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EC number: 231-146-5 | CAS number: 7440-36-0
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- Endpoint summary
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro DNA damage and/or repair study
- Remarks:
- type of assay: γH2AX assay
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- not specified
- Reliability:
- other: not rated according to Klimisch et al.
- Rationale for reliability incl. deficiencies:
- other: see adjacent "Remarks" field
- Remarks:
- The study does not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only.
Data source
Reference
- Reference Type:
- publication
- Title:
- Genotoxicity of 11 Heavy Metals Detected as Food Contaminants in Two Human Cell Lines
- Author:
- Kopp, B., Zalko, D., and Audebert, M.
- Year:
- 2 017
- Bibliographic source:
- Environ Mol Mutagen. 2018 Apr;59(3):202-210
Materials and methods
Test guideline
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Kopp et al. (2017) investigated the effect of SbCl3 on the number of γH2AX-foci in the human cell lines HepG2 and LS-174T.
- GLP compliance:
- not specified
- Remarks:
- not specified in the publication
- Type of assay:
- other: γH2AX assay
Test material
- Reference substance name:
- Antimony chloride, SbCl3
- IUPAC Name:
- Antimony chloride, SbCl3
- Test material form:
- not specified
- Details on test material:
- - Name of test material (as cited by the sutdy): SbCl3
- Source: purchased from Sigma-Aldrich (Sain Quentin Fallavier, France)
- CAS No.: 10025-91-9
Constituent 1
- Specific details on test material used for the study:
- not specified
Method
- Target gene:
- not applicable
Species / strainopen allclose all
- Species / strain / cell type:
- mammalian cell line, other: HepG2 (human hepatoblastoma cells)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: ATCC N° HB-8065
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: alphaMEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin; maintained in humified atmosphere with 5% CO2 at 37°C
- Species / strain / cell type:
- mammalian cell line, other: LS-174T (human epithelial colorectal adenocarcinoma cells)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: ATCC N° CL-188
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: alphaMEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin; maintained in humified atmosphere with 5% CO2 at 37°C
- Metabolic activation:
- not specified
- Metabolic activation system:
- not specified in the publication
- Test concentrations with justification for top dose:
- 10, 50, 100, 250, and 500 µM
- Vehicle / solvent:
- DMSO (0.2% in final cell culture medium; v/v)
Controls
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- 1 µM
- Details on test system and experimental conditions:
- CELL TREATMENT
After 16 h, the medium was replaced by 90 µL of medium without serum. Cells were exposed to 0.2% final (v/v) DMSO in the culture medium. Cells were incubated for 24 h with 10, 50, 100, 250, 500 µM. The positive control used for each treatment was 1 µM benzo[a]pyrene (BaP). The negative control was DMSO (0.2% v/v final concentration, respectively).
IN-CELL WESTERN γH2AX
The In-Cell Western technique was performed as previously described*/**/***/****/*****/******. Briefly, after the treatment, cells were fixed with 4% paraformaldehyde (Electron Microscopy Science) in phosphate buffered saline (PBS) and permeabilized with 0.2% Triton X-100. The cells were then incubated with a blocking solution (MAXblock Blocking Medium supplemented phosphatase inhibitor PHOSSTOP and RNAse A [0.1 g/L)] followed by incubation with anti-γH2AX rabbit monoclonal primary antibody (Clone 20E3, Cell signaling) in PST buffer. After 2 h of incubation at room temperature and three washes, secondary detection was carried out using an infrared fluorescent dye combined with goat antibody (CF770, Biotium). For DNA labeling, RedDot2 (Biotium) was used in combination with the secondary antibody. After 1 h of incubation, DNA and γH2AX were visualized using an Odyssey Infrared Imaging Scanner (Li-CorScienceTec, Les Ulis, France). To determine cytotoxicity, the DNA content (related to the number of cells) recorded in the treated cells was compared to the DNA content in the control cells and is expressed as relative cell count (% RCC). All experiments were performed independently at least three times.
REFERENCES
* Graillot V, Takakura N, Hegarat LL, Fessard V, Audebert M, Cravedi JP. 2012a. Genotoxicity of pesticide mixtures present in the diet of the French population. Environ Mol Mutagen 53:173–184.
**Graillot V, Tomasetig F, Cravedi JP, Audebert M. 2012b. Evidence of the in vitro genotoxicity of methyl-pyrazole pesticides in human cells. Mutat Res 748:8–16.
***Khoury L, Zalko D, Audebert M. 2013. Validation of high-throughput genotoxicity assay screening using gammaH2AX in-cell western assay on HepG2 cells. Environ Mol Mutagen 54:737–746.
****Khoury L, Zalko D, Audebert M. 2016a. Complementarity of phosphorylated histones H2AX and H3 quantification in different cell lines for genotoxicity screening. Arch Toxicol 90:1983–1995.
*****Khoury L, Zalko D, Audebert M. 2016b. Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening. Mutagenesis 31:83–96.
******Chevereau M, Glatt H, Zalko D, Cravedi JP, Audebert M. 2017. Role of human sulfotransferase 1A1 and N-acetyltransferase 2 in the metabolic activation of 16 heterocyclic amines and related heterocyclics to genotoxicants in recombinant V79 cells. Arch Toxicol - Rationale for test conditions:
- not specified
- Evaluation criteria:
- Genotoxicity was considered positive only if three criteria were all fulfilled: (1) The compound caused a 1.3-fold increase in γH2AX, (2) there was a statistically significant difference compared to controls and (3) a level of cytotoxicity below 50% of the controls was observed.
- Statistics:
- The genotoxicity of SbCl3 was compared with the results obtained with the control, using Student’s test with Excel 2016 software (*P<0.05; **P<0.01; ***P<0.001). Error bars represent SEM (standard error of the mean).
Results and discussion
Test resultsopen allclose all
- Species / strain:
- mammalian cell line, other: HepG2 (human hepatoblastoma cells)
- Metabolic activation:
- not specified
- Genotoxicity:
- other: not determinable due to methodological limitations
- Remarks:
- Please refer to "Additional information on results" and "Remarks on result"
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- mammalian cell line, other: LS-174T (human epithelial colorectal adenocarcinoma cells)
- Metabolic activation:
- not specified
- Genotoxicity:
- other: not determinable because of methodological limitations
- Remarks:
- Please refer to "Additional information on results" and "Remarks on result"
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Additional information on results:
- γH2AX ASSAY
HepG2 cells: A more than 1.5-fold increase in γH2AX was observed for SbCl3 (250 µM) and a significant but less pronounced increase was observed at 100 µM (1.3±0.16-fold). Cytotoxicity was observed from 250 µM for SbCl3.
LS-174T cells: The genotoxic effects of SbCl3 that tested positive in HepG2 cells was confirmed in the LS-174T cell line with a similar concentration-dependent induction of γH2AX at subtoxic concentrations. At a SbCl3 concentration of 250 µM, a 1.59±0.29-fold increase in number of γH2AX foci was observed in LS-174T cells. At the same concentration, the RCC was 72±28% when compared to control. - Remarks on result:
- not determinable because of methodological limitations
Applicant's summary and conclusion
- Conclusions:
- In this study, the results demonstrated that SbCl3 inhibits DNA repair.
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