloma formations were observed.Previously published in vitro studies revealed no cytotoxicity and low genotoxicity related to these CNTs (Jackson P. et al. Environ Mol Mutagen2015, 56:183-203). The results suggest that pulmonary exposure to these CNTs induces little fibrosis. A possible explanation could be the observed granuloma formation, which encapsulates the nanomaterial, shields the surrounding tissue from the nanomaterials.

7th International Symposium on Nanotechnology, Occupational and Environmental health in South Africa

Task 4.5.1Q&Ns: 10 and 14

18th-22nd of October

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53 2015 SOLUBILITY5 SOLUBILITY PART 1: OVERVIEW

Publication R. Tantra et al 46 NPL This chapter serves as a starting point for researchers to review the different analytical methods available to measure solubility of nanomaterial (in support of nano-regulation). Our findings indicate the wide variety of methods available, with the capability to measure total dissolved species or free ions but not both. Electrochemical and colorimetric based detection schemes are suitable to measure free ions (and labile fractions) whilst atomic spectrometry based techniques are more suited to measure total dissolved species. The choice of method will be dependent on the nanomaterial sample and the specific scenario that the researcher is faced with. Nonetheless, the combination of a separation with a quantification analysis platform is needed and often can be achieved by integrating individual separation method with a separate quantification method.

An important point that has been highlighted in this chapter is the need to ensure that method validation takes place for the purpose of data reliability. The case study presented explores the feasibility for CE-conductivity microchip for use to measure nanomaterial solubility. Although the study shows that this technology is suitable for detection of free ions, its use for quantitative purpose remains questionable. Obviously, there is a need to develop this state of the art technology further i.e. in establishing devices that are highly reliable and robust.

The manuscript is an extension of the solubilty review that has been published recently in Nanotoxicology and hence will answer the same regulatory questions as the previous manuscript.

54 2015 SOLUBILITY6 SOLUBILITY PART 2: COLORIMETRY

Publication R. Tantra et al 46 NPL Out of the many solubility methods that has been reviewed, the method based on electrochemical and colorimetric assays are worthy of note for the measurement of free ions. In relation to electrochemical methods, commercially available ion selective electrodes (ISEs) are an attractive option. The ISE is an established technology that has been around for several decades and measurement is very simple and fast, often involving the immersion of electrodes into a sample solution to acquire the reading. However, to date no commercial ISE exists for the quantification Zn2+; this would be particularly useful to measure the solubility of nanomaterial that contains zinc e.g. ZnO. In this case, colorimetry based assay is an attractive choice. Colorimetry has many of the advantages associated with ISE, in that it is affordable and simple to use to measure quantification of free ions. It is particularly attractive as it requires the use of an ultraviolet-visible (UV-vis) spectrometer, an instrument that nearly every laboratory has. In the book chapter, we detailed the colorimetry assay method and presented a case study to measure the amount of dissolved free zinc (Zn2+ ) from ZnO NM 110 nanomaterial when exposed to digestive juices. The exposure of this nanomaterial to digestive juices is carried out in order is important to follow the fate of the nanomaterial upon accidental ingestion in humans. The study established that there is a correlation between the amounts of free zinc that arises from the dissolution of ZnO nanomaterial w.r.t particle concentration. In addition to colorimetry based assays, Scanning Electron Microscopy (SEM), a technique that offers complementary information, have been used to establish the presence of the NM 110 at different stages of digestion. SEM findings indicate that all of the NM 110 dissolved during the acid digest. Finally, methods and results associated with mandatory tests to NanoReg are presented are supplementary material in the chapter.

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55 2015 Testing Strategy to Measure Exposure throughout the Life Cycle

Presentation A. Masion et al 7 CNRS CEREGE To address the concerns about the risks associated with nanotechnologies, regulations agencies are seeking tools for reliable and efficient decision making. While considerable efforts are undertaken to characterize possible hazards, the exposure to nanomaterials over the entire life cycle still remains a somewhat neglected research domain. However, meaningful risk assessment requires a reliable high-quality characterization of the exposure.The EU project NanoReg addresses this issue in a regulatory approach. The work package "Exposure through Life Cycle Analysis" aims at: • Characterizing real exposures (intensity and frequency) to humans (workers and consumers) and the environment during the entire life cycle of nanomaterials.• Providing companies and legislators with a set of tools for risk assessment and decision making for the short to medium-term, by gathering data and performing pilot risk assessment, including exposure monitoring and control, for a selected number of manufactured nanomaterials (MNMs) used in products.• Developing, for the long-term, new testing strategies adapted to a high number of MNMs for many factors susceptible to affect their environmental and health impact.The various tasks in this work include identifying high release scenarios and data gaps; characterize nanomaterial release qualitively and quantitatively with harmonized testing procedures relevant to all stages of the life cycle; develop predictive exposure models; assess risk management measures.This paper shows how the work developed within the NanoReg project addresses major knowledge gaps in the exposure assessment.

Workshop in Arlington

56 2015 The development of a two-box nano-specific exposure model

Presentation Laura MacCalman 9 IOM Background: The risk assessment of engineered nanoparticles (ENP) requires information on the particle size distribution (PSD) from personal exposure data that can be used in lung deposition models. It is often difficult, if not impossible, to obtain this information for ENP and as such exposure models are often used. However, there are very few models which quantitatively assess exposure to nanoparticles. We report on the development of a two-box nano-specific exposure model, developed as part of the NANoREG project.Methods: The exposure model follows the two-box theory, one box (near-field) around the worker (the primary exposure of interest in occupational research), and the other being the rest of the room (far-field). The model accounts for diffusion, dispersion and dilution losses, as well as the agglomeration of nanoparticles for specific exposure scenarios (room dimensions, ventilation, use of local controls and emission rate). Results: The model has been tested using published studies of exposure measurement as well as newly generated data and has performed well in the prediction of exposure levels. It is being further validated via experiments specifically designed to investigate the behaviour of particles over time and space, under various conditions.Conclusions: This exposure model can be used to estimate personal exposure for a given scenario and also make predictions about the effect of modifying the exposure scenario. This could allow for the evaluation of scaling-up of processes and the influence of various control measures. This model will be an important tool in the risk assessment of nanoparticles.

Conference NanOEH, Johannesburg

WP3, Q12 October 2015

57 2015 Environmental assessment of solid sorbents towards 'Safe by Design'

Presentation Andy Booth 44 SINTEF Many NMs elicit negative impacts on a range of freshwater, marine and soil organisms. However, understanding NM environmental fate and behaviour is necessary to assess the potential for exposure and risk. A simple strategy for the assessment of NM fate and behaviour in freshwater environments, and how this can be used for selecting relevant species for further toxicity (hazard) evaluation towards risk assessment, is presented. Ten carbon nanotubes (CNTs) and 3 TiO2 NMs representing candidate solid nano-sorbent materials for potential application in CO2 capture technology were selected. Studies have been conducted to investigate how physicochemical properties influence NM dispersability (concentration) and dispersion stability in aqueous media relevant to freshwater environments. A standard method for the dispersion of NMs in aquatic media has been developed to permit reproducible and comparable conditions for different test materials. A basic strategy for using such environmental data in a 'Safe by Design' approach to material development and selection will also be outlined.

Conference:European Materials Research Society (Fall Meeting), Conference and Exhibition,Warsaw, Poland

WP2, task 2.4.Q2,4,5,12

15-18th Sept 2015

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58 2015 Pooling and analysis of published in vitro data: A proof of concept study for the grouping of nanoparticles

Publication Myrtill Simkó et al 8 AIT The study aim was to test the applicability of pooling of nanomaterials-induced in vitro data for identifying the toxic capacity of specific (SiO2, TiO2, ZnO, CuO, CeO2 and CNT) nanoparticles (NP) and to test the validity for grouping. Publication selection was based on specific criteria regarding experimental conditions. Two relevant biological endpoints were selected; generation of intracellular reactive oxygen species (ROS) and viability above 90%. The correlations of the ROS ratios with the NP parameters size, concentration, and exposure time were analysed. The obtained data sets were then analysed with multiple regression analysis, ANOVA and the Tukey post-hoc test.

