Final Report Summary - NANOTEST (Development of methodology for alternative testing strategies for the assessment of the toxicological profile of nanoparticles used in medical diagnostics) Executive summary:In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it 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 cause toxicity. Nanomedicine has great potential in the development of novel treatments for human diseases. However, recently voiced concerns of scientists, politicians and the public about potential health hazards associated with NPs, even in medical therapies, need to be answered. The main objective of the Seventh Framework Programme (FP7) NANOTEST project (see http://www.nanotest-fp7.eu online) was a better understanding of mechanisms of interactions of NPs with cells, tissues and organs. Project Context and Objectives:Nanoparticles (NPs) have unique, potentially beneficial properties, but their possible impact on human health is not known. The area of nanomedicine brings humans into direct contact with NPs and it is a pressing need to understand how engineered NPs can interact with the human body following exposure. The Seventh Framework Programme (FP7) project NANOTEST (see http://www.nanotest-fp7.eu online) addresses these requirements in relation to the toxicological profile of NPs used in medical diagnostics. A better understanding of how properties of NPs define their interactions with cells, tissues and organs in exposed humans is a considerable scientific challenge but one that must be addressed to achieve responsible use of biomedical NPs. Project Results:WP1 CharacterizationAim of WP1The aim was to provide a complete and advanced physico-chemical characterization of selected engineered NPs for the assessment of their toxicological profile used in medical diagnostics and to support project partners in their toxicological evaluation.ResultsAfter a detailed survey of potential candidates, and after evaluating prons and cons of each material (availability, relevance, etc.), the following panel of commercial ENPs has been selected.All pertinent physico-chemical properties (size distribution, shape, specific surface area, porosity, chemical composition, impurities of concern, surface chemistry, surface charge, crystal structure, dispersion and stability in aqueous and biological media, aggregation state as powdered and dispersed form) of the selected ENPs have been determined prior to toxicological testing, as well as their potential interactions with culture media components, their size distributions in stock solution/dispersion and their behaviour after addition to culture media (see Deliverable 1.3). ConclusionThe comprehensive detailed physical and chemical characterization results obtained in WP1, especially the investigation on NPs behavior in culture media, greatly supported the project in deeply understanding the toxicological behavior of selected ENPs in cell and animal systems for ENPs. WP2 In vitro screening testsAim of sub WP2.0: Databases: The aim of this subWP was to coordinate in vitro studies; to develop database and create SOPs for set of in vitro tests for automation and validation; to perform statistical evaluation of NPs toxicity in different systems.Strategy: A common approach for handling experiments, analyzing and comparing data from different systems and for statistical analysis, a crucial aspect of the assessment of health risks of therapeutic NPs, has to be performed from the results of laboratory tests. To obtain experimental results which can be compared between them, the experimental conditions must be carefully described and be similar in all participating partners of the Consortium. Summary of Results: To establish and validate methods and take into consideration standardization of assays, the factors affecting inter- and intra-experimental variability, including for example, the number of replicate treatments, the numbers of cells per samples, or the amount of NPs added to the cells, have been considered in the experimental design so that statistical analysis can be reliably applied. Conclusions: In summary, all the necessary data have been provided, database was developed and validated by the different partners of the Consortium, and is ready to be uploaded to the European Commission JRC NANOhub. The information has been made accessible to the partners of NANOTEST for approval, and will be made accessible to the scientific community when approved by the European Commission.Aim of sub WP2.1: Blood: After intravenous administration of NPs, blood is primary target organ and secondary target after access of NPs to systemic circulation during other route of administration. Direct toxic effect of NPs on blood components can be the result of cytotoxicity, effect on growth and maturation of immune cells and genotoxicity. Strategy:Cytotoxicity methods used were trypan blue exclusion, cell proliferation assay, clonogenic assay, mitotic index.Genotoxicity endpoints and methods: the cytokinesis block micronucleus (CBMN) assay to detect mutagenicity, clastogenicity and aneugenicity; the comet assay (CA) to detect strand breaks, and formamydopyrimidine (FPG) modified comet assay (FPG CA) for oxidised DNA lesion especially 8-oxoGuanine.Immunotoxicity of NPs: proliferative activity of lymphocytes in vitro stimulated with mitogens and CD3 antigen to assess functionality of the lymphocytes, phagocytic activity and respiratory burst of leukocytes to evaluate function of neutrophil granulocytes and monocytes (macrophages), natural killer cell activity to determine function of natural killer cells, immunophenotypic analysis to monitor proportion of lymphocyte subsets in peripheral blood, expression of adhesion molecules to evaluate adhesive capabilities of blood cells, expression of interleukins to assess inflammatory and immune response.Summary of results and conclusionsa) Assessment of genotoxic effect: Both CBMN and the CA data on TK6 and human peripheral blood cells show consistent results. TiO2 and OA-Fe3O4 show positive effect with the CA. Micronucleus assay data show no genotoxic or clastogenic effect. For details see deliverable report and CCT report.b) Assessment of immunotoxic effect: For details see deliverable report and CCT report.Immunotoxicity induced by NPs depends on cell type, NPs, dispersion protocol and measured endpoint. Several immune biomarkers have been used to monitor the possible effects of NPs on humoral and cellular immune response. From functional assays, proliferative activity of lymphocytes demonstrated to be sensitive biomarker of specific cellular immune response. Similarly, cytotoxic activity of natural killer cells and phagocytic activity and respiratory burst of leukocytes proved to be reliable indicator of function of natural immune response.Conclusions: These findings contributed to the development of testing strategy for monitoring of potential genotoxic and immunotoxic effects of NPs on blood cells.Summary of resultsa) Evaluate uptake and transport of NPs across endothelium: The uptake of the NPs by the three endothelial cells was determined. All the solid-core NPs were well taken up by endothelial cells, with the exception of OC-Fe3O4 NPs, which are taken up only at very low levels, however, no subsequent release by or transport across the cells could be demonstrated.b) Identify effects of NPs on endothelial cells: neither U-Fe3O4 NPs nor PLGA-PEO NPs were cytotoxic, Fl-25 SiO2 NPs and TiO2 NPs were slightly cytotoxic whereas OC-Fe3O4 NPs were highly cytotoxic for all endothelial cells. The three solid-core metallic NPs, but not PLGA-PEO or Fl-SiO2 NPs, induced an oxidative stress in endothelial cells. A decrease of cellular thiol levels was observed in all three endothelial cell lines after exposure to Fe3O4 NPs, but not to TiO2 NPs. Only uncoated Fe3O4 NPs stimulated NO production by