Reproducibility of methods required to identify and characterize nanoforms of substances.

Nanoforms (NFs) of a substance may be distinguished from one another through differences in their physicochemical properties. When registering nanoforms of a substance for assessment under the EU REACH framework, five basic descriptors are required for their identification: composition, surface chemistry, size, specific surface area and shape. To make the risk assessment of similar NFs efficient, a number of grouping frameworks have been proposed, which often require assessment of similarity on individual physicochemical properties as part of the group justification. Similarity assessment requires an understanding of the achievable accuracy of the available methods. It must be demonstrated that measured differences between NFs are greater than the achievable accuracy of the method, to have confidence that the measured differences are indeed real. To estimate the achievable accuracy of a method, we assess the reproducibility of six analytical techniques routinely used to measure these five basic descriptors of nanoforms: inductively coupled plasma mass spectrometry (ICP-MS), Thermogravimetric analysis (TGA), Electrophoretic light scattering (ELS), Brunauer-Emmett-Teller (BET) specific surface area and transmission and scanning electron microscopy (TEM and SEM). Assessment was performed on representative test materials to evaluate the reproducibility of methods on single NFs of substances. The achievable accuracy was defined as the relative standard deviation of reproducibility (RSDR) for each method. Well established methods such as ICP-MS quantification of metal impurities, BET measurements of specific surface area, TEM and SEM for size and shape and ELS for surface potential and isoelectric point, all performed well, with low RSDR, generally between 5 and 20%, with maximal fold differences usually <1.5 fold between laboratories. Applications of technologies such as TGA for measuring water content and putative organic impurities, additives or surface treatments (through loss on ignition), which have a lower technology readiness level, demonstrated poorer reproducibility, but still within 5-fold differences. The expected achievable accuracy of ICP-MS may be estimated for untested analytes using established relationships between concentration and reproducibility, but this is not yet the case for TGA measurements of loss on ignition or water content. The results here demonstrate an approach to estimate the achievable accuracy of a method that should be employed when interpreting differences between NFs on individual physicochemical properties.

In vitro-in vivo correlations of pulmonary inflammogenicity and genotoxicity of MWCNT.

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.

Thermogravimetry and Mass Spectrometry of Extractable Organics from Manufactured Nanomaterials for Identification of Potential Coating Components.

Manufactured nanomaterials (MNMs) often have a surface-chemical modification in order to tailor their physicochemical properties, including also powder properties and miscibility. Surface-chemical modifications may influence the toxicological properties of the MNM, but the specific chemistry and extent are rarely described in detail in suppliers' technical data sheets. Chemical and quantitative information on any surface-chemical treatment, coating and functionalization are required for chemicals registration in Europe. Currently there is no globally accepted and documented approach to generate such data. Consequently, there is a continued research need to establish a structured approach to identify and quantify surface-chemical modifications. Here we present a tiered approach starting with screening for mass-loss during heating in a furnace or thermogravimetric analysis (TGA) followed by solvent extraction, and analysis by several mass spectrometry (MS) techniques depending on the target analytes. Thermal treatment was assumed to be able to quantify the amount of organic coating and MS was used to identify the extractable organic coatings after pressurized liquid extraction (PLE) using methanol at 200 °C. Volatile organic compounds in extracts were identified with gas chromatography and MS (GC-MS), non-volatile organic compounds with liquid chromatography MS (LC-MS), and polymeric compounds with matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF-MS). The approach was demonstrated by analysis of 24 MNM, comprising titanium dioxide, synthetic amorphous silica, graphite, zinc oxide, silver, calcium carbonate, iron oxide, nickel-zinc-iron oxide, and organoclay. In extracts of 14 MNMs a range of organic compounds were identified and the main groups were silanes/siloxanes, fatty acids, fatty acid esters, quaternary ammonium compounds and polymeric compounds. In the remaining 10 MNMs no organic compounds were detected by MS, despite the fact an organic coating was indicated by TGA.