Environmental considerations and current status of grouping and regulation of engineered nanomaterials

This article reviews the current status of nanotechnology with emphasis on application and related environmental considerations as well as legislation. Application and analysis of nanomaterials in infrastructure (construction, building coatings, and water treatment) is discussed, and in particular nanomaterial release during the lifecycle of these applications. Moreover, possible grouping approaches with regard to ecotoxicological and toxicological properties, and the fate of nanomaterials in the environment are evaluated. In terms of potential exposure, the opportunities that arise from leveraging advances in several key areas, such as water treatment and construction are addressed. Additionally, this review describes challenges with regard to the European Commission’s definition of ‘nanomaterial’. The revised REACH information requirements, intended to enable a comprehensive risk assessment of nanomaterials, are outlined

Platox in vitro and in vivo investigations (28-day inhalation) to generate valid toxicity data for risk assessment of carbon-based nanoplatelets

Carbon-based nanoplatelets (CNP) represent a new class of 2-D nanostructures in multiple variants and with interesting functional properties (e.g. material enforcement and electrical conductivity). A very high toxicity among members of the carbon family is not expected for nanoplatelets, however, hazard characterization is still incomplete. Commercial CNP candidates (ACS Material, USA) were selected, covering single layer or multi layer graphene, carboxyl graphene, single layer graphene oxide, and graphite oxide. Technical soot (Printex 90) served as particulate, non-platelet reference. The CNP were analyzed on sterility and endotoxin content; morphology (SEM pictures) and the specific surface area (BET method) were re-evaluated. As in vitro screening models both, primary rat alveolar macrophages (AM) and MRC-5 human lung fibroblast cells were analyzed on membrane damage (LDH release) and metabolic activity (AlamarBlue test). Interestingly, the two single layer graphene samples induced marked concentration-dependent membrane damage in AM after 24h of incubation, with a BMD30 of 3.2 and 2.5 µg/cm2, whereas no such effect was observed for MRC-5 cells. Some LDH release was also observed for single layer graphite oxide (BMD30: 39.3 µg/cm2). The other materials were nearly inactive. Significant effects on metabolic activity were not observed. In AM, single layer graphene CNP additionally induced direct DNA damage and release of PGE2. In conclusion, single layer graphene showed a (geno) toxic potential in vitro in AM, but not in lung fibroblasts. Based on the in vitro screening data and for validation, a single layer (highest) and a multilayer (lowest toxic potential) were selected for in vivo investigations. In a dose range finding (DRF) test, with dosing by intratracheal instillation (0.02 and 0.2 mg/rat, each) single layer graphene was confirmed as the most inflammogenic sample in bronchoalveolar lavage fluid (BALF) inducing the recruitment of neutrophils and eosinophils. In the subsequent 4-week nose-only inhalation study with the same total doses (predicted by MPPD model) the inflammatory response of single layer graphene was weaker and no eosinophils were detected in BALF. Histopathological examination is underway. PLATOX funding: FP7- ERA-NET SIINN

Approaches on MWCNT diameters and its relation to tumor development

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)

Cerium oxide and barium sulfate nanoparticle inhalation affects gene expression in alveolar epithelial cells type II

BACKGROUND: Understanding the molecular mechanisms of nanomaterial interacting with cellular systems is important for appropriate risk assessment. The identification of early biomarkers for potential (sub-)chronic effects of nanoparticles provides a promising approach towards cost-intensive and animal consuming long-term studies. As part of a 90-day inhalation toxicity study with CeO2 NM-212 and BaSO4 NM-220 the present investigations on gene expression and immunohistochemistry should reveal details on underlying mechanisms of pulmonary effects. The role of alveolar epithelial cells type II (AEII cells) is focused since its contribution to defense against inhaled particles and potentially resulting adverse effects is assumed. Low dose levels should help to specify particle-related events, including inflammation and oxidative stress. RESULTS: Rats were exposed to clean air, 0.1, 0.3, 1.0, and 3.0 mg/m(3) CeO2 NM-212 or 50.0 mg/m(3) BaSO4 NM-220 and the expression of 391 genes was analyzed in AEII cells after one, 28 and 90 days exposure. A total number of 34 genes was regulated, most of them related to inflammatory mediators. Marked changes in gene expression were measured for Ccl2, Ccl7, Ccl17, Ccl22, Ccl3, Ccl4, Il-1alpha, Il-1ss, and Il-1rn (inflammation), Lpo and Noxo1 (oxidative stress), and Mmp12 (inflammation/lung cancer). Genes related to genotoxicity and apoptosis did not display marked regulation. Although gene expression was less affected by BaSO4 compared to CeO2 the gene pattern showed great overlap. Gene expression was further analyzed in liver and kidney tissue showing inflammatory responses in both organs and marked downregulation of oxidative stress related genes in the kidney. Increases in the amount of Ce were measured in liver but not in kidney tissue. Investigation of selected genes on protein level revealed increased Ccl2 in bronchoalveolar lavage of exposed animals and increased Lpo and Mmp12 in the alveolar epithelia. CONCLUSION: AEII cells contribute to CeO2 nanoparticle caused inflammatory and oxidative stress reactions in the respiratory tract by the release of related mediators. Effects of BaSO4 exposure are low. However, overlap between both substances were detected and support identification of potential early biomarkers for nanoparticle effects on the respiratory system. Signs for long-term effects need to be further evaluated by comparison to a respective exposure setting

Organosilane-based coating of quartz species from the traditional ceramics industry: Evidence of hazard reduction using in vitro and in vivo tests

The exposure to respirable crystalline silica (RCS), e.g. quartz, in industrial settings can induce silicosis and may cause tumours in chronic periods. Consequently, RCS in the form of quartz and cristobalite has been classified as human lung carcinogen category 1 by the International Agency for Research on Cancer in 1997, acknowledging differences in hazardous potential depending on source as well as chemical, thermal, and mechanical history. The physico-chemical determinants of quartz toxicity are well understood and are linked to density and abundance of surface silanol groups/radicals. Hence, poly-2-vinylpyridine-N-oxide and aluminium lactate, which effectively block highly reactive silanol groups at the quartz surface, have formerly been introduced as therapeutic approaches in the occupational field. In the traditional ceramics industry, quartz-containing raw materials are indispensable for the manufacturing process, and workers are potentially at risk of developing quartz-related lung diseases. Therefore, in the present study, two organosilanes, i.e. Dynasylan® PTMO and Dynasylan® SIVO 160, were tested as preventive, covalent quartz-coating agents to render ceramics production safer without loss in product quality. Coating effectiveness and coating stability (up to 1 week) in artificial alveolar and lysosomal fluids were first analysed in vitro, using the industrially relevant quartz Q1 as RCS model, quartz DQ12 as a positive control, primary rat alveolar macrophages as cellular model system (75 µg cm−2; 4 h of incubation ± aluminium lactate to verify quartz-related effects), and lactate dehydrogenase release and DNA strand break induction (alkaline comet assay) as biological endpoints. In vitro results with coated quartz were confirmed in a 90-day intratracheal instillation study in rats with inflammatory parameters as most relevant readouts. The results of the present study indicate that in particular Dynasylan® SIVO 160 (0.2% w/w of quartz) was able to effectively and stably block toxicity of biologically active quartz species without interfering with technical process quality of certain ceramic products. In conclusion, covalent organosilane coatings of quartz might represent a promising strategy to increase workers’ safety in the traditional ceramics industry

ICONS - Integrated testing strategy for mechanistically assessing the respiratory toxicity of functionalized MWCNT

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)