Carbon nanotubes (CNT) are of great commercial interest. Theoretically, during processing and handling of CNT and in abrasion processes on composites containing CNT, inhalable CNT particles might be set free. For hazard assessment, we performed a 90-day inhalation toxicity study with a multiwall CNT (MWCNT) material (Nanocyl NC 7000) according to Organisation for Economic Co-operation and Development test guideline 413. Wistar rats were head-nose exposed for 6 h/day, 5 days/week, 13 weeks, total 65 exposures, to MWCNT concentrations of 0 (control), 0.1, 0.5, or 2.5 mg/m(3). Highly respirable dust aerosols were produced with a proprietary brush generator which neither damaged the tube structure nor increased reactive oxygen species on the surface. Inhalation exposure to MWCNT produced no systemic toxicity. However, increased lung weights, pronounced multifocal granulomatous inflammation, diffuse histiocytic and neutrophilic inflammation, and intra-alveolar lipoproteinosis were observed in lung and lung-associated lymph nodes at 0.5 and 2.5 mg/m(3). These effects were accompanied by slight blood neutrophilia at 2.5 mg/m(3). Incidence and severity of the effects were concentration related. At 0.1 mg/m(3), there was still minimal granulomatous inflammation in the lung and in lung-associated lymph nodes; a no observed effect concentration was therefore not established in this study. The test substance has low dust-forming potential, as demonstrated by dustiness measurements, but nonetheless strict industrial hygiene measures must be taken during handling and processing. Toxicity and dustiness data such as these can be used to compare different MWCNT materials and to select the material with the lowest risk potential for a given application.

BackgroundA standard short-term inhalation study (STIS) was applied for hazard assessment of 13 metal oxide nanomaterials and micron-scale zinc oxide.MethodsRats were exposed to test material aerosols (ranging from 0.5 to 50 mg/m3) for five consecutive days with 14- or 21-day post-exposure observation. Bronchoalveolar lavage fluid (BALF) and histopathological sections of the entire respiratory tract were examined. Pulmonary deposition and clearance and test material translocation into extra-pulmonary organs were assessed.ResultsInhaled nanomaterials were found in the lung, in alveolar macrophages, and in the draining lymph nodes. Polyacrylate-coated silica was also found in the spleen, and both zinc oxides elicited olfactory epithelium necrosis. None of the other nanomaterials was recorded in extra-pulmonary organs. Eight nanomaterials did not elicit pulmonary effects, and their no observed adverse effect concentrations (NOAECs) were at least 10 mg/m3. Five materials (coated nano-TiO2, both ZnO, both CeO2) evoked concentration-dependent transient pulmonary inflammation. Most effects were at least partially reversible during the post-exposure period.Based on the NOAECs that were derived from quantitative parameters, with BALF polymorphonuclear (PMN) neutrophil counts and total protein concentration being most sensitive, or from the severity of histopathological findings, the materials were ranked by increasing toxic potency into 3 grades: lower toxic potency: BaSO4; SiO2.acrylate (by local NOAEC); SiO2.PEG; SiO2.phosphate; SiO2.amino; nano-ZrO2; ZrO2.TODA; ZrO2.acrylate; medium toxic potency: SiO2.naked; higher toxic potency: coated nano-TiO2; nano-CeO2; Al-doped nano-CeO2; micron-scale ZnO; coated nano-ZnO (and SiO2.acrylate by systemic no observed effect concentration (NOEC)).ConclusionThe STIS revealed the type of effects of 13 nanomaterials, and micron-scale ZnO, information on their toxic potency, and the location and reversibility of effects. Assessment of lung burden and material translocation provided preliminary biokinetic information. Based upon the study results, the STIS protocol was re-assessed and preliminary suggestions regarding the grouping of nanomaterials for safety assessment were spelled out.

