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This report evaluates the training courses delivered under the contract OC/EFSA/SCER/2017/01 ‐ Lot 1. Within the period of January 2018 to February 2022 a total of 21 training courses were provided, eight on‐site training courses in Parma at EFSA, six virtual training courses during the Covid‐19 pandemic and seven eLearning courses comprising various numbers of modules. The courses covered different aspects of chemical and biological risk assessment and related tools, namely i) harmonisation of risk assessment methodologies for human health and ecological risk assessment of combined exposure to multiple chemicals (mixture assessment), ii) risk assessment of the application of nanoscience and nanotechnologies in agro/food/feed (nanotoxicity); iii) science‐based criteria for identifying endocrine disruptors in the context of EU legislation on pesticides and biocides (endocrine disruption); iv) principles on genotoxicity on scientific assessment (genotoxicity) and v) computational toxicology approaches and tools (in silico). All tutors were experts in their field and had previously performed training courses on these topics. The target participants of the training courses were members of EFSA’s Scientific Committee/Panels and their working groups as well as employees from national and international regulatory agencies associated with risk assessment of feed and food compounds. Members of the EFSA Networks as well as EFSA scientific staff also participated in the training courses. Courses were evaluated based on the feedback of participants and continuously improved also by integrating updated or new EFSA guidance documents.

Additional file 1. Radioactivity measurements in organs/tissues of female and male rats.

Additional file 1: Table S1. Summary of the MWCNT characteristics see Taylor-Just for details. Figure S1. FPLM detection of chemically purified MWCNT in absence or presence of fibroblasts. Femtosecond pulsed laser microscopy imaging of chemically purified MWCNT (red) deposited after 24 h in the absence (w/o, left) and presence (right) of MLg fibroblasts (green). Scale bars: 50 μm. Figure S2. FPLM detection of thermally purified MWCNT in absence or presence of fibroblasts. Femtosecond pulsed laser microscopy imaging of thermally purified MWCNT (red) deposited after 24 h in the absence (w/o, left) and presence (right) of MLg fibroblasts (green). Scale bars: 50 μm. Figure S3. FPLM detection of chemically purified MWCNT in absence or presence of macrophages. Femtosecond pulsed laser microscopy imaging of chemically purified MWCNT (red) deposited after 24 h in the absence (w/o, left) and presence (right) of THP-1 macrophages (green). Scale bars: 50 μm. Figure S4. FPLM detection of thermally purified MWCNT in absence or presence of macrophages. Femtosecond pulsed laser microscopy imaging of thermally purified MWCNT (red) deposited after 24 h in the absence (w/o, left) and presence (right) of THP-1 macrophages (green). Figure S5. Validation of the emission signal of the different MWCNTs. (A) Emission fingerprint of the MWCNTs under femtosecond pulsed laser illumination (excitation 810 nm, 80 MHz, about 10 mW laser power on the sample) confirming the white-light generation of each MWCNT. Intensity normalized to the highest value of each spectrum. For each MWCNT, the spectrum represents the mean of 9 spectra (three technical replicates of three experimental replicates). Spectra are recorded from the wells without cells. (B) Comparison of the mean ± standard deviation intensity values of the spectrum recorded between 400 and 410 nm. No significant difference among the MWCNTs (one-way ANOVA followed by Tukey’s multiple comparison test).

Additional file 4. XPS C1s High resolution spectra with the peak positions of the possible bonds. (PDF 176 kb)

Additional file 1. Raman spectra of MWCNTs samples showing the G (~ 1570 cm− 1) and D (~ 1346 cm− 1) mode.

Additional file 2. Curve fitting of first ordered Raman spectra.

Additional file 3. Estimation of ID1/IG ratios from Raman spectroscopy data.

