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Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vitro, other
Remarks:
genotoxic properties were investigated using a non-validated high-throughput screening (gH2AX assay) in two human cell lines (HepG2 and LS-174T)
Type of information:
experimental study
Adequacy of study:
other information
Study period:
no information
Reliability:
other: genotoxic properties were investigated using a non-validated high-throughput screening (gH2AX assay) in two human cell lines (HepG2 and LS-174T)
Rationale for reliability incl. deficiencies:
other: genotoxic properties were investigated using a non-validated high-throughput screening (gH2AX assay) in two human cell lines (HepG2 and LS-174T)
Qualifier:
no guideline followed
Principles of method if other than guideline:
The genotoxic and cytotoxic properties of different heavy metals were investigated using a non-validated high-throughput screening (gH2AX assay) in two human cell lines (HepG2 and LS-174T).
HepG2 and LS174-T cells cultured in aMEM Medium (10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin) were seeded in 96-well microplates at a density of 3.23 x10E4 cells per well, 16 h prior to treatment. After 16 h, the medium was replaced by 90 mL of medium without serum. Next, 10 mL of metalloids solubilized in pure water were placed directly in the treated well. Cells were incubated for 24 h with different range of concentration for each metalloid depending on its solubility and cytotoxicity: 1, 10, 100, 500, 1000 µM for K2TeO3.
The positive control used for each treatment was 1 µM benzo[a]pyrene (BaP). The negative control was pure water (10% v/v final concentration).
Briefly, after the treatment, cells were fixed and DNA and gH2AX were visualized using the In-Cell Western technique (ICW assay).
GLP compliance:
not specified
Type of assay:
other: High-throughput screening (gH2AX assay)
Specific details on test material used for the study:
Heavy metals (purity>95%) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). All stock solutions were prepared in pure water (10X) (EMD Millipore ElixTM Essential 5 Water Purification System)
Target gene:
The phosphorylation of histone H2AX (gH2AX) was determined as an early and sensitive marker of genotoxicity induced by different types of DNA lesions including notably DNA adducts, DNA abasic sites, DNA strand breaks and DNA replication or transcription blocking lesions and including those hypothesized to be induced by heavy metals.
Species / strain / cell type:
mammalian cell line, other: HepG2 human hepatoblastoma cells
Details on mammalian cell type (if applicable):
ATCC N° HB-8065
Cells were cultured in aMEM medium supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Cultures were maintained in a humidified atmosphere with 5% CO2 at 37°C and the medium was refreshed every 2–3 days during subculturing.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
mammalian cell line, other: LS-174T human epithelial colorectal adenocarcinoma cell
Details on mammalian cell type (if applicable):
ATCC N° CL-188
Cells were cultured in aMEM medium supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Cultures were maintained in a humidified atmosphere with 5% CO2 at 37°C and the medium was refreshed every 2–3 days during subculturing.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
depending on its solubility and cytotoxicity: 1, 10, 100, 500, 1000 µM
Vehicle / solvent:
pure water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
HepG2 and LS174-T cells cultured in aMEM Medium (10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin, maintained in a humidified atmosphere with 5 % CO2 at 37 °C) were seeded in 96-well microplates at a density of 3.23 x10E4 cells per well, 16 h prior to treatment. After 16 h, the medium was replaced by 90 µL of medium without serum. Next, 10 µL of metalloids solubilized in pure water were placed directly in the treated well. Cells were incubated for 24 h with different range of concentration for each metalloid depending on its solubility and cytotoxicity: 1, 10, 100, 500, 1000 µM for K2TeO3.
The positive control used for each treatment was 1 µM benzo[a]pyrene (BaP). The negative control was pure water (10% v/v final concentration).
All experiments were performed independently at least three times.
After the treatment, cells were anaylsed using the In-Cell Western technique (ICW). Briefly, after the treatment cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) and permeabilized with 0.2% Triton X-100. The cells were then incubated with a blocking solution followed by incubation with anti-gH2AX 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 gH2AX were visualized using an Odyssey Infrared Imaging Scanner.
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).
Rationale for test conditions:
Study design is based on validation of high-throughput genotoxicity assay screening using gammaH2AX in-cell western assay on HepG2 cells and other publications using the same or similar test conditions.
Evaluation criteria:
Genotoxicity was considered positive only if three criteria were all fulfilled: (1) The compound caused a 1.3-fold increase in gH2AX, (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 each metalloid was compared with the results obtained with the controls, 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).
Species / strain:
mammalian cell line, other: HepG2 cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Remarks:
results of negative control are not shown
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not specified
Remarks:
results of positive control are not shown
Species / strain:
mammalian cell line, other: LS-174T cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Remarks:
results of negative control are not shown
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not specified
Remarks:
results of positive control are not shown
Additional information on results:
Response of HepG2 Cells:
Cytotoxicity was observed from 100 µM and 500 µM for K2TeO3. A satistically significant increase was observed at 500 and 1000 µM, which was linked with marked cytotoxicity. At 500 µM K2TeO3 induced a more than 1.5-fold increase in gH2AX of 1.60 +/- 0.8 with RCC (%): 61+/- 30.