rodent endothelial cells. Conclusions: In summary, only OC-Fe3O4 NPs were cytotoxic, while U-Fe3O4 NPs and OC-Fe3O4 NPs and TiO2 NPs induced an oxidative stress in endothelial cells. Only TiO2 NPs induced damage to DNA. Even if endothelial cells were able to internalize NPs, no direct transport across an endothelial cell layer and no modification for the adhesion of cells of the immune lineage were demonstrated. Aim of sub WP 2.3: the liver: The liver is a heterogeneous organ and the major site for biotransformation and defense against foreign materials and xenobiotics. Thus, in assessing the risk of hepatotoxicity of NPs, in particular cytotoxicity, oxidative stress and genotoxicity, the in vitro interactions between the selected NPs and the most representative liver cells were studied.Strategy: Hepatocytes, Kupffer cells and liver sinusoidal endothelial cells (LSEC) were the cell types chosen for hepatocytoxicity studies. Hepatocytes represent more than 70% of liver cells while Kupffer cells and LSEC are also relevant due to their phagocytic phenotype and fenestrated structure, respectively. Summary of results:a) Liver cell isolation: the preparation of hepatocytes and Kupffer cells were set up with an acceptable purity as revealed by immunofluorescence staining. However the culture of Kupffer cells was limited to 48h. LSEC were not efficiently isolated, even after improving the cell isolation procedures of published protocols.b) Basal cytotoxicity: Comparison of basal cytotoxicity of PLGA-PEO and Fl-SiO2 NPs in liver macrophages and hepatocytes showed that Kupffer cells were more susceptible to NPs toxicity than hepatocytes. The differences may be due to a different internalization pattern of the NPs. c) Measurement of NP-induced oxidative stress with the DCFH-DA probe resulted in interference of the particles with the dye. Despite that free radical formation could be observed for both Fe3O4 NPs in the two cell types while TiO2 NPs only induced oxidative stress in Kupffer cells.d) Genotoxicity: DNA damage was also determined in these cells. TiO2 NPs were the only NPs inducing genotoxicity in a dose-dependent manner in hepatocytes. In contrast, all tested NPs but PLGA-PEO NPs caused a genotoxic effect in Kupffer cells even after a short time exposure (4h).Conclusions: In summary, Kupffer cells are the liver cells initially exposed to NPs and the most susceptible to NP toxicity. Oxidative stress was shown for Fe3O4 and TiO2 NPs in hepatocytes and Kupffer cells, respectively. DNA damage was more relevant in Kupffer cells than in hepatocytes even after exposure to low concentrations of NPs and for a short period of time. Aim of sub WP2.4: the respiratory system: Lung epithelial cells are the first target cells after inhalation but also secondary targets after injection of NPs especially at the alveolar level due to the small distance between the epithelial cells and the capillary.Strategy: The effects of NPs (up to 75 μg/cm2) were determined after 24 and 48 h of exposure o the bronchial (16HBE) and alveolar (A549) cells. Endocytosis was evaluated by measuring the right angle scattering of the flow cytometer laser, oxidative stress by DHE oxidation in flow cytometry, anti-oxidant enzyme mRNA expression by RT-qPCR, metabolic activity and membrane integrity, by WST-1 and propidium iodide uptake, respectively, genotoxicity by the CA, and the inflammatory response by measuring cytokine mRNA expression by RT-qPCR. Summary of results: First the suitability of the assays to test the toxicity of NPs was controlled. ELISA and neutral red uptake are prone to interferences with NPs, thus the WST-1 and flow cytometric analysis methods were used to avoid interferences. The comparison of the dose-response analysis (WST-1, PI uptake, DHE oxidation) revealed that OC-Fe3O4 NPs induced the greatest responses whereas U-Fe3O4 NPs are only slightly toxic, suggesting a coating effect. Conclusions: In summary, based on the results of the thorough dose response studies a testing strategy could be proposed for the lung. PLGA-PEO NPs can be used as negative control NPs. TiO2 NPs are relevant positive controls but SiO2 NPs revealed even better candidates as they induce greater responses and interfere less with the assays. Aim of sub WP2.5: the placenta: The objective of this sub-WP was to determine whether NPs can cross the placenta to reach the fetus and to evaluate the interaction of NPs with placental cells in terms of cytotoxicity, oxidative stress, and DNA damage, in order to consider potential impact of exposure to engineered NPs on the fetus during pregnancy.Results: The human BeWo b30 placental cell line was selected for in vitro study utilizing the Transwell model (polyester membrane, 3 μm pore diameter) for transport studies (up to 24h) and compared results using the ex vivo isolated dually perfused human placenta model (up to 6h). For toxicity studies, cells were exposed in complete culture medium to dispersed NPs from 0.12 - 75 μg/cm2 and analyzed after 0.5h to 48h of exposure. Summary of resultsa) Placental uptake and transport of NPs: OC-Fe3O4 NPs were rapidly transported across BeWo cells in a dose-dependent manner whereas there was negligible transport of U-Fe3O4 NPs. Both NPs were internalized by cells and increased concentration led to increased cell uptake of U-Fe3O4 but no effect on OC-Fe3O4 uptake. Both sizes of SiO2 NPs were rapidly transported but particle size and concentration did not affect extent of transport or NP uptake. b) NP-induced cytotoxicity, inflammation, oxidative stress and genotoxicity in placental cells: All NPs tested caused significant LDH release but only at 75 μg/cm2 for SiO2 NPs. With WST-1, Fe3O4 were cytotoxic at 3-75 μg/cm2, SiO2 at 75 μg/cm2 and TiO2 and PLGA-PEO NPs were not cytotoxic. IL-6 release was increased with SiO2 (75 ug/cm2) and U-Fe3O4 (15-75 μg/cm2) NPs, decreased with OC-Fe3O4 (75 μg/cm2) and not affected by TiO2 or PLGA-PEO NPs. Exposure to the panel of NPs did not affect apoptosis or necrosis. Conclusion: In summary NPs can cross the placental barrier in vitro but transport is reduced ex-vivo. Overall, OC-Fe3O4 NPs were most cytotoxic and TiO2 most genotoxic. Given the importance of oxidative stress pathways, BeWo cells may not be suitable for toxicity testing.Aim of sub WP 2.6: the gastrointestinal tract: The gastrointestinal tract is important to assess the oral absorption of NPs. Therefore, the in vitro absorption and cytotoxicity of the selected NPs were determined in several in vitro intestinal models that mimic the physiological conditions of the human intestine.Strategy: Caco-2 cells, CacoReady and Caco-Goblet cell systems were selected for this evaluation. When grown in Transwells® devices the cells form polarized monolayers after 21-day of culture. In addition, CacoGoblet are able to secrete mucus mimicking the terminal part of the intestine. Under those culture conditions, two independent compartments are formed, the apical (upper compartment) and the basal (lower compartment) compartments. NPs were applied to the apical compartment and samples from the basal compartment were analyzed at different time-points. Summary of results:a) Uptake studies: The transport of the metallic NPs was analyzed by ICP-MS/MS. Preliminary results showed that only very low amount of Fl-SiO2 NPs were detectable in both cell models. However, neither TiO2, nor U-Fe3O4, nor OC-Fe3O4 NPs could be detected in the basal compartment, probably due to the technical characteristics of the Transwell membranes rather than the incapacity of the NPs to cross the cellular barriers.b) Basal cytotoxicity: Cell viability studies performed with cells seeded in plastic dishes showed that OC-Fe3O4 NPs were cytotoxic for CacoReady cells after 4h and 24h of exposure and that only Fl-25 SiO2 NPs and synthetic amorphous silica NPs were cytotoxic after 24h of