The results show that this method is applicable for the selected metal oxide NP, but might need reconsideration and increase of the data set for CNT. Several statistically significant correlations and results were obtained and thus validating the method. Furthermore, the relevance of the combination of ROS release with a cell viability test was shown. The data also show that it is recommendable to compare ROS production of professional phagocytic with non-phagocytic cells.

In conclusion, this is the first systematic analysis showing that pooling of available data into groups is a useful method for data evaluation leading to an insight of NP induced toxicity in vitro,independent of the biological endpoint.

International Journal of Molecular Sciences

Tasks 1.3; 1.4; 1.7; Q&N: 4

59 2015 New steps towards the standardization of nanoecotoxicity testing: natural organic matter ‘camouflages’ the adverse effects of TiO2 and CeO2 nanoparticles on green microalgae

Publication Cristina Cerrillo et al 51 Tekniker The emission of CeO2 and TiO2 nanoparticles (NPs) into the environment is raising serious concerns about their emerging adverse effects on environmental and human health in the last few years. Aquatic organisms constitute one of the most important pathways for their entrance and transfer throughout the food webs, and the pressing need for standardization of methods to analyze the ecotoxicity of these NPs requires aquatic media which represent realistic environmental conditions. The present study aims to determine the usefulness of Suwannee River natural organic matter (SR-NOM) in the assessment of the agglomeration kinetics and ecotoxicity of CeO2 and TiO2 NPs towards green microalgae Pseudokirchneriella subcapitata. As observed for other algal species and types of natural organic matter in the literature, SR-NOM alleviated the adverse effects of NPs on algal growth, completely in the case of TiO2 NPs and partially in the case of CeO2 NPs. Furthermore, it markedly increased the stability of the NPs in algal medium, which led to a better reproducibility of their toxicity testing. The electrophoretic mobility provided by SR-NOM to these NPs was similar to that previously reported in various river and groundwaters. Thus, SR-NOM might be a representative sample of what is found in many different ecosystems, and the ‘camouflage’ of toxicity observed would not pose a limitation to its use in harmonized ecotoxicological assessment of CeO2 and TiO2 NPs.

Environmental Toxicology & Chemistry Journal

Task 4.6Q&N 1,3

60 2015 Biological Interactions of Carbon-Based Nanomaterials: From Coronation to Degradation

Publication Kunal Bhattacharya, et al

15 KI Carbon-based nanomaterials including single- and multi-walled carbon nanotubes, graphene oxide, fullerenes and nanodiamonds are potential candidates for various applications in medicine such as drug delivery and imaging. However, the successful translation of nanomaterials for biomedical applications is predicated on a detailed understanding of the biological interactions of these materials. Indeed, the potential impact of the so-called bio-corona of proteins, lipids, and other biomolecules on the fate of nanomaterials in the body should not be ignored. Enzymatic degradation of carbon-based nanomaterials by immune-competent cells serves as a special case of bio-corona interactions with important implications for the medical use of such nanomaterials.In the present review, we highlight emerging biomedical applications of carbon-based nanomaterials. We discuss recent studies on nanomaterial „coronation‟ and how this impacts onbiodistribution and targeting along with studies on the enzymatic degradation of carbon-based nanomaterials, and the role of surface modification of nanomaterials for these biologicalinteractions.

Nanomedicine: Nanotechnology, Biology, and Medicine

Task 5.5 dec-15

http://www.mdpi.com/1422-0067/16/11/26211

http://www.mdpi.com/1422-0067/16/11/26211

http://www.mdpi.com/1422-0067/16/11/26211

http://www.mdpi.com/1422-0067/16/11/26211

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61 2015 CARBON NANOMATERIALS THERMAL DEGRADATION: POTENTIAL RISK OF NANOWASTE COMBUSTION

Publication Danihelka Pavel, et al. 67 VSB New nanomaterials are used in nearly every industrial area and provide many benefits. Nevertheless, with new properties of new nanomaterials come new potential risks. Social demands to ensure the safety of nanomaterials enters to forefront, taking into account risks, caused by nanomaterials production and use, and are connected with whole nanomaterial life cycle. Potential risks associated with the end-of-life of nanomaterials are an issue that needs to be addressed. Nanoparticles or transformed nanomaterials can be released during disposal methods and enter environment, what should be monitored.Carbon based nanomaterials are interesting for incineration tests, because they might be eliminated totally. As the case study of nanoparticles release potential during incineration, multiwalled carbon nanotubes (MWCNTs) and carbon black were tested. The primary objective of tests described in this study consisted in validating, whether nanoparticles can be released in the gas phase during the combustion in static air and low-oxygen atmosphere. Nanoparticles (28 – 462 nm) were released from 875°C, resp. 925°C, to 1000°C for two types of MWCNTs. A comparison of nanoparticles size distribution and raw MWCNTs diameters indicates that fibres disintegrate during combustion. The highest nanoparticles concentrations were around diameter 30 – 70 nm. In nitrogen atmosphere thermogravimetric experiments showed mass decrease. There is a hypothesis that even very low-oxygen concentration leads to mass loss, probably due to oxidation of nanomaterials. SEM analysis of nanomaterials residue showed modified and variously deformed fibres with conical ends. So, there is hypothesis that needle-like fibre shapes of CNT are more toxic than other carbon nanomaterials.

Environmental Toxicology & Chemistry Journal

Task 3.3. Q/Ns 3

62 2015 Keeping it real: The importance of material characterization innanotoxicology

Publication Bengt Fadeel, et al. 15 KI Nanomaterials are small and the small size and corresponding large surface area of nanomaterials confers specific properties, making these materials desirable for various applications, not least in medicine. However, it is pertinent to ask whether size is the only property that matters for the desirable or detrimental effects of nanomaterials? Indeed, it is important to know not only what the material lookslike, but also what it is made of, as well as how the material interacts with its biological surroundings. It has been suggested that guidelines should be implemented on the types of information required in terms of physicochemical characterization of nanomaterials for toxicological studies in order to improve thequality and relevance of the published results. This is certainly a key issue, but it is important to keep in mind that material characterization should be fit-for-purpose, that is, the information gathered should be relevant for the end-points being studied.

Biochemical and Biophysical Research Communications

WP2,WP5.1Q&N 1

63 2015 Toxicological effects of nanoparticles following oral administration in rat

Presentation Francesca Maranghi, et al.

17 ISS A repeated-dose 90-day oral toxicity study in rat with synthetic amorphous silica (SiO2) nanoparticle (NM203 from JRC Repository) has been carried out (OECD guideline 408) in the frame of the NANoREG project (http://nanoreg.eu/), with specific focus on reproductive-endocrine-immune/inflammatory related parameters and genotoxicity. The aim was the identification of hazards and to obtain dose-response data to characterize the risk of nanomaterials highly relevant for food safety. Six groups of Sprague-Dawley rats were treated by gavage with 0 (control, vehicle only - ultrapure water), 2, 5, 10, 20 and 50 mg/kg body weight per day. Dispersion protocol from the Nanogenotox project has been used and specific characterization of dispersions has been performed before the study. General toxicity data showed no differences between treated and control rats. Death incidence was not increased by the treatments. Preliminary immunotoxicity data showed alterations in blood count, PHA-induced lymphocyte proliferation (spleen, MLN) and LPS-induced NO and cytokine production (peritoneal resident macrophages). Preliminary genotoxicity (micronucleus in target organs and pig-a tests in blood cells) and reproductive toxicity data didn’t show any significant effect. Clinical biochemistry showed no alteration in both sexes concerning liver biomarkers. Kidney biomarkers showed significant alteration in creatinine in males at 10, 20 and 50 mg/kg and in females in all treated groups. Comet assay in male bone marrow showed significant increased DNA damage at 2, 10 and 50 mg/kg, in ovaries at 5 and 50 mg/kg and in male spleen at 5, 10 and 50 mg/kg. No genotoxic effects at the gastrointestinal level have been recorded. Liver and spleen showed tissue alterations in both sexes at different dose levels. Histopathological analyses, biodistribution/bioaccumulation and electron microscopy observations in target tissues are in progress. Preliminary data showed in general the presence of gender differences and the difficulty in individuating a critical dose (effective in different parameters) and a dose-response relationship.

