Genotoxicity of commercial colloidal and laboratory-synthesized silica nanoparticles was tested using the single cell gel electrophoresis or Comet assay. By using a carefully developed protocol and careful characterization of the nanoparticle dispersions, Comet assays were performed on 3T3-L1 fibroblasts with 3, 6, and 24 h incubations and 4 or 40 microg/ml of silica nanoparticles. No significant genotoxicity was observed for the nanoparticles tested under the conditions described, and results were independently validated in two separate laboratories, showing that in vitro toxicity testing can be quantitatively reproducible.

The comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However, few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In this study, 12 laboratories analysed the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation (0-10 Gy) and used to construct a calibration curve to calculate the number of lesions per 10(6) base pair. All laboratories detected dose-response relationships in the coded samples irradiated with ionizing radiation (1.5-7 Gy), but there were overt differences in the level of DNA damage reported by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P < 0.05, Levene's test). A large fraction of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level of DNA damage measured by the alkaline version of the comet assay.

There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.

The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet assay end points to number of lesions/106 bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their own comet assay protocols. Nine of 10 laboratories reported the same ranking of the level of damage in the coded samples. The variation in assessment of oxidatively damaged DNA was largely due to differences in protocols. After conversion of the data to lesions/106 bp using laboratory-specific calibration curves, the variation between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variation in net FPG-sensitive sites increased from 49 to 73%, whereas the inter-laboratory variation decreased. The participating laboratories were successful in finding a dose–response of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories.

Inter-laboratory comparison of nanoparticle size measurements using dynamic light scattering and differential centrifugal sedimentation. NanoImpact, (2018).

The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found dose-response relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.

Synthetic amorphous silica nanomaterials (SAS) are extensively used in food and tire industries. In many industrial processes, SAS may become aerosolized and lead to occupational exposure of workers through inhalation in particular. However, little is known about the in vivo genotoxicity of these particulate materials. To gain insight into the toxicological properties of four SAS (NM-200, NM-201, NM-202, and NM-203), rats are treated with three consecutive intratracheal instillations of 3, 6, or 12 mg/kg of SAS at 48, 24, and 3 hrs prior to tissue collection (cumulative doses of 9, 18, and 36 mg/kg). Deoxyribonucleic acid (DNA) damage was assessed using erythrocyte micronucleus test and the standard and Fpg-modified comet assays on cells from bronchoalveolar lavage fluid (BALF), lung, blood, spleen, liver, bone marrow, and kidney. Although all of the SAS caused increased dose-dependent changes in lung inflammation as demonstrated by BALF neutrophilia, they did not induce any significant DNA damage. As the amount of SAS reaching the blood stream and subsequently the internal organs is probably to be low following intratracheal instillation, an additional experiment was performed with NM-203. Rats received three consecutive intravenous injections of 5, 10, or 20 mg/kg of SAS at 48, 24, and 3 hrs prior to tissue collection. Despite the hepatotoxicity, thrombocytopenia, and even animal death induced by this nanomaterial, no significant increase in DNA damage or micronucleus frequency was observed in SAS-exposed animals. It was concluded that under experimental conditions, SAS induced obvious toxic effects but did cause any genotoxicity following intratracheal instillation and intravenous injection.

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