exposure. Similar information was obtained for CacoGoblet cells, including cytotoxicity of Fl-50 SiO2 NPs after 24h of exposure. Cell viability studies for cells grown on Transwells were technically not possible.Conclusions: In summary, despite the low cytotoxicity of this set of NPs in these model cell systems, the presence of mucus could protect NP-induced cytotoxicity but does not play any role in NP transport. However, Transwell devices impair transport studies for some types of NPs. A testing strategy is proposed to evaluate the interactions of NPs with intestine cells.Aim of sub-WP2.7: the central nervous system: The central nervous system (CNS) is very sensitive to insults, in particular to oxidative stress, but xenobiotics must be transported across the blood-brain barrier (BBB) to reach the brain parenchyma. Therefore it is of particular interest and relevance to evaluate if and which NPs can get access to this organ, and what might be the consequences of the interactions of NPs with cells forming the BBB, the immune defensive cells of the CNS, the microglial cells, or with cells of the astrocytic lineage.Strategy: For the experiments human brain-derived endothelial cells, murine N11 microglial cells and human LN229 glioblastoma cells were selected as representative cells of the brain. The cells were exposed in complete culture medium to increasing concentrations from 0.4 μg/ml (0.12 μg/cm2) to 235 μg/ml (75 μg/cm2) of dispersed NPs and analyzed after up to 72h of exposure. Summary of resultsa) Interaction of NPs with cerebral endothelial cells: Human brain endothelial cells dose-dependently and time-dependently internalized all NPs with the exception of OC-Fe3O4 NPs, but none were released or transported by the cells following uptake. TiO2 and Fe3O4 NPs were cytotoxic, induced an oxidative stress and/or DNA damage in endothelial cells.b) Evaluation of the effects of NPs on microglial and astrocytic cells: Only U-Fe3O4 NPs, but neither OC-Fe3O4 nor Fl-SiO2 NPs were taken up by N11 microglial cells and LN229 glioblastoma cells, but only OC-Fe3O4 NPs displayed cytotoxicity for N11 and LN229 cells. No oxidative stress reaction could be demonstrated with these cells.Conclusion: In summary, NPs can gain access to the CNS if the BBB is disrupted, since brain endothelial cells can internalize NPs but cannot transport them. The internalization of the NPs by brain endothelial cells, in particular for metallic-core NPs, results in cytotoxicity, the induction of an oxidative stress and potential genotoxicity for the cells.Aim of sub WP2.8: the kidney: The accumulation and elimination of engineered NPs may be via the kidney. The objective of this sub-WP was to evaluate the interactions of the selected NPs with representative cells of the kidney addressing cytotoxicity, oxidative stress, DNA damage, cell uptake and subsequent release and transport of the NPs across epithelial kidney cell layers.Strategy: From several possible cell lines available, the MDCK (distal tubule) and LLC-PK (proximal tubule) porcine kidney epithelial cells, human embryonic kidney HEK293 cells and the Cos-1 monkey kidney fibroblasts were selected. The cells were exposed in complete culture medium to concentrations from 0.4 μg/ml (0.12 μg/cm2) to 235 μg/ml (75 μg/cm2) dispersed NPs and analyzed after 0.5h to 72h of exposure.Summary of resultsa) Uptake, subsequent release and transport of NPs by kidney cells: The uptake by the MDCK and LLC-PK cells of U-Fe3O4 NPs, but not OC-Fe3O4 NPs was time dependent, cell line dependent and proportional to the amount of NPs added. Significant release of U-Fe3O4 NPs by MDCK cells but not by LLC-PK cells was observed, following uptake. The uptake, but not the release by the cells of Fl-25 SiO2 NPs, but not Fl-50 SiO2 NPs, was observed only at the highest concentration tested. b) NP-induced cytotoxicity, oxidative stress and genotoxicity in kidney cells: OC-Fe3O4 NPs were the most cytotoxic NPs among all tested NPs, already cytotoxic at the highest tested concentration after 24 h. TiO2 NPs were the only NPs tested inducing the production of high amounts of ROS in exposed MDCK and LLC-PK cells, whereas U-Fe3O4 NPs induced a significant decrease of cellular thiol content in MDCK cells and in LLC-PK cells. Conclusion: In summary epithelial renal cells can take up and release, but not transport, NPs following their uptake. OC-Fe3O4 NPs and TiO2 NPs were the most damaging NPs for renal cells, considering cytotoxicity, oxidative stress and DNA damage. A testing strategy is proposed to evaluate the interactions of NPs with representative cells of the kidney.Aim of sub WP2.9: automation. In vitro assays proposed by the NANOTEST partners had to be evaluated for automation feasibility and implemented on the robotic platform for high throughput screening (HTS) with data generation on all NPs identified by NANOTEST.Strategy: The robotic platform was tested for automation and simultaneous screening of several NPs, including the development of a generic dispersion protocol for the different NPs, an accurate serial dilution of the NPs, treatment of cells and measurement of relevant endpoints.Summary of resultsa) Oxidative stress (ROS production): PLGA-PEO NPs and Endorem showed no cytotoxicity and did not induce oxidative stress while with TiO2 NPs a trend towards cell toxicity was observed possibly due to the significant increase in ROS. SiO2 NPs had a clear cytotoxicity effect but not ROS mediated. Fl-25 SiO2 and Fl-50 SiO2 NPs were both cytotoxic with the 25 nm NP having a bigger effect. b) Genotoxicity (double strand breaks): PLGA-PEO NPs and Endorem revealed no cytotoxicity or genotoxicity at the time points and concentrations tested. TiO2 NPs had no effect on cell viability but induced DNA breaks to different extents. SiO2 NPs led to variable results between experiments but induced a clear DNA damaging effect at high exposure. Conclusion: The data generated via HTS and HCI for the endpoints oxidative stress and genotoxicity are in concordance with the observations of the NANOTEST partners: OC-Fe3O4, Fl-25 SiO2 and TiO2 NPs are damaging for cells in various degrees and the cause may be attributed in some cases to oxidative stress or DNA damage. Aim of CCT-1: Cytotoxicity: The objective of this CCT was to determine whether NPs induce cytotoxicity and which assay is the most sensitive and appropriate for in vitro toxicity testing of NPs. Cytotoxicity is basal toxicity where basic functions of cells are affected and thus result in cellular damage. Strategy: The cytotoxicity and determination of LC50 can be evaluated by measuring different endpoints. We used three types of assays to measure:a) cellular metabolic activity;b) membrane integrity; andc) cell number and cell proliferation.Membrane integrity was determined by LDH assay, Trypan blue exclusion, Neutral red uptake, and Propidium Iodide uptake.Summary of resultsa) Development of sets of cytotoxic tests for NPs cytotoxicity testing. Comparison of assays: All cytotoxicity assays were able to detect dose response. However, results showed that colorimetric assays such as MTT, WST-1, LDH, Neutral red uptake are likely to interfere with NPs and need protocol adaptation and additional control to avoid false positive results. On the other hand cell number - proliferation assays (DNA synthesis, RGA and PE assays) do not show any interference, but are more tedious. b) Evaluation of the cytotoxic effects of NPs and cell sensitivity: All NPs except PLGA-PEO NPs exhibited some level of cytotoxicity. OC-Fe3O4 NPs were the most cytotoxic NPs followed by TiO2, U-Fe3O4 Fl-SiO2 25, Fl-SiO2 50, PLGA-PO NPs and Endorem. Conclusion: In summary, the cytotoxicity induced by NPs depends on the NPs, the cytotoxicity test and the cell type. Testing strategy of NPs should contain at least three different cytotoxicity tests and 2 representative