Nanoitaly 2015 Task 4.55 September, 21-24 2015

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64 2015 Life Cycle Assessment as a tool for environmental sustainability of nanomaterials and nanotechnologies

Presentation Simona Scalbi, et al. 18 ENEA The presentation gives an overview on how LCA methods can contribute to the sustainability assessment of nanotechnologies and nanomaterials as proposed in the NANoREG Framework. It is preliminary step for task 1.4 for developing a "FRAMEWORK FOR SAFETY ASSESSMENT OF NANOMATERIALS"

NanotechItaly 2015 Workshop "State of art of Italian partner-ship in NANoREG project and the link with ProSafe Coordination Action"

Task 1.4 25-27 novembre 2015, Bologna

65 2015 Mechanism-based genotoxicity screening of metal oxide nanoparticles using the ToxTracker panel of reporter cell lines

Publication A. Gliga, B. Fadeel and HL Karlsson

15 KI BACKGROUND:The rapid expansion of manufacturing and use of nano-sized materials fuels the demand for fast and reliable assays to identify their potential hazardous properties and underlying mechanisms. The ToxTracker assay is a recently developed mechanism-based reporter assay based on mouse embryonic stem (mES) cells that uses GFP-tagged biomarkers for detection of DNA damage, oxidative stress and general cellular stress upon exposure. Here, we evaluated the ability of the ToxTracker assay to identify the hazardous properties and underlying mechanisms of a panel of metal oxide- and silver nanoparticles (NPs) as well as additional non-metallic materials (diesel, carbon nanotubes and quartz).METHODS:The metal oxide- and silver nanoparticles were characterized in terms of agglomeration and ion release in cell medium (using photon cross correlation spectroscopy and inductively coupled plasma with optical emission spectroscopy, respectively) as well as acellular ROS production (DCFH-DA assay). Cellular uptake was investigated by means of transmission electron microscopy. GFP reporter induction and cytotoxicity of the NPs was simultaneously determined using flow cytometry, and genotoxicity was further tested using conventional assays (comet assay, γ-H2AX and RAD51 foci formation).RESULTS: We show that the reporter cells were able to take up nanoparticles and, furthermore, that exposure to CuO, NiO and ZnO nanoparticles as well as to quartz resulted in activation of the oxidative stress reporter, although only at high cytotoxicity for ZnO. NiO NPs activated additionally a p53-associated cellular stress response, indicating additional reactive properties. Conventional assays for genotoxicity assessment confirmed the response observed in the ToxTracker assay. We show for CuO NPs that the induction of oxidative stress is likely the consequence of released Cu ions whereas the effect by NiO was related to the particles per se. The DNA replication stress-induced reporter, which is most strongly associated with carcinogenicity, was not activated by any of the tested nanoparticles.CONCLUSIONS: We conclude that the ToxTracker reporter system can be used as a rapid mechanism-based tool for the identification of hazardous properties of metal oxide NPs. Furthermore, genotoxicity of metal oxide NPs seems to occur mainly via oxidative stress rather than direct DNA binding with subsequent replication stress.

Particle and Fibre Toxicology

WP5, Task 5.5, 5.6 Q&N 8

66 2015 Size-dependent cytotoxicity of silver nanoparticles in human lung cells: the role of cellular uptake, agglomeration and Ag release

Publication A. Gliga, B. Fadeel and HL Karlsson

15 KI BACKGROUND: Silver nanoparticles (AgNPs) are currently one of the most manufactured nanomaterials. A wide range of toxicity studies have been performed on various AgNPs, but these studies report a high variation in toxicity and often lack proper particle characterization. The aim of this study was to investigate size- and coating-dependent toxicity of thoroughly characterized AgNPs following exposure of human lung cells and to explore the mechanisms of toxicity.

METHODS: BEAS-2B cells were exposed to citrate coated AgNPs of different primary particle sizes (10, 40 and 75 nm) as well as to 10 nm PVP coated and 50 nm uncoated AgNPs. The particle agglomeration in cell medium was investigated by photon cross correlation spectroscopy (PCCS); cell viability by LDH and Alamar Blue assay; ROS induction by DCFH-DA assay; genotoxicity by alkaline comet assay and ãH2AX foci formation; uptake and intracellular localization by transmission electron microscopy (TEM); and cellular dose as well as Ag release by atomic absorption spectroscopy (AAS).

RESULTS: The results showed cytotoxicity only of the 10 nm particles independent of surface coating. In contrast, all AgNPs tested caused an increase in overall DNA damage after 24 h assessed by the comet assay, suggesting independent mechanisms for cytotoxicity and DNA damage. However, there was no ãH2AX foci formation and no increased production of intracellular reactive oxygen species (ROS). The reasons for the higher toxicity of the 10 nm particles were explored by investigating particle agglomeration in cell medium, cellular uptake, intracellular localization and Ag release. Despite different agglomeration patterns, there was no evident difference in the uptake or intracellular localization of the citrate and PVP coated AgNPs. However, the 10 nm particles released significantly more Ag compared with all other AgNPs (approx. 24 wt% vs. 4-7 wt%) following 24 h in cell medium. The released fraction in cell medium did not induce any cytotoxicity, thus implying that intracellular Ag release was responsible for the toxicity.

CONCLUSIONS: This study shows that small AgNPs (10 nm) are cytotoxic for human lung cells and that the toxicity observed is associated with the rate of intracellular Ag release, a 'Trojan horse' effect.

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67 2015 Influence of salinity on fate and behavior of silver standardized nanomaterial and toxicity effects in the estuarine bivalve Scrobicularia Plana.

Publication Carole Bertrand, et al. 32 UCO Silver nanomaterials (AgNM) are included in many products because of their antibacterial properties. In this experiment, a standardized AgNM (NM-300K) was used with a mean diameter < 20 nm. NM-300K are uncoated but supplied with a stabilizing agent (NM-300K DIS). The aim of this study was to investigate the behavior of NM-300K in estuarine-like medium at two salinities (15 and 30 psu). Uptake as well as sublethal effects of Ag (NM-300K, AgNO3) at a low concentration (10 µg Ag L-1) of exposure during 7 days of the euryhaline endobenthic clam Scrobicularia plana were assessed. Two different tools were used to measure the release of dissolved Ag from NM-300K: Diffusive Gradient in Thin films (DGT) and ultrafiltration. Bioaccumulation of Ag in the digestive gland was measured. Sublethal effects were assessed using a multimarker approach including several biological responses at sub-individual and individual levels. Homo-aggregation of NM-300K was observed at both tested salinities. For all treatments, Ag bioaccumulation was significantly greater at 15 psu compared with 30 psu. Burrowing of clams was affected by the stabilizing agent NM-300K DIS in function of the salinity tested with stronger effects at 15 psu