Background Nanomaterials like cerium oxide and barium sulfate are frequently processed in industrial and consumer products and exposure of humans and other organisms is likely. Generally less information is given on health effects and toxicity, especially regarding long-term exposure to low nanoparticle doses. Since inhalation is still the major route of uptake the present study focused on pulmonary effects of CeO2NM-212 (0.1, 0.3, 1.0, 3.0 mg/m3) and BaSO4NM-220 nanoparticles (50.0 mg/m3) in a 90-day exposure setup. To define particle-related effects and potential mechanisms of action, observations in histopathology, bronchoalveolar lavage and immunohistochemistry were linked to pulmonary deposition and clearance rates. This further allows evaluation of potential overload related effects. Results Lung burden values increased with increasing nanoparticle dose levels and ongoing exposure. At higher doses, cerium clearance was impaired, suggesting lung overload. Barium elimination was extremely rapid and without any signs of overload. Bronchoalveolar lavage fluid analysis and histopathology revealed lung tissue inflammation with increasing severity and post-exposure persistency for CeO2. Also, marker levels for genotoxicity and cell proliferation were significantly increased. BaSO4 showed less inflammation or persistency of effects and particularly affected the nasal cavity. Conclusion CeO2 nanoparticles penetrate the alveolar space and affect the respiratory tract after inhalation mainly in terms of inflammation. Effects at low dose levels and post-exposure persistency suggest potential long-term effects and a notable relevance for human health. The generated data might be useful to improve nanoparticle risk assessment and threshold value generation. Mechanistic investigations at conditions of non-overload and absent inflammation should be further investigated in future studies.

Multiwalled carbon nanotubes (MWCNT) show promising technical properties (e.g. composite enforcement; electrical conductivity). MWCNT, meeting WHO criteria, exhibit straight or curved/tangled morphology, depending on length and diameter. Evident fiber toxicity was observed for the rigid fiber type, i.e carcinogenicity in the rat model after intraperitoneal injection. Tangled type MWCNT (not carcinogenic) may show an altered toxic potential regarding fibrotic or genotoxic effects following surface modification with functional groups such as -NH2 or -COOH. The ERA-NET SIINN project ICONS (International Collaboration On Nanotube Safety) aimed at mechanistically evaluating and ranking the pro-fibrotic and genotoxic potential of tangled MWCNT, focusing on impact of core purification and surface functionalization. A batch of industrially relevant Nanocyl NC7000 was therefore chemically or thermally purified and surface functionalized (-COOH and -NH2). At Fraunhofer ITEM, the eight resulting MWCNT (pristine, milled, purified, and functionalized) were tested for sterility and endotoxin. For in vitro use, they were dispersed using an ultrasound-based protocol, and characterized by light and scanning electron microscopy. Subsequent in vitro (geno)toxicity testing with MRC-5 primary human lung fibroblasts revealed differential inhibition of proliferation (RICC, mitotic index), induction of membrane damage and micronuclei, and loss of chromosomes. Based on these results and existing in vivo data (generated by LTAP and NCSU; oro-pharyngeal aspiration tests), two functionalized MWCNT will be selected for a 4-wk inhalation test in rats (based on OECD TG 412), including a 4-wk recovery (validation test). Completed pre-trials demonstrated feasibility of generating respirable MWCNT aerosols by dry dispersion with pressurized air. This project is funded by the German BMBF (FKZ: 03XP0063).

Exposure to certain long and straight multiwalled carbon nanotubes (MWCNT) has been implicated in adverse biological effects, including induction of malignant mesothelioma. Besides morphology, functionalization seems to affect the toxic potential of MWCNT. The ERA-NET SIINN project ICONS (""International Collaboration On Nanotube Safety"") therefore aimed at mechanistically evaluating and ranking the pro-fibrotic and genotoxic potential of seven differentially purified (chemically or thermally) and functionalized (-COOH and -NH2), or milled MWCNT, made from one batch of industrially relevant Nanocyl NC7000 (tangled morphology). As a prerequisite, initial tests indicated sterility and lack of relevant endotoxin contamination of the materials. In a subproject performed by Fraunhofer ITEM, human peritoneal mesothelial LP9 cells (target cells for mesothelioma development) were incubated for 24 h with 5 µg/cm2 of the diverse MWCNT, followed by microarray analysis to evaluate material-specific transcriptomic signatures. In subsequent bioinformatic analyses, significant number of differentially-regulated genes (DEGs) was noted for all eight MWCNT (range: 555 to 2524 genes), with the amount of differentially-regulated genes and the observed fold-change values being strongly dependent on the MWCNT type. Interestingly, number of DEGs was about two- to five-fold higher for the surface-modified samples than for pristine Nanocyl NC7000. Furthermore, number of induced genes seemed to correlate with the cytotoxic potential of the different MWCNT (e.g. membrane damage and disturbance of the tubulin network). Comparison analyses found IL-8 signaling as the top-scoring activated canonical pathway, and a broad panel of cytokines, chemokines and growth factors as top-scoring upregulated genes in all data sets. Inflammatory response and mechanisms pertaining to carcinogenesis were shown to be significantly activated. In conclusion, surface modification of tangled MWCNT can adversely influence transcriptomic signature in primary mesothelial cells, with enhancement of the pro-inflammatory potential of pristine MWCNT. This project part was funded by the German BMBF (FKZ: 03XP0063).