cells per organ.Aim of CCT-2: Oxidative stress: The objective of this CCT was to determine whether NPs can generate an oxidative stress and which assays permit the best evaluation of ROS production, thiol depletion and antioxidant enzymes expression.Strategy: The intracellular production of ROS was evaluated using two methods: DHE oxidation in fluorescence multiwell plate reader or flow cytometry and DCFH-DA assay in fluorescence plate reader. Thiol depletion was measured using the monobromobimane assay. The induction of anti-oxidant enzymes (SOD-2 and HO-1) mRNA expression was measured by RT-qPCR. Summary of results- Lung: The flow cytometric protocol for measuring DHE oxidation was used successfully unlike the fluorescent plate reader to evaluate the intracellular production of ROS induced by the panel of NPs. TiO2 NPs induce an oxidative stress in both cell lines only at cytotoxic concentrations. In comparison PLGA-PEO NPs, despite their non-cytotoxicity, induced a weak production of intracellular ROS. For the other NPs, all of them were able to induce a dose-dependent increase of ROS. - Kidney: DCFH-DA and monobromobimane assays were used. TiO2 NPs were the only NPs tested inducing the production of high amounts of ROS in exposed MDCK and LLC-PK cells whereas U-Fe3O4 NPs induced a significant decrease of cellular thiol content in MDCK cells and in LLC-PK cells.- CNS: TiO2 and Fe3O4 NPs induced an oxidative stress in endothelial cells but not in microglial and astrocytic cells.- Vascular system: The three solid-core metallic NPs, but not PLGA-PEO or SiO2 NPs, induced an oxidative stress in endothelial cells. A decrease of cellular thiol levels was observed in all three endothelial cell lines after exposure to Fe3O4 NPs, but not to TiO2 NPs.- Liver: Measurement of NP-induced oxidative stress with the DCFH-DA probe resulted in interference of the particles with the dye. Conclusions: In summary,1. The oxidative stress induced by NPs depends on the cell type and on the NPs. PLGA-PEO NPs induced no or very weak oxidative stress in the entire cell types instead of solid-core metallic NPs which generally induced ROS production.2. The detection of fluorescent probes by flow cytometry avoids interferences of free NPs but takes more time than detection by fluorescence plate readers and could not be used for high-throughput screening. DHE assay is well adapted to flow cytometry measurement and DCFH-DA and bromobimane assays to microfluorimetry. Aim of CCT-3: Transport: The objective of this CCT was to determine whether NPs can cross specific barriers to be further disseminated throughout the body (e.g. via the gut) or to reach particularly sensitive areas e.g. brain or fetus. Strategy: The following organs were evaluated for NP barrier function using representative cell lines: lung (16HBE, NCl-H292, Calu 3); placenta (BeWo b30); gut (CacoReady and CacoGoblet); blood brain barrier (BBB) (HCEC); kidney (MDCK and LLC-PK).Summary of resultsa) Lung: Only Calu 3 cultures were suitable for transport showing a network of tight junctions (TJ) and elevated TEER values. With fl-TiO2 NPs greater than50% were trapped in the membrane and only 1% detected in basal compartment demonstrating that Transwell inserts are not suitable for TiO2 NP study.b) Placenta: rapid dose-dependent transport in vitro of OC-Fe3O4 but not U-Fe3O4 NPs. Both NPs were internalized by cells and increased dose led to increased cell uptake of U-Fe3O4 but no effect on OC-Fe3O4 uptake. Both sizes of SiO2 NPs were rapidly transported but particle size and concentration did not affect extent of transport or NP uptake. NP exposure did not affect barrier permeability. In the perfused placenta, there was no transport of OC-Fe3O4 NPs and very limited transport of SiO2 NPs.c) BBB: Human brain endothelial cells dose-dependently and time-dependently internalized all NPs with the exception of OC-Fe3O4 NPs, but none were released or transported by the cells following uptake.d) Gut: both Caco models transported SiO2 NPs but transport was not evident for Fe3O4 NPse) Kidney: epithelial renal cells could take up and release, but not transport, NPs following their uptake.Conclusion: In summary NP transport is most affected by tightness of the cell barrier: transport across BBB less than kidney less than gut less than placenta.Aim of CCT-4: Immunotoxicity: The objective of this CCT was to determine whether NPs may induce immunotoxic effects and which assays are sensitive and appropriate for in vitro immunotoxicity testing of NPs. Strategy: Immune system is complex network of cooperating cells, therefore panel of assays have been proposed for monitoring: Natural cellular immune response - phagocytic activity and respiratory burst of leukocytes, natural killer cell activity. Acquired cellular immune response: proliferative activity of lymphocytes in vitro stimulated with mitogens and CD3 antigen. Non-functional assays: immunophenotypic analysis of leukocytes, expression of adhesion molecules on leukocytes. Humoral immune response: interleukins.Cell components used to evaluate the immunotoxicity induced by NPs:1) human peripheral blood cells from volunteers were exposed in complete culture medium to increasing concentrations of dispersed NPs from 0.12 μg/cm2 to 75 μg/cm2 and analyzed after up to 72h of exposure.2) Rat peripheral blood and spleen cells from exposed animals. Female Wistar rats have been intravenously exposed to single dose of TiO2 NPs: 0.59 mg/kg or 3 doses of OC-Fe3O4 NPs: 0.03642 mg/kg, 0.3642 mg/kg, 3.642 mg/kg and killed 1 day, 1 week, 2 weeks and 1 month after exposure. Summary of results: Comparison of in vitro and in vivo findingsPhagocytic activity of granulocytes: In vitro, both tested NPs stimulated phagocytic activity of granulocytes. In vivo - significant stimulatory effect of TiO2 and no significant alterations in animals exposed to OC-Fe3O4. Stimulatory effect of in vivo exposure to TiO2 NPs is in agreement with in vitro studies. Phagocytic activity of monocytes: in vitro, high dose of OC-Fe3O4 NPs suppressed the cell function. Proliferative response of lymphocytes: Basal response: in vitro as well as in vivo exposure to TiO2 NPs significantly stimulated basal proliferative activity of peripheral blood cells. T-lymphocyte response: stimulatory effect of TiO2 NPs on proliferative activity of T-lymphocytes (stimulated with Con A) is in agreement with in vitro study. Conclusion: In vitro model of human peripheral blood cells reflected to the certain extent in vivo response of animal peripheral blood immune cells to TiO2 and OC-Fe3O4 NPs exposure seen in exposed rats.Human peripheral blood cells can be used as in vitro model for assessment of immunotoxicity. In first tier, cytotoxicity assays should be used to identify non-cytotoxic concentrations of the NPs for in vitro studies. Moreover, including positive and negative controls in both in vitro and in vivo models is strongly recommended. Aim of CCT-5: Genotoxicity: The objective of this CCT was to investigate the potential genotoxic effects of NPs and to evaluate suitable models for genotoxicity. Genotoxic compounds can be mutagenic and thus potentially carcinogenic. Strategy: the following assays and endpoints were used to evaluate genotoxicity of NPs - the CBMN assay to detect mutagenicity, clastogenicity and aneugenicity; the CA to detect strand breaks, FPG modified comet (FPG CA) for oxidised DNA lesions (oxoGuanine and Fapy derivates); phosphorylation of Histone2A.X (pH2AX) assay for double strand breaks. The following organ-representative cells were evaluated for NPs-induced genotoxicity: blood (TK6, human peripheral blood lymphocytes); kidney (MDCK and LLC-PK, Cos-1 and HEK293); vascular system (endothelial cell line Ecp23 and EC219); lung (16HBE); placenta (BeWo); liver (hepatocytes and Kupffer cells); CNS (HCEC); 3T3 mouse fibroblasts for automation