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International audience; The genotoxicity of nano-structured synthetic amorphous silica (SAS), a common food additive, was investigated in vivo in rats. A 90-day oral toxicity study was performed according to OECD test guideline 408 and the genotoxicity of pyrogenic SAS nanomaterial NM-203 was assessed in several organs, using complementary tests. Adult Sprague-Dawley rats of both sexes were treated orally for 90 days with 0, 2, 5, 10, 20, or 50 mg SAS/kg bw per day. Dose levels were selected to approximate expected human dietary exposures to SAS. DNA strand breaks were evaluated by the comet assay in blood, bone marrow, liver, and spleen according to OECD test guideline 489; mutations induced in bone marrow precursors of erythrocytes were assessed by the Pig-a assay and chromosome/ genome damage by the micronucleus assay in blood (OECD test guideline 474) and colon. No treatment-related increases of gene (Pig-a) or chromosome/genome (micronucleus) mutations were detected in the blood. The percentage of micronucleated cells was not increased in the colon of treated rats. Among the organs analyzed by the comet assay, the spleen was the only target showing a weak but biologically relevant genotoxic effect.

Due to several gaps remaining in the toxicological evaluation of nanomaterials (NMs), consumers and public health agencies have shown increasing concern for human health protection. In addition to aluminum (Al) microparticles, Al-containing nanomaterials (Al NMs) have been applied by food industry as additives and contact materials. Due to the limited amount of literature on the toxicity of Al NMs, this study aimed to evaluate the in vivo genotoxic potential of Al0 and Al2O3 NMs after acute oral exposure. Male Sprague-Dawley rats were administered three successive gavages at 6, 12.5 and 25 mg/kg bw. A comparison with AlCl3 was done in order to assess the potential effect of dissolution into Al ions. Both DNA strand breaks and oxidative DNA damage were investigated in six organs/tissues (duodenum, liver, kidney, spleen, blood and bone marrow) with the alkaline and the Fpg-modified comet assays. Concomitantly, chromosomal damage was investigated in bone marrow and colon with the micronucleus assay. The comet assay only showed DNA damage with Al2O3 NMs in bone marrow (BM), while AlCl3 induced slight but non-significant oxidative DNA damage in blood. No increase of chromosomal mutations was observed after treatment with the two Al MNs either in the BM or in the colons of rats.

Synthetic amorphous silica (SAS) in its nanosized form is now used in food applications although the potential risks for human health have not been evaluated. In this study, genotoxicity and oxidative DNA damage of two pyrogenic (NM-202 and 203) and two precipitated (NM-200 and -201) nanosized SAS were investigated in vivo in rats following oral exposure. Male Sprague Dawley rats were exposed to 5, 10, or 20 mg/kg b.w./day for three days by gavage. DNA strand breaks and oxidative DNA damage were investigated in seven tissues (blood, bone marrow from femur, liver, spleen, kidney, duodenum, and colon) with the alkaline and the (Fpg)-modified comet assays, respectively. Concomitantly, chromosomal damage was investigated in bone marrow and in colon with the micronucleus assay. Additionally, malondialdehyde (MDA), a lipid peroxidation marker, was measured in plasma. When required, a histopathological examination was also conducted. The results showed neither obvious DNA strand breaks nor oxidative damage with the comet assay, irrespective of the dose and the organ investigated. Similarly, no increases in chromosome damage in bone marrow or lipid peroxidation in plasma were detected. However, although the response was not dose-dependent, a weak increase in the percentage of micronucleated cells was observed in the colon of rats treated with the two pyrogenic SAS at the lowest dose (5 mg/kg b.w./day). Additional data are required to confirm this result, considering in particular, the role of agglomeration/aggregation of SAS NMs in their uptake by intestinal cells.