With advancements in nanotechnology, the application of multi-walled carbon nanotubes (MWCNTs) in commercial products has been increasingly expanding. Consequently, increase in exposure of humans to MWCNTs raised questions about potential risks of such materials. The efforts to characterize specific features of MWCNTs that may potentially be associated with adverse health effects have been challenging as variations in the purification process and surface functionalization alter physicochemical properties. As part of the ERA-NET SIINN project ICONS (""International Collaboration On Nanotube Safety""), we examined the consequence of purification, followed by surface functionalization, on MWCNT-induced production of interleukin 1 beta (IL-1 beta) by human macrophages in vitro and in the lungs of mice in vivo. A library of eight differently purified (chemically or thermally) and functionalized (-COOH or -NH2) MWCNT samples were prepared from a single batch of industrially relevant Nanocyl NC7000. THP-1 monocytes were PMA-differentiated to macrophages (40 ng/mL, 48 hrs) and exposed to 1, 10, 100 µg/cm2 MWCNTs. Cell supernatants were analyzed for IL-1 beta via ELISA. Mice (C57BL6 strain, N=4 per group) were exposed via oropharyngeal aspiration to the same library of MWCNTs at doses of 1.6 and 4 mg/kg. After 3 days, BALF was collected and IL-1 beta analyzed via ELISA. THP1 cells exposed to thermally-purified MWCNTs functionalized with -COOH or -NH2 produced more IL-1 beta than when exposed to NC7000. Thermally-purified MWCNTs functionalized with either -COOH or -NH2 produced greater IL-1 beta in the BALF from mice compared to NC7000, whereas chemically purified MWCNTs functionalized with either -COOH or -NH2 produced less IL-1 beta in BALF compared to NC7000. These data suggest that the purification method used prior to surface functionalization is an important determinant in mediating inflammasome activation and IL-1 beta release as part of the innate immune response to MWCNTs. Funding: Supported by NSF Grant 15-022 and NIEHS Training Grant T32ES007046

Characterization of carbon nanotube dispersions requires measurement of both, concentration and surface area.

Question: Some forms of carbon nanotubes (CNT) have been found to exhibit carcinogenic potential depending on their morphological characteristics and catalyst impurities. The use of CNTs therefore requires appropriate risk and safety assessment. Correlation of hazards of biodurable fibres to specific morphologies is an approach that has been successfully developed for microscale fibres. The derived "fibre toxicological paradigm" achieves morphology-based hazard classification for a broad class of materials. The present study aims at contributing to an extension of this paradigm to the world of nanoscale fibres. Methods: CNTs of different diameters will be compared with respect to tumor development after intraperitoneal (i.p.) injection. It will be studied whether a diameter based threshold for CNT carcinogenicity can be identified. Rats will be administered a single i.p. injection. The tumor incidence will be histopathologically approximately evaluated performed after two years post application. Additionally, an interim sacrifice will be inflammatory three month to investigate potential effects in the peritoneum. Five different types of CNT will be tested in two concentrations (0.1 and 1.0 x 109 WHO-fibers): One single-walled CNT type, one multi-walled CNT type with an average diameter of 10 nm and a length of > 5 µm, two custom-synthesized multi-walled CNTs with average diameters of 20 and of 30 nm and a length > 5 µm, and one short MWCNT (max. 4-5 µm) with a diameter of > 40 nm. Amosite will be used as positive control. All nanomaterials will be comprehensively characterized before and after dispersion to reliable determine administered diameter distributions together with other characteristics that potentially influence toxicity, including length, purity and rigidity. Conclusion: The generated data is expected to provide information on morphology induced modes-of-action of CNTs including inflammation and carcinogenicity. The data is expected to contribute to the development of a morphology-based classification approach for CNTs and other inert nanofibres. This project is funded by the German FederalInstitute for Occupational Safety and Health (F2376).

Additional file 2: Table S2. Analyzed genes per array. List of all analyzed genes (short and long name) separated per PCR profiler array.