procedures.Summary of resultsBlood: TK6 cells and peripheral blood lymphocytes were used to evaluate genotoxicity (using CBMN and CA/ FPG CA assays). The CA: a) Exposure of TK6 cells to U-Fe3O4, PLGA-PEO, Fl-25 SiO2 and Fl-50 SiO2 and Endorem NPs did not cause genotoxic effect. TiO2 NPs induced DNA damage in TK6 cells dependent on the dispersion protocol used. OC-Fe3O4 NPs increased SBs and oxidized bases. b) CA on peripheral blood lymphocytes confirmed the results o TK6 cells.Vascular system: TiO2 NPs induced DNA damage, but U-Fe3O4, OC-Fe3O4 and both-sized Fl-SiO2 NPs induced only slight DNA damage whereas PLGA-PEO NPs were negative.Liver: TiO2 NPs were the only NPs dose-dependently inducing genotoxicity in hepatocytes. All tested NPs but PLGA-PEO NPs caused a genotoxic effect in Kupffer cells even after a short time exposure (4h). DNA damage was also more relevant in Kupffer cells than in hepatocytes.Lung: DNA damage was significantly induced only after Fl-25 SiO2 NPs exposure.Placenta: CA results showed a dose related response to TiO2 NPs in BeWo cells. U-Fe3O4 (75 ug/cm2) and Fl-25 SiO2 NPs induced only mild DNA damage. OC-Fe3O4 NPs did not induce genotoxicity.Central Nervous system: TiO2 and Fe3O4 NPs induced DNA damage in endothelial cells.Kidney: PLGA-PEO and U-Fe3O4 NPs did not cause genotoxic effects using the CA in Cos-1 cells. In contrary OC-Fe3O4 NPs induced a mild but significant increase of DNA damage and oxidative DNA lesions. Fl-25 SiO2 and Fl-50 SiO2 NPs caused slight increase in SBs and oxidized bases in Cos-1 cells. TiO2 NPs caused DNA damage (HEK293, Cos-1) but the effect was dependent on the NPs dispersion protocol used.Conclusion: Genotoxicity induced by NPs depends on the cell type, the NPs, the dispersion protocol and the measured endpoint. It is crucially important to use non-cytotoxic concentrations when assessing genotoxicity. Both CBMN and CA, especially with lesion specific enzymes, can give a reliable picture of potential genetic instability as they measure different endpoints, while pH2AX assay is an interesting end-point for automated procedures.WP3 In vivo studies to validate the in vitro findingsAim of WP3: The objective of the in vivo study was to validate the findings of the alternative in vitro assessment of the toxicological profile of NPs used in diagnostics or therapeutics, as planned by WP2, by experiments on animals.Strategy: First the acute toxicity study was performed in order to define the LD50 for TiO2 NPs and OC-Fe3O4 NPs after intravenous (i.v.) administration to adult female rats. The study was performed according to OECD guidelines 425. LD50 for TiO2 NPs was established to be 59.22 mg/kg with confidence interval from 55 to 70 mg/kg. For OC-Fe3O4 NPs, the LD50 was 36.42 mg/kg with confidence interval (0 - 20 000 mg/kg). In the in vivo experiment, animals were divided into five groups: negative control (vehicle); reference control - i.v. administered TiO2 NPs in a dose equal to 1% of LD50/ kg body weight; and three exposed groups receiving OC-Fe3O4 NPs in doses: 0.1 1 and 10% of LD50/kg body weight (established in the acute toxicity study). The following biomarkers were measured:- cardiotoxicity and hepatotoxicity (oxidative phosphorylation parameters in isolated heart and liver mitochondria: oxygen consumption after stimulation by ADP, basal oxygen consumption, rate of oxidative phosphorylation, mitochondrial membrane integrity, coupling of oxidation with phosphorylation, measurement of complex I activity by the use of NAD substrate glutamate or malate, concentrations of oxidised forms of coenzymes Q- CoQ9ox and CoQ10ox, and alpha-tocopherol levels in isolated myocardial and liver mitochondria, contents of cholesterol and triacylglycerols in the liver tissue);- damage in lung tissue cells (inflammatory bronchoalveolar lavage (BAL) biomarkers: count of alveolar macrophages (AM), differential count of cells (AM, granulocytes and lymphocytes), immature forms of AM, multinucleated lung cells and total amount of protein; cytotoxic BAL parameters: phagocytic activity of AM, viability of AM, lactate dehydrogenase activity (in cell - free lavage fluid), acid phosphatase activity (in cell - free lavage fluid and in BAL suspension), cathepsin D activity (in cell - free lavage fluid and in BAL suspension);- nephrotoxicity (plasma concentration of glucose, albumin, creatinine, urea, bilirubin, total cholesterol, triacylglycerols, sodium, potassium, calcium, magnesium, phosphate, iron; enzyme activities of AST, ALT, GTT, lipase, and creatinine kinase), and urine chemistry (concentration of urea, creatinine, sodium, potassium, calcium, magnesium, phosphate), creatinine and urea clearance was calculated, renal excretion of urea, creatinine, minerals, and ions and protein excretion per 24 h was calculated, fractional excretion of ions and minerals was calculated, plasma advanced oxidation protein products (AOPPs), advanced glycation end-products, plasma immunoreactive insulin, rat-specific hsCRP, carbonyl-proteins and rat-specific asymmetric dimethylarginine and kidney injury molecule-1 (KIM-1) and calbindin concentrations in urine were measured, TGF- 1, collagen IV and TNF-a gene expression in kidney cortex homogenates were measured);- basic haematological examination was undertaken at sacrifice (erythrocytes, leukocytes, platelets, hemoglobin, hematocrit)- organ weight (kidneys, liver, spleen, brain, heart, lungs), organ-to-body weight ratio was determined;- genotoxicity (CBMN test in bone marrow, CA detecting strand breaks (SBs), oxidative DNA damage and sensitivity of DNA to hydrogene peroxide in white blood cells by the CA;- oxidative stress (concentrations of malondialdehyde (MDA) and activities of antioxidant enzymes (glutathione peroxidase (GPx), catalase, glutathione S-transferase, superoxide dismutase (SOD) and ceruloplasmin oxidase) in liver, lung, brain, heart tissues and in blood, levels of reduced glutathione (GSH) and vitamins tocopherol, tocopherol, carotene, retinol, xanthophyll and lycopene ) as non-enzymatic antioxidants in blood);- immunotoxicity - natural cellular immunity: phagocytic activity of granulocytes and monocytes and respiratory burst of phagocytes, specific cellular immunity: lymphoproliferative assay (LTT), lymphocyte subsets in peripheral blood using flow cytometry, expression of adhesion molecules CD11b and CD54 on peripheral blood leukocytes, humoral immunity: levels of tumour necrosis factor alpha, interleukin-10 and interleukin-4 in blood and spleen cell supernatants);Summary of results:Cardiotoxicity and hepatotoxicity: Changes in functional parameters of heart mitochondria, CoQ and a-tocopherol content in the groups of 0.1% and 10% OC-Fe3O4 NPs could be caused by adaptation of the organism to short time exposure (1 day, 1 week). After longer exposure, there were no significant changes in these parameters. Our results did not indicate severe damage of heart mitochondria after all.Oxygen consumption after stimulation by ADP, basal oxygen consumption and rate of oxidative phosphorylation (rate of ATP production) were significantly increased after 2 weeks of exposure in liver mitochondria in the group of 10 % OC-Fe3O4 NPs. However, according to cholesterol and triacylglycerol concentrations in the liver tissue, the administered NPs did not cause any serious toxic liver damage.Damage in lung tissue cells: The most expressive response after exposure to NPs compared with the corresponding control was found in acute phase (1 day after exposure). 7 and 14 days after exposure, less pronounced response of bronchoalveolar lavage (BAL) parameters was found, but still the response was comparable with that, found one day after exposure. 