International audience; Volume 280, Supplement 1, Pages S1-S346 (20 October 2017)Abstracts of the 53rd Congress of the European Societies of Toxicology (EUROTOX)Bratislava, Slovakia, 10th–13th September, 2017Edited by Mumtaz Iscan and Helena KandarovaP-03-02-16 Aluminum on different forms is found in several goods: food addi- tives, medication, beverage, water treatment as well as cooking utensils. EFSA has established a tolerable weekly intake (TWI) of 1 mg/kg bw for human oral exposure (EFSA, 2008). Since the 2000s, due to the increasing use of nanoparticles (NP), aluminum NPs are expected to be more largely involved in human exposure although their effects on human health has not been fully characterized. Nevertheless, aluminum was depicted to cross epithelial barriers and to include neurotoxicity and embryotoxic- ity. As aluminum NPs may not behave as Al ions, their toxic effects must be investigated. In this study, we investigated the genotoxicity of Al, Al 2 O 3 NPs and the ionic form AlCl 3 on intestine, the contact organ fol- lowing oral exposure, and on liver, the main target organ for Al accumulation using both the alkaline comet assay and the Fpg- modified comet assay for highlighting potential oxidative damage. We observed DNA damage on the human intestinal Caco2 and hepatic HepaRG cells (exposure from 0.6 to 256 g/cm 2 ). How- ever, no genotoxicity was detected in duodenum and liver of male Sprague Dawley rats after 3 gavages as well as after 28 days oral treatment with 6, 12 and 25 mg/kg bw/day. This project was funded by Agence Nationale de la Recherche (ANR-13-IS10-0005-01).

Microcystin-LR (MC-LR), involved in human and animal poisonings by cyanobacteria, has been shown to be both a potent tumour promoter in rat liver and an inhibitor of serine/threonine protein phosphatases, specifically PP1 and PP2A. The research on the genotoxic potential of MC-LR counts only few in vivo studies. In order to determine the target organs for DNA-damage induction by MC-LR, the single-cell gel electrophoresis (SCGE) or comet assay was performed in mice. Following a single oral administration of 2 and 4mg/kg bw of MC-LR, a statistically significant induction of DNA damage in blood cells was obtained after 3h. However, after an intra-peritoneal injection (ip), DNA lesions were mainly induced in the liver, but were also reported in the kidney, the intestine and the colon. The sensitivity of the ip route compared to the oral route suggested a difference in the bio-disponibility of the toxin. In any case, DNA damage was induced by MC-LR irrespective of the administration route. Among the target organs, the DNA damage induced in the intestinal tissues (ileum and colon) may contribute to an increased cancer risk.

International audience; Silica (SiO2) in its nanosized form is now used in food applications although the potential risks for human health need to be evaluated in further detail. In the current study, the uptake of 15 and 55 nm colloidal SiO2 NPs in the human intestinal Caco-2 cell line was investigated by transmission electron microscopy. The ability of these NPs to induce cytotoxicity (XTT viability test), genotoxicity (γH2Ax and micronucleus assay), apoptosis (caspase 3), oxidative stress (oxidation of 2,7-dichlorodihydrofluorescein diacetate probe) and proinflammatory effects (interleukin IL-8 secretion) was evaluated. Quartz DQ12 was used as particle control. XTT and cytokinesis-block micronucleus assays revealed size- and concentration-dependent effects on cell death and chromosome damage following exposure to SiO2 nanoparticles, concomitantly with generation of reactive oxygen species (ROS), SiO2-15 nm particles being the most potent. In the same way, an increased IL-8 secretion was only observed with SiO2-15 nm at the highest tested dose (32 μg/ml). TEM images showed that both NPs were localized within the cytoplasm but did not enter the nucleus. SiO2-15 nm, and to a lower extent SiO2-55 nm, exerted toxic effects in Caco-2 cells. The observed genotoxic effects of these NPs are likely to be mediated through oxidative stress rather than a direct interaction with the DNA. Altogether, our results indicate that exposure to SiO2 NPs may induce potential adverse effects on the intestinal epithelium in vivo.

