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Toxicological information

Genetic toxicity: in vitro

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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:
2017
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
Name:
Unnamed
Type:
Constituent
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
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.