28 days after exposure, BAL parameters were moderately changed in comparison with control; significant differences between rats exposed to 0.1%, 1% and 10% LD50 OC-Fe3O4 NPs and control animals disappeared. The highest biological activity was recorded in rats administered with high dose 10% LD50 OC-Fe3O4 NPs. TiO2 NPs influenced BAL parameters in less extent - but its biological effect is not negligible. Significance of differences in inflammatory BAL parameters after longer period of time after exposure (28 days) was decreasing. The cytotoxic parameters showed increasing trend with time after exposure. The lack of significance could be affected by large interindividual differences in animals.Basic haematological examinations and renal toxicity: Administration of NPs did not affect significantly the metabolic parameters studied (glycaemia, insulin-to-glucose ratio, lipid profile, liver enzyme activities and creatine kinase activity). No clinically significant differences among the groups were observed in parameters characterizing renal function (plasma urea and creatinine concentration, creatinine and urea clearance regardless of the way of their expression, renal excretion of urea, creatinine, proteins, minerals and ions, as well as in fractional excretion of ions and minerals). Our findings suggest some disturbance in ions and minerals handling one week after exposure to NPs, but these changes were not systematic, and appeared to be within the normal ranges. Genotoxicity: Neither mutagenic (incidence of micronucleated immature erythrocytes) nor cytotoxic (decrease in the proportion of immature erythrocytes) effects were found when testing the selected NPs in mammalian erythrocyte micronucleus test in bone marrow of animals. The selected NPs in tested doses were non mutagenic or the negative result was a consequence of the fact that the NPs did not reach the target tissue (bone marrow) and this test is not appropriate to use in this case.Oxidative stress: Most significant differences after OC-Fe3O4 NPs exposure were observed after 1 day in brain and after 1 week in liver and lung. Most sensitive biomarkers were the activities of GPx and SOD. Regarding the results of measurements in blood the decreases in concentrations of non- enzymatic antioxidants were most notable after 4 weeks of exposure.Immunotoxicity: In exposed animals, immunomodulatory effect of TiO2 and OC-Fe3O4 NPs manifested very early, from one day to one week after exposure. In first phase, phagocytic activity and respiratory burst of leukocytes was significantly altered by the exposure to NPs. While stimulatory effect of TiO2 NPs on phagocytic function of granulocytes and monocytes and respiratory burst of phagocytes was found, high dose of OC-Fe3O4 NPs suppressed phagocytic activity of monocytes. Moreover, one week after exposure, treatment of animals with OC-Fe3O4 NPs increased percentage of (CD8+) cytotoxic T-cells in peripheral blood of exposed rats. Enhanced levels of interleukin-10 (produced primarily by monocytes) found in spleen cell cultures in rats exposed to low dose of OC-Fe3O4 NPs might indicate the effort to regulate immune response. Later, 4 weeks after exposure, functionality of the lymphocytes was changed measured by proliferative activity after stimulation of the cells with panel of mitogens and CD3 antigen. Conclusion: Our in vivo study documented that single i.v. administration of TiO2 NPs and OC-Fe3O4 NPs in above mentioned doses to young female rats did not elicit overt acute or subacute toxicity. Subtle differences in some parameters between the control and NPs administered groups were revealed mainly short time after exposure to NPs. These findings indicate possible immunomodulatory effect of single intravenous exposure to OC-Fe3O4 less than TiO2 NPs in exposed animals.WP4 Structure Activity and PBPK modelingAim of sub WP4.1: Structure-Activity Modelling: The objective of this task was to explore the feasibility of developing structure-activity models for NPs. This was interpreted in a broad sense to include any relationship between the structure of NPs (using suitable descriptors) and their (toxicological) effects at any level of biological organisation.Results: Development and evaluation of a theoretical model for predicting the oxidative stress potential of metal oxide NPs. Following a preliminary review of the literature, it was clear that traditional statistically-based QSAR modelling, which requires multiple chemical descriptors and a reasonable number of high quality biological datapoints, would not be feasible. Therefore, a more theoretical approach was adopted. Building on previous findings (Meng et al, 2009) linking the oxidative stress potential of NPs with adverse outcomes, including inflammation, cytotoxicity, and in vivo toxicities, a theoretical model was developed (Burello and Worth, 2011) for predicting the reactivity of metal oxide NPs as well as their ability to cause oxidative stress through the generation of ROS. Conclusion: The theoretical model for predicting oxidative stress potential could be used in the assessment of NPs by prioritising metal oxides for further evaluation, and by forming part of a more extensive battery of models and in vitro tests for characterising metal oxide toxicity.Resultsa) Integration of a lung transport & deposition model and a lung clearance/retention model to predict internal doses by inhalation exposure data: The combination of the deposition model with the clearance model allows for the connection of the external exposure to the internal dose, though further research is required on the extension of this model to account for the distribution of the inhaled particles to other targets organs in the human body. This will be feasible in time, as the particle transport mechanisms into the body become clearer and the experimental data increase. Overall, the presented study demonstrates that the combination of representative measurements of the full size spectrum of the inhaled aerosol, mechanistic mathematical modeling of deposition and of clearance/retention and in vitro toxicological assays can lead to derivation of exposure limits and risk assessment.b1) Employment of CFPD to estimate particle behavior in the respiratory system: Transport and deposition of particles in a physiologically based bifurcation created by the 3rd and 4th lung generations, was calculated for different flow conditions and particles sizes using an in-house computational model. The study showed that total deposition fraction increases with increasing particle size, but does not change significantly for the different flow conditions. These results are in agreement with earlier experimental findings. Moreover, it was shown that there is significant deposition both at the bifurcation and the walls of the daughter tubes. In the latter case, the deposition sites and the particle concentration profiles change significantly between the different flow cases. In the next step, particle transport and deposition will be studied in real lung geometries obtained by imaging techniques (CT-scans, MRI).b2) Employment of CFPD to estimate particle behavior in the cardiovascular system: The computational model was used to predict the transport and deposition of superparamagnetic particles suspended in a liquid under the influence of an external magnetic field. Particle deposition fraction was calculated for different liquid velocities and viscosities, as well as for different magnetic fields and the results were compared to previous experimental findings. In all examined cases the fully Eulerian model describes successfully the qualitative characteristics of the experimental deposition fraction curves, although in some cases underestimates deposition. This is an indication of missing deposition mechanism and further studies are in order.Conclusion: In summary, all the above studies showed that with proper refinement the developed computational models and methodologies, may serve as an alternative testing strategy, replacing