Synthetic amorphous silica (SAS) in its nanosized form is now used in food applications although the potential risks for human health have not been evaluated. In this study, genotoxicity and oxidative DNA damage of two pyrogenic (NM-202 and 203) and two precipitated (NM-200 and -201) nanosized SAS were investigated in vivo in rats following oral exposure. Male Sprague Dawley rats were exposed to 5, 10, or 20 mg/kg b.w./day for three days by gavage. DNA strand breaks and oxidative DNA damage were investigated in seven tissues (blood, bone marrow from femur, liver, spleen, kidney, duodenum, and colon) with the alkaline and the (Fpg)-modified comet assays, respectively. Concomitantly, chromosomal damage was investigated in bone marrow and in colon with the micronucleus assay. Additionally, malondialdehyde (MDA), a lipid peroxidation marker, was measured in plasma. When required, a histopathological examination was also conducted. The results showed neither obvious DNA strand breaks nor oxidative damage with the comet assay, irrespective of the dose and the organ investigated. Similarly, no increases in chromosome damage in bone marrow or lipid peroxidation in plasma were detected. However, although the response was not dose-dependent, a weak increase in the percentage of micronucleated cells was observed in the colon of rats treated with the two pyrogenic SAS at the lowest dose (5 mg/kg b.w./day). Additional data are required to confirm this result, considering in particular, the role of agglomeration/aggregation of SAS NMs in their uptake by intestinal cells.

Cylindrospermopsin (CYN), a cyanobacterial hepatotoxin mainly produced by Cylindrospermopsis raciborskii, has been involved in human intoxications and livestock deaths. The widespread occurrence of CYN in the water supplies lead us to investigate its genotoxicity to assess potential chronic effects. This study reports evaluation of CYN-induced in vivo DNA damage in mice using alkaline comet assay (ACA) and micronucleus assay (MNA) concomittantly. ACA measures DNA breakage from single and double strand breaks as well as alkali labile sites. Conversely, MNA detects chromosome damage events such as chromosomal breakage and numeric alterations. Male Swiss mice were treated with CYN concentrations of 50, 100, and 200 μg/kg by a single intraperitoneal (ip) injection or with 1, 2, and 4 mg/kg by gavage. Methyl methane sulfonate (MMS) was used as positive control at 80 mg/kg. Twenty-four hours after treatment, samples of liver, blood, bone marrow, kidney, intestine, and colon were taken to perform ACA, the bone marrow and the colon were also used for MNA. Parameters used to quantify DNA damage were % Tail DNA for ACA and both micronucleated immature erythrocytes and epithelial colon cells for MNA. DNA breaks and chromosome damage were significantly increased by MMS in all the organs evaluated. Significant DNA damage was detected within the colon by ACA after ip injection of 100 and 200 μg/kg CYN (P < 0.01). DNA damage was also detected in colon samples after 4 mg/kg oral administration of CYN and in bone marrow after 1 and 2 mg/kg of orally administered CYN. Histological examination showed foci of cell death within the liver and the kidney from mice that received the two highest doses of CYN by either route of administration. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

The food dyes amaranth, sunset yellow and tartrazine were administered twice, at 24h intervals, by oral gavage to mice and assessed in the in vivo gut micronucleus test for genotoxic effects (frequency of micronucleated cells) and toxicity (apoptotic and mitotic cells). The concentrations of each compound and their main metabolites (sulfanilic acid and naphthionic acid) were measured in faeces during a 24-h period after single oral administrations of the food dyes to mice. Parent dye compounds and their main aromatic amine metabolites were detected in significant amounts in the environment of colonic cells. Acute oral exposure to food dye additives amaranth, sunset yellow and tartrazine did not induce genotoxic effect in the micronucleus gut assay in mice at doses up to 2000 mg/kg b.w. Food dyes administration increased the mitotic cells at all dose levels when compared to controls. These results suggest that the transient DNA damages previously observed in the colon of mice treated by amaranth and tartrazine by the in vivo comet assay [Sasaki, Y.F., Kawaguchi, S., Kamaya, A., Ohshita, M., Kabasawa, K., Iwama, K., Taniguchi, K., Tsuda, S., 2002. The comet assay with 8 mouse organs: results with 39 currently used food additives. Mutat. Res. 519, 103-119] are unable to be fixed in stable genotoxic lesions and might be partly explained by local cytotoxicity of the dyes.