Background Multi-walled carbon nanotubes (MWCNT) have received attention due to extraordinary properties, resulting in concerns for occupational health and safety. Costs and ethical concerns of animal testing drive a need for in vitro models with predictive power in respiratory toxicity. The aim of this study was to assess pro-inflammatory response (Interleukin-8 expression, IL-8) and genotoxicity (DNA strand breaks) caused by MWCNT with different physicochemical properties in different pulmonary cell models and correlate these to previously published in vivo data. Seven MWCNT were selected; two long/thick (NRCWE-006/Mitsui-7 and NM-401), two short/thin (NM-400 and NM-403), a pristine (NRCWE-040) and two surface modified; hydroxylated (NRCWE-041) and carboxylated (NRCWE-042). Carbon black Printex90 (CB) was included as benchmark material. Human alveolar epithelial cells (A549) and monocyte-derived macrophages (THP-1a) were exposed to nanomaterials (NM) in submerged conditions, and two materials (NM-400 and NM-401) in co-cultures of A549/THP-1a and lung fibroblasts (WI-38) in an air-liquid interface (ALI) system. Effective doses were quantified by thermo-gravimetric-mass spectrometry analysis (TGA-MS). To compare genotoxicity in vitro and in vivo, we developed a scoring system based on a categorization of effects into standard deviation (SD) units (< 1, 1, 2, 3 or 4 standard deviation increases) for the increasing genotoxicity. Results Effective doses were shown to be 25 to 53%, and 21 to 57% of the doses administered to A549 and THP-1a, respectively. In submerged conditions (A549 and THP-1a cells), all NM induced dose-dependent IL-8 expression. NM-401 and NRCWE-006 caused the strongest pro-inflammatory response. In the ALI-exposed co-culture, only NM-401 caused increased IL-8 expression, and no DNA strand breaks were observed. Strong correlations were found between in vitro and in vivo inflammation when doses were normalized by surface area (also proxy for diameter and length). Significantly increased DNA damage was found for all MWCNT in THP-1a cells, and for short MWCNT in A549 cells. A concordance in genotoxicity of 83% was obtained between THP-1a cells and broncho-alveolar lavaged (BAL) cells. Conclusion This study shows correlations of pro-inflammatory potential in A549 and THP-1a cells with neutrophil influx in mice, and concordance in genotoxic response between THP-1a cells and BAL cells, for seven MWCNT.

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alpha-1-antichymotrypsin (ACT), is an acute phase protein and a protease inhibitor produced by the liver and brain. ACT is involved in the pathogenesis of Alzheimer's disease (AD), since elevated ACT concentration was found in cerebrospinal fluid (CSF) and brain from AD. ACT has also been shown to influence amyloid deposition in vitro and in animal models of AD. In this investigation 830 healthy controls, 69 subjects with cognitive impairment and not dementia (CIND), 53 patients with severe clinical AD and 142 patients with mild AD were investigated. Plasma levels of ACT were measured with a new competitive immune enzyme linked immune-assay (ELISA). ACT levels were higher in AD patients than in CIND or controls. An age dependent increase of plasma ACT was present in both healthy elderly and CIND. Patients with mild clinical AD were followed up for two years and stratified according to the rate of clinical deterioration. CT plasma levels were elevated in AD patients that showed an accelerated rate of cognitive deterioration during the follow up; this increment being prominent in AD with the Apolipoprotein E (APOE) epsilon 4 allele. Therefore, increased peripheral ACT levels in APOE 4 positive patients appear to predict an accelerated clinical progression. Plasma ACT might be used as a surrogate marker to monitor the conversion of pre-dementia stages to AD and the progression of the disease. The development of compounds able to interfere with the ACT biological activity (protease inhibition and/or promotion of amyloid deposition) might have therapeutic relevance for the disease.

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Background The EU-project GRACIOUS developed an Integrated Approach to Testing and Assessment (IATA) to support grouping high aspect ratio nanomaterials (HARNs) presenting a similar inhalation hazard. Application of grouping reduces the need to assess toxicity on a case-by-case basis and supports read-across of hazard data from substances that have the data required for risk assessment (source) to those that lack such data (target). The HARN IATA, based on the fibre paradigm for pathogenic fibres, facilitates structured data gathering to propose groups of similar HARN and to support read-across by prompting users to address relevant questions regarding HARN morphology, biopersistence and inflammatory potential. The IATA is structured in tiers, allowing grouping decisions to be made using simple in vitro or in silico methods in Tier1 progressing to in vivo approaches at the highest Tier3. Here we present a case-study testing the applicability of GRACIOUS IATA to form an evidence-based group of multiwalled carbon nanotubes (MWCNT) posing a similar predicted fibre-hazard, to support read-across and reduce the burden of toxicity testing. Results The case-study uses data on 15 different MWCNT, obtained from the published literature. By following the IATA, a group of 2 MWCNT was identified (NRCWE006 and NM-401) based on a high degree of similarity. A pairwise similarity assessment was subsequently conducted between the grouped MWCNT to evaluate the potential to conduct read-across and fill data gaps required for regulatory hazard assessment. The similarity assessment, based on expert judgement of Tier 1 assay results, predicts both MWCNT are likely to cause a similar acute in vivo hazard. This result supports the possibility for read-across of sub-chronic and chronic hazard endpoint data for lung fibrosis and carcinogenicity between the 2 grouped MWCNT. The implications of accepting the similarity assessment based on expert judgement of the MWCNT group are considered to stimulate future discussion on the level of similarity between group members considered sufficient to allow regulatory acceptance of a read-across argument. Conclusion This proof-of-concept case-study demonstrates how a grouping hypothesis and IATA may be used to support a nuanced and evidence-based grouping of ‘similar’ MWCNT and the subsequent interpolation of data between group members to streamline the hazard assessment process.