experiments that are expensive both in time and resources.Strategy and suggested battery of testsOverall aimThe overall aim of NANOTEST was to provide a testing strategy for NPs used in medical diagnostics. The specific objective of the project was to develop a set of Master SOPs for at least 2 assays for each type of toxicity. The most advanced and standardised techniques will be adapted for automation and prepared for validation. NANOTEST aimed to provide testing strategies for hazard identification and risk assessment of NPs, and to propose recommendations for evaluating potential risks associated with new medical NPs. A battery of assays that can be directly applied to fulfill regulatory requirements (REACH) will also help to decide whether new regulations are needed for risk assessment of NPs. In vitro and in silico methods will have an impact on the use of animals for toxicity testing. The development of these methods and strategies can also be utilised for the assessment of health effects of NPs used and applied in other areas (cosmetics, etc.) and thus can have wider impact on all 3 Rs (Replacement, Reduction and Refinement).Testing strategyOne of the main obstacles for assessing the toxicity of nanomaterials is the lack of knowledge of how physicochemical properties relate to the interaction of NPs with biological system and the mechanism of toxicity. It is clear that physical and chemical properties can influence NP behaviour and may have an impact on toxicity; they must therefore be an integral part of toxicity testing. This is one of the key aspects of toxicity screening strategies (Dusinska et al. 2009, 2011, 2012, 2013). Both primary and secondary characterisation of tested NPs are crucial, including in situ characterisation during exposure. The physico-chemical properties that should be considered for assessing toxic effects of nanomaterials include as a minimum chemical composition, particle size, shape, surface properties, size distribution, agglomeration state and crystal structure. Regarding the likelihood of biomolecular corona formation, it is also important to set up experimental conditions that can mimic exposure in humans. As NPs change their properties depending on surrounding milieu we recommend at least two different exposure conditions for testing the NPs effects (Magdolenova et al., 2012a).From the evaluation of the different cell models, depending on route of exposure and use of NPs there should be several organ models used for testing. Blood is an important model both as a direct target as well as surrogate target tissue and gives an indication of toxicity. Peripheral blood lymphocytes are suitable cells but unfortunately not always accessible, thus the TK6 (lymphoblastic) cell line is an alternative. We additionally propose that commercially available human cell lines for each representative organ be included in the testing strategy e.g. for lung cells available cell lines (A549 cells is one alternative), CaCo2 cells (colon), LN229 cells (glioblastoma), and HEK293 or MDCK (porcine kidney). The strategy proposed is:1) to determine possible cytotoxicity and induction of oxidative stress- For cytotoxicity studies: basal cellular toxicity tests such as RGA and PE and the MTT and WST-1 assays and a time course of 24 h and 72 h, using OC-Fe3O4 NPs as positive control NPs and PLGA-PEO NPs as negative control NPs. However, the cytotoxicity of Fe3O4NPs seems to depend on the coating rather than the iron oxide core itself.- For oxidative stress: the thiol depletion measured by monobromobimane assay (and possibly DCFDA) and the induction of antioxidant enzyme mRNA expression measured by RT-qPCR (Guadagini et al., 2013b), 4 h and 24 h time-course, using uncoated TiO2 NPs as positive control NPs and PLGA-PEO NPs as negative control NPs2) then to determine the uptake and possible release, following uptake, of the NPs by relevant cells of the different organs, at non cytotoxic concentrations of the NPs,- For uptake and release studies: 24 h uptake followed by 24 h release, using U-Fe3O4 NPs as positive control NPs.- For transport studies: 24 h time-course, using OC-Fe3O4 NPs as positive control NPs, limiting these experiments to NPs which do not agglomerate in the culture conditions.3) then, to determine possible genotoxic effect (see review Magdolenova et al., 2012b,2013)- For DNA damage: cells exposed for 24h to NPs, using the Comet assay for DNA SBs and oxidized DNA lesions (TiO2 or OC-Fe3O4 as positive control NPs and PLGA-PEO NPs as negative control NPs, at non-cytotoxic concentrations of NPs).- For mutagenicity and clastogenicity: CBMN modified protocol for NPs genotoxicity testing. However, positive and negative controls should be further specified.4) The H2AX assay is an interesting end-point for automated procedures, but little information so far exists about predicting NP-induced genotoxicity using this test. The following experimental testing strategy is proposed for assessment of immunosafety of newly developed NPs. Human peripheral whole blood or isolated peripheral blood mononuclear cells (PBMC) as representatives of human blood cell model are proposed for in vitro screening of the immunotoxic potential of nanoproducts. The main strength is the complexity of the model containing several cell components in a relatively intact environment. Testing strategy for assessment of immunotoxic effect of NPs should contain a panel of immune assays to cover several aspects of immune response.Technical limitations of the assays:a) interference with specific assays was observed for metallic oxide solid core NPs. Thus, the evaluation of possible interference is required to ensure reliable results. This is mainly relevant for cytotoxicity assays, oxidative stress responses of cells, and the production by the cells of bio-molecules such as peptides, proteins, or others (Guadagnini et al., 2013a).b) the release and transport studies are limited to NPs that can be detected at low concentrations in buffers, and are also limited by the physical properties of the membranes used to develop 2-chamber models and to NPs which do not agglomerate under cell culture conditions.Conclusions and remarksFor NP toxicity testing the primary and secondary characteristics of NPs should be included as an integral part of the testing strategy. We proposed that at least 3 cytotoxicity tests (the MTT, WST-1 and plating efficiency assays or RGA), a set of (at least 3-5) representative cells and 5 NPs concentrations are used for each NPs. Initially, cytotoxicity response to the NPs must be determined, then oxidative stress response using at least 2 assays. A testing strategy for assessment of immunotoxic effects of NPs should contain assays covering several aspects of immune response (inducible proliferative response, phagocytic and respiratory burst). For genotoxicity, the modified comet assay for DNA damage (strand breaks as well as oxidised DNA lesions) should be included in the testing strategy together with the micronucelus assay, optionally the H2AX for automated procedures. Potential Impact:The rapid growth in the use of nanomaterials in medicine has led to a concern about possible health risks. There is a serious lack of information available to predict health effects due to nanomaterial exposure. In a regulatory context, nanoscale materials are still mostly treated in the same way as conventional chemicals and there is no consensus or clear nano-specific guideline for their regulation (Dusinska et al. 2009; 2011; 2012; 2013). The NANOTEST project (see http://www.nanotest-fp7.eu online) was one of the initiatives set up by European Commission to fill the knowledge gaps in this area, by studying the interactions of representative therapeutic nanomaterials with living cells, and developing and validating appropriate high-throughput toxicity testing protocols using in