WP3, Task 3.2, Q&N3

68 2015 CELL TRANSFORMATION ASSAY FOR GENOTOXIC AND NON-GENOTOXIC CARCINOGENS

Publication Dusinska M, et al. 16 NILU Cell transformation assays (CTAs) are in vitro carcinogenicity tests measuring morphological transformation of cells either as transformed colonies (SHE cells) or foci (C3H/10T1/2 and BALB/c 3T3 including Bhas 42 cells) derived from a single cell. CTAs such as Bhas 42 CTA can detect both genotoxic and non-genotoxic carcinogens. When used as an initiation assay to test tumour-initiating activity, cells at low-density are treated with a test chemical for three days, whereas a promotion assay to test for tumour-promoting activity, near-confluent cells are treated with a test chemical for a period of 10 days. The Bhas 42 CTA has advantages compared with BALB/c 3T3 and other CTAs due to its simplicity, higher sensitivity, less time needed for assay performance, and robustness (exemplified by its adaptation to a high throughput method). The Bhas 42 CTA has been validated together with other CTAs and recommended for development of an OECD guideline. The assay has already been applied in testing various chemical and physical agents including particles and nanomaterials. Protocols for both 6 and 96-well plate formats of initiation and promotion assays are described in detail.

Genotoxicity and Repair

WP 5 task 5.5 and 5.6. WP 1 Task 1.4, 1.7 WP6.2 Q9 and Q10, Q12

dec-14 on hold until MC 18

69 2015 Can the comet assay be used reliably to detect nanoparticle-induced genotoxicity?

Publication Dusinska M., et al. 16 NILU The comet assay is a sensitive assay that is used to detect DNA strand breaks as well as oxidatively damaged DNA at the level of single cells. Today the assay is commonly used in the nano-genotoxicology field. In this review we critically discuss possible interactions between nanoparticles (NPs) and the comet assay. Concerns for such interactions have arisen from the occasional observation of NPs in the “comet head”, which implies that NPs may be present during performance of the assay. This could give rise to false positive or false negative results, depending on the type of comet assay endpoint and NP. For most NPs, an interaction that substantially can impact the comet assay results is unlikely. For photocatalytically active NPs such as TiO2 on the other hand, exposure to light containing UV can lead to increased DNA damage. Samples should therefore not be exposed to such light. By comparing studies in which both the comet assay and the micronucleus assay have been used, a good consistency between the assays was found in general (69%) and it was even higher when excluding the studies on TiO2 NPs (81%). The strong consistency between the comet assay and the micronucleus test applied to a range of different NPs – even though the two tests measure different endpoints – implies thatboth can be trusted in the assessment of genotoxicity of NPs, and that both could usefully be included in a standard battery of test methods.

Environ Mol Mutagen

WP 5 task 5.5 and 5.6. WP 1 Task 1.4, 1.7 WP6.2 Q9 and Q10, Q12

2015 on hold until MC 18

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70 2015 Is the toxic potential of nanosilver dependent on its size?

Publication Dusinska M , et al. 16 NILU Background: The main goal of this research was to study the interactions of a fully characterized set of silver nanomaterials (Ag ENMs) with cells in vitro, according to the standards of Good Laboratory Practices (GLP), to assure the quality of nanotoxicology research. We were interested in whether Ag ENMs synthesized by the same method, with the same size distribution, shape and specific surface area, but with different charges and surface compositions could give different biological responses.Methods: A range of methods and toxicity endpoints were applied to study the impacts of interaction of the Ag ENMs with TK6 cells. As tests of viability, relative growth activity and trypan blue exclusion were applied. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. The mutagenic potential of Ag ENMs was investigated with the in vitro HPRT gene mutation test on V79-4 cells according to the OECD protocol. Ag ENM agglomeration, dissolution as well as uptake and distribution within the cells were investigated as crucial aspects of Ag ENM toxicity. Ag ENM stabilizers were included in addition to positive and negative controls.Results: Different cytotoxic effects were observed including membrane damage, cell cycle arrest and cell death. Ag ENMs also induced various kinds of DNA damage including strand breaks and DNA oxidation, and caused gene mutation. We found that positive Ag ENMs had greater impact on cyto- and genotoxicity than did Ag ENMs with neutral or negative charge, assumed to be related to their greater uptake into cells and to their presence in the nucleus and mitochondria, implying that Ag ENMs might induce toxicity by both direct and indirect mechanisms.Conclusion: We showed that Ag ENMs could be cytotoxic, genotoxic and mutagenic. Our experiments with the HPRT gene mutation assay demonstrated that surface chemical composition plays a significant role in Ag ENM toxicity.

Particle and Fibre Toxicology 2014

WP 5 task 5.5 and 5.6. WP 1 Task 1.4, 1.7 WP6.2 Q3, Q4, Q6, Q9, Q10, Q12


71 2015 Nanoparticles in food. epigenetic changes induced by nanomaterials and possible impact on health

Publication Dusinska M, et al. 16 NILU Disturbed epigenetic mechanisms, which developmentally regulate gene expression via modifications to DNA, histone proteins, and chromatin, have been hypothesized to play a key role in many human diseases.Recently it was shown that engineered nanoparticles (NPs), that already have a wide range of applications in various fields including food production, could dramatically affect epigenetic processes, while their ability to induce diseases remains poorly understood. Besides the obvious benefits of the newtechnologies, it is critical to assess their health effects before proceeding with industrial production. In this article, after surveying the applications of NPs in food technology, we review recent advances in the understanding of epigenetic pathological effects of NPs, and discuss their possible health impact withthe aim of avoiding potential health risks posed by the use of nanomaterials in foods and food-packaging.

Food Chem Toxicol WP 5 task 5.5 and 5.6. WP 1 Task 1.4, 1.7 WP6.2 Q9, Q10, Q13, Q14


72 2015 Critical factors to be considered when testing nanomaterials for genotoxicity with the comet assay.

Publication Dusinska M. , et al. 16 NILU The comet assay is widely used to test the genotoxicity of engineered nanomaterials (ENMs) but outcomes may vary when results from different laboratories, or even within one laboratory, are compared. We address some basic methodological considerations, such as the importance of carrying out physico-chemical characterisation of the ENMs in test-medium, performing uptake and cytotoxicity tests, and testing several genotoxicity-related endpoints. In this commentary, we discuss the different ways in which concentration of ENMs can be expressed, and stress the need to include appropriate controls and reference standards to monitor variation and avoid interference. Treatment conditions, including cell number, cell culture plate format and volume of treatment medium on the plate are crucial factors that may impact on results and thus should be kept constant within the study.

Mutagenesis WP 5 task 5.5 and 5.6. WP 1 Task 1.4, 1.7 WP6.2 Q3, Q4, 9, Q10, Q12

2015 January on hold until MC 18

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73 2015 Biological impact assessment of nanomaterial used in nanomedicine

Publication Dusinska M. et al. 16 NILU Therapeutic nanoparticles (NPs) are used in nanomedicine as drug carriers or imaging agents, providing increased selectivity/specificity for diseased tissues. The first NPs in nanomedicine were developed for increasing the efficacy of known drugs displaying dose-limiting toxicity and poor bioavailability and for enhancing disease detection. Nanotechnologies have gained much interest owing to their huge potential for applications in industry and medicine. It is necessary to ensure and control the biocompatibility of the components of therapeutic NPs to guarantee that intrinsic toxicity does not overtake the benefits. In addition to monitoring their toxicity in vitro, in vivo and in silico, it is also necessary to understand their distribution in the human body, their biodegradation and excretion routes and dispersion in the environment. Therefore, a deep understanding of their interactions with living tissues and of their possible effects in the human (and animal) body is required for the safe use of nanoparticulate formulations. Obtaining this information was the main aim of the NanoTEST project, and the goals of the reports collected together in this special issue are to summarise the observations and results obtained by the participating research teams and to provide methodological tools for evaluating the biological impact of NPs.

