Genotoxicity of synthetic amorphous silica nanoparticles in rats following short-term exposure. Part 2: intratracheal instillation and intravenous injection.

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.

Assessment of the genotoxic and carcinogenic potentials of 3-aminothiophene derivatives using in vitro and in silico methodologies.

Thiophene derivatives, a class of compounds widely used in products such as pharmaceuticals, agrochemicals or dyestuffs, represent chemicals of concern. Indeed, the thiophene ring is often considered as a structural moiety that may be involved in toxic effects in humans. We primarily focus on the genotoxic/mutagenic and carcinogenic potentials of the methyl 3-amino-4-methylthiophene-2-carboxylate (1), a precursor of the articaine local anesthetic (4) which falls within the scope of the European REACH (Registration, Evaluation, Authorisation and restriction of CHemicals) legislation. To discern some structure-toxicity relationships, we also studied two related compounds, namely the 3-amino 4-methylthiophene (2) and the 2-acetyl 4-chlorothiophene (3). Techniques employed to assess mutagenic and DNA-damaging effects involved the Salmonella mutagenicity assay (or Ames test) and the single-cell gel electrophoresis assay (or Comet assay). In the range of tested doses, none of these derivatives led to a positive response in the Ames tests and DNA damage was only observed in the Comet assay after high concentration exposure of 2. The study of their carcinogenic potential using the in vitro SHE (Syrian Hamster Embryo) cell transformation assay (CTA) highlighted the activity of compound 2. A combination of experimental data with in silico predictions of the reactivity of thiophene derivatives towards cytochrome P450 (CYP450), enabled us to hypothesize possible pathways leading to these toxicological profiles.

Prevalidation study of the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0 for assessment of carcinogenic potential of chemicals.

The European Centre for the Validation of Alternative Methods (ECVAM) has organised an interlaboratory prevalidation study on the Syrian hamster embryo (SHE) cell transformation assay (CTA) at pH 7.0 for the detection of rodent carcinogens. The SHE CTA at pH 7.0 has been evaluated for its within-laboratory reproducibility, transferability and between-laboratory reproducibility. Four laboratories using the same basic protocol with minor modifications participated in this study and tested a series of six coded-chemicals: four rodent carcinogens (benzo(a)pyrene, 3-methylcholanthrene, 2,4-diaminotoluene and o-toluidine HCl) and two non-carcinogens (anthracene and phthalic anhydride). All the laboratories found the expected results with coded chemicals except for phthalic anhydride which resulted in a different call in only one laboratory. Based on the outcome of this study, it can be concluded that a standardised protocol is available that should be the basis for future use. This protocol and the assay system itself are transferable between laboratories and the SHE CTA at pH 7.0 is reproducible within- and between-laboratories.

Recommended protocol for the Syrian hamster embryo (SHE) cell transformation assay.

The Syrian hamster embryo (SHE) cell transformation assay (CTA) is a short-term in vitro assay recommended as an alternative method for testing the carcinogenic potential of chemicals. SHE cells are "normal" cells since they are diploid, genetically stable, non-tumourigenic, and have metabolic capabilities for the activation of some classes of carcinogens. The CTA, first developed in the 1960s by Berwald and Sachs (1963,1964) [3,4], is based on the change of the phenotypic feature of cell colonies expressing the first steps of the conversion of normal to neoplastic-like cells with oncogenic properties. Pienta et al. (1977) [22] developed a protocol using cryopreserved cells to enhance practicality of the assay and limit sources of variability. Several variants of the assay are currently in use, which mainly differ by the pH at which the assay is performed. We present here the common version of the SHE pH 6.7 CTA and SHE pH 7.0 CTA protocols used in the ECVAM (European Centre for the Validation of Alternative Methods) prevalidation study on CTA reported in this issue. It is recommended that this protocol, in combination with the photo catalogues presented in this issue, should be used in the future and serve as a basis for the development of the OECD test guideline.

Photo catalogue for the classification of cell colonies in the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0.

This catalogue is a display of Syrian hamster embryo (SHE) cell colony photos representative of the cell transformation assay (CTA) carried out at pH 7.0. It is intended as a visual aid for the identification and the scoring of cell colonies in the conduct of the assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the assay results.