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BackgroundCarbon nanotubes, graphene, graphite nanoplatelets and carbon black are seemingly chemically identical carbon-based nano-materials with broad technological applications. Carbon nanotubes and carbon black possess different inhalation toxicities, whereas little is known about graphene and graphite nanoplatelets.MethodsIn order to compare the inhalation toxicity of the mentioned carbon-based nanomaterials, male Wistar rats were exposed head-nose to atmospheres of the respective materials for 6 hours per day on 5 consecutive days. Target concentrations were 0.1, 0.5, or 2.5 mg/m3 for multi-wall carbon nanotubes and 0.5, 2.5, or 10 mg/m3 for graphene, graphite nanoplatelets and low-surface carbon black. Toxicity was determined after end of exposure and after three-week recovery using broncho-alveolar lavage fluid and microscopic examinations of the entire respiratory tract.ResultsNo adverse effects were observed after inhalation exposure to 10 mg/m3 graphite nanoplatelets or relatively low specific surface area carbon black. Increases of lavage markers indicative for inflammatory processes started at exposure concentration of 0.5 mg/m3 for multi-wall carbon nanotubes and 10 mg/m3 for graphene. Consistent with the changes in lavage fluid, microgranulomas were observed at 2.5 mg/m3 multi-wall carbon nanotubes and 10 mg/m3 graphene. In order to evaluate volumetric loading of the lung as the key parameter driving the toxicity, deposited particle volume was calculated, taking into account different methods to determine the agglomerate density. However, the calculated volumetric load did not correlate to the toxicity, nor did the particle surface burden of the lung.ConclusionsThe inhalation toxicity of the investigated carbon-based materials is likely to be a complex interaction of several parameters. Until the properties which govern the toxicity are identified, testing by short-term inhalation is the best option to identify hazardous properties in order to avoid unsafe applications or select safer alternatives for a given application.

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Two Ceria nanomaterials (NM-211 and NM-212) were tested for inhalation toxicity and organ burdens in order to design a chronic and carcinogenicity inhalation study (OECD TG No. 453). Rats inhaled aerosol concentrations of 0.5, 5, and 25 mg/m3 by whole-body exposure for 6 h/day on 5 consecutive days for 1 or 4 weeks with a post-exposure period of 24 or 129 days, respectively. Lungs were examined by bronchoalveolar lavage and histopathology. Inhaled Ceria is deposited in the lung and cleared with a half-time of 40 days; at aerosol concentrations higher than 0.5 mg/m3, this clearance was impaired resulting in a half-time above 200 days (25 mg/m3). After 5 days, Ceria (>0.5 mg/m3) induced an early inflammatory reaction by increases of neutrophils in the lung which decreased with time, with sustained exposure, and also after the exposure was terminated (during the post-exposure period). The neutrophil number observed in bronchoalveolar lavage fluid (BALF) was decreasing and supplemented by mononuclear cells, especially macrophages which were visible in histopathology but not in BALF. Further progression to granulomatous inflammation was observed 4 weeks post-exposure. The surface area of the particles provided a dose metrics with the best correlation of the two Ceria’s inflammatory responses; hence, the inflammation appears to be directed by the particle surface rather than mass or volume in the lung. Observing the time course of lung burden and inflammation, it appears that the dose rate of particle deposition drove an initial inflammatory reaction by neutrophils. The later phase (after 4 weeks) was dominated by mononuclear cells, especially macrophages. The progression toward the subsequent granulomatous reaction was driven by the duration and amount of the particles in the lung. The further progression of the biological response will be determined in the ongoing long-term study.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-014-1349-9) contains supplementary material, which is available to authorized users.

Metabolomics is a widely used technology in academic research, yet its application to regulatory science has been limited. The most commonly cited barrier to its translation is lack of performance and reporting standards. The MEtabolomics standaRds Initiative in Toxicology (MERIT) project brings together international experts from multiple sectors to address this need. Here, we identify the most relevant applications for metabolomics in regulatory toxicology and develop best practice guidelines, performance and reporting standards for acquiring and analysing untargeted metabolomics and targeted metabolite data. We recommend that these guidelines are evaluated and implemented for several regulatory use cases.

SummaryGrouping of substances and utilizing read-across of data within those groups represents an important data gap filling technique for chemical safety assessments. Categories/analogue groups are typically developed based on structural similarity and, increasingly often, also on mechanistic (biological) similarity. While read-across can play a key role in complying with legislation such as the European REACH regulation, the lack of consensus regarding the extent and type of evidence necessary to support it often hampers its successful application and acceptance by regulatory authorities. Despite a potentially broad user community, expertise is still concentrated across a handful of organizations and individuals. In order to facilitate the effective use of read-across, this document presents the state of the art, summarizes insights learned from reviewing ECHA published decisions regarding the relative successes/pitfalls surrounding read-across under REACH, and compiles the relevant activities and guidance documents. Special emphasis is given to the available existing tools and approaches, an analysis of ECHA's published final decisions associated with all levels of compliance checks and testing proposals, the consideration and expression of uncertainty, the use of biological support data, and the impact of the ECHA Read-Across Assessment Framework (RAAF) published in 2015.