Nanotoxicology. WP 5 task 5.5 and 5.6. WP 1 Task 1.4, 1.7 WP6.2 Q9, Q10, Q12, Q13, Q14


74 2015 Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classic in vitro tests.

Publication Dusinska M. et al. 16 NILU 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 ofinterference 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, 3H-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.

Nanotoxicology WP 5 task 5.5 and 5.6. WP 1 Task 1.4, 1.7 WP6.2 Q3, Q9, Q10, Q12

2013 Jul 27 on hold until MC 18

75 2015 Coating-dependent induction of cytotoxicity and genotoxicity of iron oxide nanoparticles

Publication Dusinska M. et al. 16 NILU Surface coatings of nanoparticles (NPs) are known to influence advantageous features of NPs as well as potential toxicity. Iron oxide (Fe3O4) NPs are applied for both medical diagnostics and targeted drug delivery. We investigated the potential cytotoxicity and genotoxicity of uncoated iron oxide (U-Fe3O4) NPs in comparison with oleate-coated iron oxide (OC-Fe3O4) NPs. Testing was performed in vitro in human lymphoblastoid TK6 cells and in primary human blood cells.For cytotoxicity testing, relative growth activity, trypan blue exclusion, 3H-thymidine incorporation and cytokinesis-block proliferation index were assessed. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. Particle characterization was performed in the culture medium. Cellular uptake, morphology and pathology were evaluated by electron microscopy. U-Fe3O4 NPs were found not to be cytotoxic (considering interference of NPs with proliferation test) or genotoxic under our experimental conditions. In contrast, OC-Fe3O4 NPs were cytotoxic in a dose-dependent manner, and also induced DNA damage, indicating genotoxic potential. Intrinsic properties of sodium oleate were excluded as a cause of the toxic effect. Electron microscopy data were consistent with the cytotoxicity results. Coating clearly changed the behaviour and cellular uptake of the NPs, inducing pathological morphological changes in the cells.



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76 2015 Immunotoxicity and genotoxicity testing of PLGA-PEO nanoparticles in human blood cell model.

Publication Dusinska M. et al. 16 NILU A human blood cell model for immunotoxicity and genotoxicity testing was used to measure the response to polylactic-co-glycolic acid (PLGA-PEO) nanoparticle (NP) (0.12, 3, 15 and 75 mg/cm2 exposure in fresh peripheral whole blood cultures/isolated peripheral blood mononuclear cell cultures from human volunteers (n ¼ 9–13). PLGA-PEO NPs were not toxic up to dose 3 mg/cm2; dose of 75 mg/cm2 displays significant decrease in [3H]-thymidine incorporation into DNA of proliferating cells after 4 h (70% of control) and 48 h (84%) exposure to NPs. In non-cytotoxic concentrations, in vitro assessment of the immunotoxic effects displayed moderate but significant suppression of proliferative activity of T-lymphocytes and T-dependent B-cell response in cultures stimulated with PWM4CON A, and no changes in PHAcultures. Decrease in proliferative function was the most significant in T-cells stimulated with CD3 antigen (up to 84%). Cytotoxicity of natural killer cells was suppressed moderately (92%) but significantly in middle-dosed cultures (4 h exposure). On the other hand, in low PLGA-PEO NPs dosed cultures, significant stimulation of phagocytic activity of granulocytes (119%) 4 monocytes (117%) and respiratory burst of phagocytes (122%) was recorded. Genotoxicity assessment revealed no increase in the number of micronucleated binucleated cells and no induction of SBs or oxidised



77 2015 Health effects of selected nanoparticlesin vivo: Liver function and hepatotoxicity following intravenous injection of titanium dioxide and Na-oleate coated iron oxide nanoparticles in rodents.

Publication Dusinska M. et al. 16 NILU The study determined the effect of intravenous administration of acutely toxic or sub-lethaldoses of Na-oleate-coated Fe3O4 (OC-Fe3O4) nanoparticles (NPs) on liver structure and function in Wistar rats, compared to titanium dioxide (TiO2) NPs and saline-injected controls. The acute study, using a modified OECD 425 progressive dosing procedure, found LD50 values of 59.22 and 36.42 mg/kg for TiO2 and OC-Fe3O4 NPs, respectively. In the sub-lethal study, rats were either injected with saline (negative controls), a sub-lethal reference (0.592 mg/kgTiO2 NPs, equal to 1% of LD50 on a body weight basis) or OC-Fe3O4 NPs in doses equivalent to 0.1, 1 or 10% of the LD50, respectively (corresponding to 0.0364, 0.364 and 3.64mg Fe3O4/kg body weight). Animals were sampled 24 h, 1, 2 and 4 weeks post-injection for adverse effects. Mitochondrial respiration was significantly increased 2 weeks after injection of 10% OC-Fe3O4 NPs compared to controls, but the effect was transient. Cholesterol and triacylglycerol concentrations in the liver tissue did not increase in any treatment. There were some disturbances to antioxidant enzymes after OC-Fe3O4 NPs treatment in the livers of animals 1 week post-exposure; with the most sensitive changes occurring in glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities. Lipidosis and mild necrosis with changes in sinusoid space were also observed in histological sections of the liver. Overall, these data suggest that the liver likely retains functional integrity with acute and sub-lethal doses of OC-Fe3O4 NPs, albeit with some stimulation of redox defences and evidence of some tissue

Nanotoxicology WP 5 task 5.5 and 5.6. WP 1 Task 1.4, 1.7 WP6.2 Q3, Q6, Q7, Q9, Q10, Q12


78 2015 Iron oxide nanoparticle toxicity testing using high throughput analysis and high content imaging

Publication Dusinska M. et al. 16 NILU Applying validated in vitro assays to the study of nanoparticle toxicity is a growing trend in nanomaterial risk assessment. Precise characterisation of reference nanomaterials and a well regulated in vitro testing system are required to determine the physicochemical descriptors which dictate the toxic potential of nanoparticles. The use of automated, high-throughput technologies to facilitate the identification and prioritisation of nanomaterials which could posea risk is desirable and developments are underway. In this study, two mammalian fibroblast lines (Balb/c 3T3 and COS-1 cells) were treated with a range of concentrations of iron oxide nanomaterials manufactured for use in medical diagnostics, using an automated platform and high-content-imaging endpoints for cell viability, oxidative stress and DNA damage (double strand breaks). At the same time, the high-throughput comet assay was employed to measureDNA strand breaks and oxidised bases. Our results show that these methods provide a fast way to determine the toxicity of coated and uncoated iron oxide nanoparticles and, furthermore, to predict the mechanism of toxicity in vitro.

Nanotoxicology WP 5 task 5.5 and 5.6. WP 1 Task 1.4, 1.7 WP6.2 Q5, Q9, Q10, Q12,Q13,Q14

2013 Jul 17 on hold until MC 18

79 2015 Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles

Publication Dusinska M. et al. 16 NILU 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 50nm (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.

Nanotoxicology WP 5 task 5.5 and 5.6. WP 1 Task 1.4, 1.7 WP6.2 Q5, Q9, Q10, Q12,Q13,Q14


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80 2015 Towards an alternative testing strategy for nanomaterials used in nanomedicine: Lessons from NanoTEST

Publication Dusinska M. et al. 16 NILU 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 forNPs 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 forhazard 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.

Nanotoxicology WP 5 task 5.5 and 5.6. WP 1 Task 1.4, 1.7 WP6.2 Q9, Q10, Q12,Q13,Q14


81 2015 Impact of nanosilver on various DNA lesions and HPRT gene mutations.