vitro models. Keeping in mind the 3 Rs (refine, reduce, replace), the testing strategy should reduce in vivo experiments. To achieve this, a comparison of in vitro and in silico with in vivo results is an important and critical aspect of the validation of in vitro and in silico methods. One of the main problems encountered in testing NPs for possible human hazard include the lack of appropriate standard protocols.In order to develop suitable alternative testing strategies, it was essential to bring together a panel of experts from different disciplines in order to maximise the benefit that can be obtained from different and novel approaches to this problem. This was an opportunity for the cosortium of European researchers to push this important research area forward. This was possible only through the collaboration of highly competent experts in an interdisciplinary network combining the highest levels of European expertise. For NANOTEST to be successful it was essential that we had the necessary critical mass in terms of resources, knowledge and facilities and this could not be achieved at a national level. NANOTEST outcomes are remarkable and their potential impacts are listed below:Impact on toxicity testing of NPs by developing tests and testing strategyThe main goal of the NANOTEST was to develop alternative testing strategies and high-throughput toxicity-testing protocols using in vitro and in silico methods for the assessment of the toxicological profile of NP used in medical diagnostics. The NANOTEST approach focused on eight different target tissues and organs (blood, vascular system, liver, kidney, lung, placenta, digestive, renal and central nervous systems) and crucial toxicology pathways oxidative stress, inflammation, immunotoxicity and genotoxicity. Additionally detailed characterization of NPs was performed. Toxicity and uptake studies, methods development and high-throughput and assays automation allowed us to deliver a battery of assays relevant to targeted organ for the development and validation of suitable biomarkers. Additionally, NANOTEST research highlighted problems connected with interaction of NPs with toxicity tests, proteins and other components in biological fluids or with detection analysis which may impede their detection for the assessment of the biological effects of NP exposure. Contribution to 3RsThe information on toxicity of NPs can be obtained using animals and in vivo experimentation but it can be difficult to isolate the exact mechanisms and toxicological pathways involved in relation to specific NP characteristics in addition to ethical concerns regarding animal use. NANOTEST focused on development of in vitro and ex vivo models in a more efficient manner to define the markers which can determine the toxicological potential of NP, before pre-clinical evaluation of new biomedical nanomaterials. Such an approach clearly decreases the numbers of animals necessary for pre-clinical validation and spares a lot of suffering for animals, and results in a drastic decrease of the number of animals which would be necessary to obtain this information. Extrapolation from in vivo studies in nanotoxicity testing is even more challenging than with chemical toxicology, and due to the enormous variety of NPs being produced, alternative in vitro toxicity tests will have to be considered further. Within NANOTEST the key toxicology endpoints, uptake and transport studies have been addressed together with detailed in situ characterization in tested media using a broad range of human and mammalian cells in vitro. Contribution to European regulation and legislationThe ambition of the proposal was to develop suitable methods and testing strategies which could be further developed as robust automated assays and after validation, integrated into any new regulatory to complement existing European legislation. An approach to the safe, integrated and responsible introduction of nanotechnology into medical practice should thus be included at a fundamental scientific level, to assess all aspects of risk and to contribute to appropriate regulations for this new technology. This approach is fully in line with the Commissions European strategy for nanotechnology set out in the Communication Towards a European Strategy for Nanotechnology and its associated Action Plan. NANOTEST investigated 6 selected NPs and collected new knowledge addressing the key toxicity endpoints. A better understanding of NP kinetics, molecular and cellular mechanisms, pathways of action, and their associations with health effects in exposed cells, organs, animals, and human blood achieved in NANOTEST will benefit medical sciences, provides knowledge required for risk assessments, and will support the definition of guidelines for the safe production, use, and disposal of NP.Contribution to communityNanotechnology is a promising tool for the development of innovative treatment strategies allowing us to overcome obstacles encountered by classical drug delivery. This has led to the development of nanomedicine. Nanomaterials may allow the controlled release of therapeutics, protection of drugs against degradation, targeted drug delivery and facilitated transport across barriers. While a lot of attention has been paid to the development of new engineered nanomaterials and to new applications of nanotechnologies, comparatively less research has been performed to assess the potential hazard of these new materials. There is public concern about the potential health hazards of these new materials, especially as a variety of nanoparticles have been shown to induce toxicity related to their nanometer size leading to the new field of nanotoxicology.Impact on the state of the art and future researchThe NANOTEST project has already had, and will continue to have, impact far beyond its size and budget, in many areas of nanotoxicology research as it has facilitated the development of several insights that will have a durable impact on the toxicology and other research fields. Among the key scientific developments resulting from the project are the understanding of the interaction of NPs with biological systems, interference of NPs with methods detection systems, uptake and transport studies, the different effects of aggregation and agglomeration on toxicity outcomes, development and modifications of methods for NP toxicity testing, etc.Database development and its impactA large database was developed with all results reported on the same template. Data were stored in the project database which comprised a list of the 303 datafiles, along with their contextual information. This database will be included into NANOhub hosted by European Commission's JRC as a source of information for the scientific community and regulators and for later metaanalysis. The NANOhub database is based on the OECD endpoint-related templates. It provides an inventory of information on NPs from various projects.Training and dissemination activitiesDuring the NANOTEST project, several training courses (harmonisation of procedures, handling of nanomaterials), practical courses (genotoxicity course), thematic workshops (as part of annual meetings), scientific workshops (with ENPRA, NanoImpactNet and other projects) and dissemination activities (workshop with stakeholders, conferences, publications) have been provided and stimulated beyond the project itself. This comprises practical laboratory science between disciplines and poles of excellence, e.g. the NPs preparation dispersion protocols, the transfer of suitable methods for toxicological in vitro assessment, database (NANOhub) training.List of Websites:http://www.nanotest-fp7.eu Related documents Final Report - NANOTEST (Development of methodology for alternative testing strategies for the assessment of the toxicological profile of nanoparticles used in medical diagnostics)