Publication Dusinska M. et al. 16 NILU Background: The main goal of this research was to study the interactions of a fully characterized set of silver nanomaterials (Ag ENMs) with cells in vitro, according to the standards of Good Laboratory Practices (GLP), to assure the quality of nanotoxicology research. We were interested in whether Ag ENMs synthesized by the same method, with the same size distribution, shape and specific surface area, but with different charges and surface compositions could give different biological responses.Methods: A range of methods and toxicity endpoints were applied to study the impacts of interaction of the Ag ENMs with TK6 cells. As tests of viability, relative growth activity and trypan blue exclusion were applied. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. The mutagenic potential of Ag ENMs was investigated with the in vitro HPRT gene mutation test on V79-4 cells according to the OECD protocol. Ag ENM agglomeration, dissolution as well as uptake and distribution within the cells were investigated as crucial aspects of Ag ENM toxicity. Ag ENM stabilizers were included in addition to positive and negative controls.Results: Different cytotoxic effects were observed including membrane damage, cell cycle arrest and cell death. Ag ENMs also induced various kinds of DNA damage including strand breaks and DNA oxidation, and caused gene mutation. We found that positive Ag ENMs had greater impact on cyto- and genotoxicity than did Ag ENMs with neutral or negative charge, assumed to be related to their greater uptake into cells and to their presence in the nucleus and mitochondria, implying that Ag ENMs might induce toxicity by both direct and indirect mechanisms.Conclusion: We showed that Ag ENMs could be cytotoxic, genotoxic and mutagenic. Our experiments with the HPRT gene mutation assay demonstrated that surface chemical composition plays a significant role in Ag ENM toxicity.

Particle and Fibre Toxicology

WP 5 task 5.5 and 5.6. WP 1 Task 1.4, 1.7 WP6.2 Q3, Q5, Q9, Q10


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Date Comment


82 2015 Impact of Storage Conditions and Storage Time on Silver Nanoparticles’ Physicochemical Properties and Implications for their Biological Effects, RCS

Paper Dusinska M. et al. 16 NILU Background: It is increasingly recognized that nanoparticles (NPs) can ‘age’ while stored, and that the impact of this may lead to divergent results in terms of the observed toxicity of nominally the same particles. An understanding of the ‘aging’ process of NPs during storage may be crucial for interpretation of toxicity test results, and may also provide insights into optimal storage conditions for such dispersions in order to optimize their long-term stability against dissolution and/or agglomeration.The main motivation for this study was to investigate whether changes in silver (Ag) NPs’ properties occur over time, and to what extent, and whether storage of the dispersions under different conditions and with different surface coatings impact their stability, as a function of the NPs’ surface coating/charge. Additionally, the impact of the NPs’ surface chemistry on their cytotoxicity was examined, and alterations of the observed cytotoxicity were related to the changes of the NPs’ physicochemical properties over time.

Methods: The stability of the AgNPs was investigated by zeta potential measurements, DLS, TEM, UV-Vis, ICP-MS and DCS. The NPs’ physicochemical properties were examined immediately after their synthesis and repeatedly within 6 months of storage at room temperature in the presence or absence of light, and at 4°C in the absence of light (in a fridge). Viability and cytotoxicity tests were performed using A549 cells, shortly after the NPs’ synthesis and after 6 months of storage, using conventional assays such as plating efficiency and relative growth activity, and by high content screening in order to assess multiple markers of cell viability and cytotoxicity in parallel. Results: The storage time and conditions influenced the properties of NPs with all types of coatings studied. The fastest and the most toxicologically significant changes in AgNPs’ properties occurred at room temperature under daylight exposure, as expected. The observed changes were dependent on the NPs’ coating/charge: the positively charged NPs agglomerated the most, while the neutral NPs were the most stable. The storage conditions and the NPs’ surface chemistry/charge also had an impact on the toxicity of the all tested NPs. Observed changes in Ag NPs’ toxicity were related to different, subsequent processes such as particle agglomeration, dissolution, and oxidation as well as stabilizer degradation.Conclusions: Both storage time/conditions and surface chemistry of AgNPs influence the evolution of the NP properties over time, and the resulting changes in the NPs’ physicochemical properties influence their toxicity. Storage of AgNPs at lower temperature in the dark can, to some extent, protect the NP dispersion from rapid transformation (agglomeration and/or dissolution). Thus, NP ‘aging’ effects as described here can also contribute to contradictory results of toxicity of the same nanomaterial observed in the literature.

WP 5 task 5.5 and 5.6. WP 1 Task 1.4, 1.7 WP6.2 Q1, Q2, Q3, Q12, Q13

on hold until MC 18

83 2015 Fingerprint Characterisation of Graphene based products

Publication A. Prina-Mello, et al. 14 TCD Graphene based Elicarb© in their dry carbon and liquid dispersion formulations of exfoliated graphene were characterised for the purpose of material technical datasheets and fingerprint characterisation. The techniques used to characterise were TEM, EDX, XRD, Raman, TGA, and FTIR. The sheets could be easily exfoliated for imaging by TEM, under mild conditions, and were sub- micron in size. It was found by XRD that the dry carbon corresponded to the expected hexagonal close packed structure, with an extra peak, characteristic of extended separation between sheets. Raman also confirmed sheet separation in the dry product. High temperature annealing under N2 could remove the alkyl functionalities characteristic of the surfactant, which was further confirmed by FTIR performed pre-and post-TGA, for functional group vibration analysis. The sheets were predominantly sub-micron in length and area, as found by TEM size analysis. This particular formulation opens the door for new industrial applications, particularly in biomedical research, due to non-toxic intercalating surfactant.

Nature Publishers – Scientific Reports

WP2, Q&N 1,2

of 28 - 3-12-2015


Author(s) Ben. Nr.


TaskQ&Ns

Date Comment


84 2015 In vivo and in vitro studies of pulmonary nanoparticle uptake and translocation with label-free techniques

Presentation I. Estrela-Lopis, et al. 45 ULEI Increasing applications of engineered nanomaterials (ENMs) in the industry and private consumption demand the thoroughly identification of hazards and potential adverse effects on human and environment. These adverse effects include the ability of ENM to induce damage at the cellular, tissue, or organism levels by interacting with cellular structures. Modifying the surface chemistry of nanomaterials is of prime importance to control and understand the potentially toxic impact of nanomaterials, their translocation, localization and interactions with biomolecules at cellular levels.The translocation of engineered nanomaterials (ENMs) across plasma membranes was studied in human lung adenocarcinoma epithelial cells (A549) by means of label-free imaging methods based on elemental and molecule analysis. Proton induced X-ray emission (PIXE) and Rutherford backscattering (RBS) were used simultaneously at the Leipzig Ion Nanoprobe LIPSION for cell analysis. These two IBM techniques provide unique and powerful tools for element dosimetry and spatially resolved elemental analysis. Cellular response, quantification of ENM uptake and their distribution at the single cell level were performed for CeO2 as well as for CuO-Core, CuO-NH3+ , PEGylated and carboxylated CuO nanoparticles. It was shown that the uptake of the CuO nanoparticles by cells is influenced by their surface chemical properties. Additionally the confocal Raman microscopy (CRM) was applied for 3D visualization of ENMs at subcellular level. The co-localization of ENMs with cell compartments as well as with biomolecules was studied. Correlation of the intracellular uptake with the toxicological response was studied. Furthermore, the results of CeO2 ENM uptake in vitro were compared and correlated with in vivo study. For this purpose CeO2 uptake, localization and distribution in lung tissues of Wistar rats exposed to 25 mg/m3 CeO2 over 28 days were analysed.

12th International Conference on Nanosciences & Nanotechnologies, Thessaloniki, Greece,

WP4 - Task4.4 WP 5 – Task 5.6 Q&N 3, 5 and 6

July 2015

85 2015 Case study on environmental sustainability assessment of QDsLED with Life Cycle Assessment

Presentation Simona Scalbi 18 ENEA The presentatation describes Instruments for the evaluation of the sustainability of nanomaterials life cycle. Among others it addresses the EU strategies for new materials and technology, the sustainability assessment tools, the NANoREG project. A case study is presented on on environmental sustainability assessment of QDsLED with Life Cycle Assessment

NanoItaly 2015 nov-15

86 2015 No genotoxicity in rat blood cells upon 3- or 6-month inhalation exposure to CeO2 or BaSO4 nanomaterials

Publication Eugenia Cordelli, et al. 18 ENEA In the course of a 2-year combined chronic toxicity - carcinogenicity study performed according to Organisation for Economic Co-operation and Development (OECD) Test Guideline 453, systemic (blood cell) genotoxicity of two OECD representative nanomaterials, CeO2 NM-212 andBaSO4 upon 3- or 6-month inhalation exposure to rats was assessed. DNA effects were analysed in leukocytes using the alkaline Comet assay, gene mutations and chromosome aberrations were measured in erythrocytes using the flow cytometric Pig-a gene mutation assay and the micronucleus test (applying both microscopic and flow cytometric evaluation), respectively. Since nano-sized CeO2 elicited lung effects at concentrations of 5 mg/m³ (burdens of 0.5 mg / lung) in the preceding range-finding study, whereas nano-sized BaSO4 did not induce any effect, female rats were exposed to aerosol concentrations of 0.1 up to 3 mg/m3 CeO2 or 50 mg/m3 BaSO4 nanomaterials (6 hours / day; 5 days / week; whole-body exposure). The blood of animals treated with clean air served as negative control, whereas blood samples from rats treated orally with three doses of 20 mg/kg body weight ethylnitrosourea at 24 hours intervals were used as positive controls. As expected, ethylnitrosourea elicited significant genotoxicity in the alkaline Comet and Pig-a gene mutation assays and in the micronucleus test. By contrast, 3- and 6-month CeO2 or BaSO4 nanomaterial inhalation exposure did not elicit significant findings in any of the genotoxicity tests. The results demonstrate that subchronic inhalation exposure to different low doses of CeO2 or to a high dose of BaSO4 nanomaterials does not induce genotoxicity on the rat hematopoietic system at the DNA, gene or chromosome levels.

Mutagenesis WP 4, Task 4.2Q&N 6,7,10,12,14

of 28 - 3-12-2015


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87 2015 Fate of TiO2 and ZnO nanoparticles in wastewater treatment plant

Presentation Y. Kim, et al. KR KWU Abstract : Over the past decade, an increasing number of manufactured nanoparticles (NPs) have been incorporated into products and manufacturing processes due to the rapid innovation and commercialization in the field of nanotechnology. Because of their unique antibacterial, antifungal, and partially antiviral properties, NPs have been one of the most promising engineered nanoparticles (ENPs) for applications such as medicines, clothing, and cosmetics. The intentional or accidental release of NPs to the environment is hence largely unavoidable. The potential toxicity and bioaccumulation of NPs make it necessary to probe their fates and transport in the environment. Municipal wastewater treatment plants (WTPs) therefore act as the “gateways” controlling release of ENPs from domestic and/or industrial sources to the aquatic environment via treated effluent that is discharged into surface waters. In general, wastewater effluent is discharged primarily into surface waters (rivers, lakes, oceans) and represents a significant potential point source for pollutants into the environment. In the present study, we found that even the highest AgNPs spiking, STP and WTP are not a potential point source. Both the mechanical and biological treatments can reduce the NPs from wastewater. Totally, more than 90~95% of the NPs that entered municipal WWTPs are reduced through the wastewater treatment processes, which gives rise to extremely low concentrations of AgNPs in the effluents (e.g., some ng/L). On the other hand, the removed NPs are likely to be accumulated in the wastewater biosolids that are usually used as agricultural land amendments, placed in landfills, or incinerated. The biosolids may represent a potential source for NPs release into the environment that is very different from WWTP liquid discharge. Hence, future investigation will have to further enlighten the biosolids releases and resulting ecosystem exposures.

International Union of Pure and Applied Chemistry 2015, Bexco, Busan, Korea

WP3, task 3.4 Aug. 8-14, 2015

88 2015 Nanomaterial Characterization in NANoREG: Results and Challenges

Presentation Alessandro Ponti 31 CNR

CNR performed several tasks related to nanomaterial (NM) characterization within the WP2 of NANoREG. First, extensive characterization of several NMs in order to build a database of physico-chemical data and material datasheets. CNR was also involved in the development of standard operational procedures (SOP) and templates for endpoint data recording.

NanotechItaly 2015; workshop

tasks 2.1, 2.3, and 2.4Q&Ns: 1, 2, and 3

nov-15

89 2015 Contamination with LPS: an issue in nanomaterial toxicity testing in NANoREG

Presentation Diana Boraschi and Paola Italiani

31 CNR Within NANoREG, CNR has contributed to the characterisation of biologically active contaminants of nanomaterials (NM), with specific focus on the ubiquitous bacterial-derived contaminant LPS. First, we have evaluated the suitability of commercially available assays for assessing LPS contamination of NM. We found that one of the assays could be applied to NM, provided a series of controls is performed. Then, we have tested such contamination in the NANoREG core materials. CNR has developed a standard operational procedure (SOP) for reliable LPS detection in NM.

NanotechItaly 2015; workshop

task 2.6 and 5.5 nov-15

90 2015 Towards Safety by Design: Building a database of nano(eco)toxicity studies to establish the impact of physical-chemical properties

Presentation Yehia S. El-Temsah and Fern Wickson

38 genøk Nanosafety research is expanding rapidly. This rapid expansion has, however, lead to a hugely diverse set of methods, protocols and results, making it difficult to draw any conclusions on the safety of ENMs. Within the large-scale FP7 project “NANoREG”, there is an aim to both evaluate the current challenges and data needs for conducting toxicological and safety evaluations for ENMs, and to develop an inventory of the impact of physical-chemical properties on eco toxicological endpoints. This inventory is planned to be developed through the assembly of a database of nano(eco)toxicity research and will be used for the identification of structure activity relationships (QSAR) that can

10th Intern. Conf. on the Environmental Effects of Nanoparticles and Nanomaterials,

Task 6.3Q&Ns 15

sep-15

91 2015 Toxicological effects of synthetic amorphous silica nanoparticles following repeated-dose oral administration in rat: the EFSA approach

Presentation Maranghi F 17 ISS the hazard identification of SAS nanomaterial International Conference NanotechItaly2015 (http://www.nanotechitaly.it/).

Task 4.5.5 25-27 novembre 2015, Bologna

92 2015 NP fate in human exposurerelevant matrices

Presentation SabellaStefania

28 IIT While research community has suggested that characterization of NPs is key for risk assessment (1), most of the present publications however show a detailed characterization of NPs in water or physiological buffer only at time zero, do not considering exposure conditions relevant for the assay such as the matrix composition and the exposure time.It has been demonstrated that a proper in situ characterization of NPs (2,3) may help a more accurate measure of the effective dose and a better correlation of the NP properties to the induced effect in relevant exposure conditions (e.g., ROS production, cytokines release, etc). Strictly connected to this argument are also the multiple interactions between NPs and matrix components (proteins biomolecules salts etc ) that impact on NP colloidal stability and solubility

Nanotechitaly 2015 25-27 novembre 2015, Bologna

of 28 - 3-12-2015


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93 2015 Extra-pulmonary Translocation of CeO2 Nanoparticles: From Lung to Liver after Subacute and Chronic Low Dose Inhalation

Presentation P Laux 6 BfR Cerium dioxide (CeO2), a representative of granular, biopersistent particles without known specific toxicity (GBP), was examined on lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure in a chronic inhalation study according to OECD TG 453. So far, no definite conclusions on the biokinetics of GBP nanoparticles under the conditions of chronic inhalation could be drawn, despite the numerous in vitro and in vivo studies. Furthermore, concerns about adverse impacts on extra-pulmonary organs, especially metabolizing organs like liver, are emerging for CeO2 due to its alternating redox character. For that reason we determined the burden of the lungs and extra-pulmonary organs according to their contents of CeO2 nanoparticles (NM-212) resulting either from subacute inhalation (study according to OECD TG 412) or from chronic low-dose inhalation (study according to OECD TG 453). The aim of these studies was to examine the clearance, translocation and excretion of CeO2.The maximum CeO2 lung burden was determined with 5.88 mg after 24 months of exposure to an aerosol concentration of 3 mg/m³. Additionally, tracheobronchial and mediastinal lymph nodes contained high levels of CeO2, possibly because these lymph nodes drain the lung. Since in liver a maximum content of 0.0167 mg CeO2 was detected, we currently investigate the potential biotransformation of these nanoparticles, motivated by the scientific discussion on subcellular effects.

SOT 2016 on thetranslocation of CeO2 Nanoparticles https://www.toxicology.org/events/am/AM2016/

March 13-17, 2016

94 2015 Multi-walled carbon nanotubes (NM401) induce ROS mediated HPRT mutations in Chinese hamster lung fibroblasts

Publication Laura Rubio, et al. 28 IIT Although there is an important set of data showing potential genotoxic effects of nanomaterials (NMs) at the DNA (comet assay) and chromosome (micronucleus test) levels, few studies have been conducted to analyze their potential mutagenic effects at gene level. We have determined the ability of multi-walled carbon nanotubes (MWCNT, NM401), to induce mutations in the HPRT gene in Chinese hamster lung (V79) fibroblasts. NM401, characterized in the EU NanoGenotox project, were further studied within the EU Framework Programme Seven (FP7) project NANoREG. We have been able to observe significant cellular uptake of MWCNT by using transmission electron microscopy (TEM), as well as a concentration-dependent induction of intracellular reactive oxygen species. In addition, a clear concentration-dependent increase in the induction of HPRT mutations was observed. Data support a potential genotoxic/ carcinogenic risk associated with MWCNT exposure.

Environmental Research journal

WP5, Q1, Q9 and Q10

on hold

95 2015 Can the comet assay be used reliably to detect nanoparticle-induced genotoxicity?

Publication Hanna Karlsson, et al. 15 KI The comet assay is a sensitive method to detect DNA strand breaks as well as oxidatively damaged DNA at the level of single cells. Today the assay is commonly used in nano-genotoxicology. In this review we critically discuss possible interactions between nanoparticles (NPs) and the comet assay. Concerns for such interactions have arisen from the occasional observation of NPs in the "comet head", which implies that NPs may be present while the assay is being performed. This could give rise to false positive or false negative results, depending on the type of comet assay endpoint and NP. For most NPs, an interaction that substantially impacts the comet assay results is unlikely. For photocatalytically active NPs such as TiO2 , on the other hand, exposure to light containing UV can lead to increased DNA damage. Samples should therefore not be exposed to such light. By comparing studies in which both the comet assay and the micronucleus assay have been used, a good consistency between the assays was found in general (69%); consistency was even higher when excluding studies on TiO2 NPs (81%). The strong consistency between the comet and micronucleus assays for a range of different NPs-even though the two tests measure different endpoints-implies that both can be trusted in assessing the genotoxicity of NPs, and that both could be useful in a standard battery of test methods.

Environ Mol Mutagen

WP5, Task 5.5, Q&N10

March 2015 on hold mail PO 19-10-2015 few questions - 3-11-2015 no answer yet, rappel to author

96 2015 RELEVANCE OF BARRIERS (NANoREG, WP5 TASK 5.3): SET-UP OF EXPERIMENTAL CONDITIONS FOR NANOPARTICLES CHARACTERIZATION AND INTESTINAL BARRIER CROSSING

Presentation Isabella De Angelis, et al.

17 ISS In WP 5 of NANoREG project, task 5.3 is focused on the evaluation of capability of NMs to cross five biological barriers (i.e. blood-brain barrier, skin, intestine, lung and oral mucosa) using in vitro models.ISS is co-leader of task 5.3 and it is mainly involved in the subtask 5.3.1 where are utilized differentiated Caco-2 cells as in vitro intestinal barrier model. Some of the main results obtained up to now are:• Good inter-laboratory reproducibility between all Caco-2 partners, both in terms of epithelial barrier formation and cellular differentiation.• Viability MTS test was set up as common cytotoxicity protocol.• DLS data of Z-Average and PDI showed a good reproducibility of NM batch dispersion in MilliQ-BSA (0.05%) using SOP for probe-sonicator calibration and de-agglomeration efficiency.• The NM dispersions in culture medium showed higher PDI and Z-Average than the corresponding values of the batch dispersion, indicating polydisperse and unstable dispersions.

International Conference NanotechItaly2015 (http://www.nanotechitaly.it/)

WP5, task 5.3, Q&N Q1, Q2, Q7, Q10

nov-15

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97 2015 NANoREG Safe by Design approach

Presentation Federico Benetti 41 VN

workshop on “State of Art of Italian Partnership in NANoREG Project and the Link with ProSafe Coordination Action” during the.International Conference NanotechItaly2015

nov-15 approved

98 2015 Towards a general physiologically based pharmacokinetic (PBPK) model for intravenously injected nanoparticles

Publication Ulrika Carlander1 15 KI This manuscript links to NANoREG Task 4.5.8 Development of physiologically based pharmacokinetic models (PBPK). The aim is to address regulatory questions 6, 7, and 8 (fate, kinetics, long-term effects, determination, and extrapolation). A recent model for pegylated polyacrylamide nanoparticles was extended to other non-degradable nanoparticles injected intravenously into rats. Published data on biodistribution were used in combination with anatomical and physiological data. Three studies covering four nanoparticle types were found suitable for PBPK modelling and covered; polyethylene glycol coated polyacrylamide nanoparticles, polyacrylamide nanoparticles, gold nanorods and titanium dioxide nanoparticles. Our model describes the biokinetic behavior of all four types of nanoparticles adequately, despite extensive differences in this behavior, as well as in their physicochemical properties. Our simulations show that: phagocytosis should be incorporated into nano-PBPK models, dose exerts a profound impact on the biokinetics, and more informative experimental data are needed to make models useful for risk assessment.

International Journal of Nanomedicine

WP 4 Task 4.5.8 Q&N Q6, Q7 and Q8.

99 2015 High throughput screening methods applied to the study of toxicity of nanomaterials

Publication Maria Dusinska 16 NILU Introduction to high throughput screening of nanomaterialsExperimental design for effective high throughput screening: accelerating toxicity analysisLabel-free cellular screening of NP uptakeHigh content analysisHigh throughput screening for NM-induced cytotoxicityHigh throughput flow cytometryCell death (apoptosis/necrosis determination)Reactive oxygen productionSpecific cellular uptakeImpedance-based monitoringHigh throughput comet assayIncreasing throughputAutomated scoringHigh throughput in vitro micronucleus assayThe γH2AX assayCost-effectiveness of high throughput screening of nanomaterials

RSC Advances (RSC), WIRES nanotechnology (Wiley) or High Throughout Screening (Dove press).

WP5, Task 5.6, Q&N Q5, Q10 and Q14

on hold - concise abstract requested (mail 3-11)

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