Responses of LS-174T Cells:
The genotoxic effects of the metalloids that tested positive in HepG2 cells was confirmed in the LS-174T cell line with a similar concentration-dependent induction of gH2AX at subtoxic concentrations. A satistically significant increase was observed at 500 and 1000 µM, which was linked with marked cytotoxicity. At 500 µM K2TeO3 induced gH2AX fold induction of 1.37 +/- 0.23 with an RCC (%) of 69+/-11.

Conclusively, K2TeO3 induced an increasing concentration-response of gH2AX with marked cytotoxicity at the highest concentrations tested. Thereby, K2TeO3 was exceeding the threshold for positivity at a concentration showing marked cytotoxicity already.
Conclusions:
K2TeO3 was found to induce an increasing concentration-response of gH2AX and marked cytotoxicity at the highest concentrations tested in the two human cell lines HepG2 and LS-174T. The investigated heavy metals tested positive in this assay were ranked based on their gentoxic potency as indicated by the lowest observed adverse effect concentration. Thereby, K2TeO3 was in second last position as K2TeO3 was only slightly exceeding the non-validated threshold value (1.3 fold) for positivity (1.37 fold in LS-174T cells) and at this concentration marked cytotoxicty (RCC 69 %) was observed already.
Executive summary:

In this study, the genotoxic and cytotoxic properties of 11 different heavy metals were investigated using high-throughput screening (gH2AX assay) in two human cell lines (HepG2 and LS-174T).

HepG2 and LS174-T cells were seeded in 96-well microplates at a density of 3.23 x10E4 cells per well, 16 h prior to treatment. After 16 h, the medium was replaced by 90 µL of medium without serum. Next, 10 µL of metalloids solubilized in pure water were placed directly in the treated well. Cells were incubated for 24 h with different range of concentration for each metalloid depending on its solubility and cytotoxicity: 1, 10, 100, 500, 1000 µM for K2TeO3. The positive control used for each treatment was 1 µM benzo[a]pyrene (BaP). The negative control was pure water (10% v/v final concentration). However, the results for positive and negative controls were not shown.

Briefly, after the treatment, cells were fixed and DNA and gH2AX were visualized using the In-Cell Western technique (ICW assay).

K2TeO3 was found to induce an increasing concentration-response of gH2AX and marked cytotoxicity at the highest concentrations tested in the two human cell lines HepG2 and LS-174T. The investigated heavy metals tested positive in this assay were ranked based on their gentoxic potency as indicated by the lowest observed adverse effect concentration. Thereby, K2TeO3 was in second last position as K2TeO3 was only slightly exceeding the non-validated threshold value (1.3 fold) for positivity (1.37 fold in LS-174T cells) and at this concentration marked cytotoxicty (RCC 69 %) was observed already.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames Test:


In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains TA 1535, TA 1537, TA98 and TA100 of S. typhimurium and E. coli WP2uvrA were exposed to Tellurium, (powder 99.95 % a.i.), at concentrations up to 5000 µg/plate in the presence and absence of mammalian metabolic activation, (S9 mix; phenobarbital/β-naphthoflavone induced rat liver).


Tellurium had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.


 


Chromosomal Aberration Test with Tellurium dioxide


The read-across substance Tellurium dioxide was tested according to OECD guideline 479 in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells . The test item was examined up to the cytotoxic concentrations according to the relevant OECD guideline covering the range from cytotoxicity to no or little cytotoxicity.


Treatment with the test item did not result in a repeatable, statistically and biologically significant increase in the frequency of the cells with structural chromosome aberrations without gaps either in the presence or absence of a metabolic activation system.


 


Mammalian Cell Gene Mutation Test with Tellurium dioxide:


An in vitro mammalian cell assay according to OECD Guideline 476 was performed in mouse lymphoma L5178Y TK+/- cells at the tk locus to test the potential of Tellurium dioxide to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation. The evaluated concentration ranges covered the range from cytotoxicity to no or little cytotoxicity.


 


Statistical significant increase was observed, however taking into account the global evaluation factor no biological relevant mutagenic effect of tellurium dioxid, was found either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.


 


 


Justification for read-across is outlined in document attached to section 13 of the dossier.


 



Justification for selection of genetic toxicity endpoint
Reliable data are available from the full in-vitro test set required under REACH regulation.

Short description of key information:
Negative data are available from a gene mutation assay in bacteria with tellurium. As well negative data were obtained from a chromosomal aberration test and a mammalian cell gene mutation assay with the read-across substance tellurium dioxide.


Negative data are available from a gene mutation assay in bacteria with tellurium. As well negative data were obtained from a chromosomal aberration test and a mammalian cell gene mutation assay with the read-across substance tellurium dioxide.  


 


Tellurium was subject to CLH process in 2019 and 2020. The RAC in its assessment and comparison with the classification criteria agrees that no indication for a mutagenic potential of neither tellurium nor tellurium dioxide can be derived from the presented in vitro studies.


On this basis and in the absence of any in vivo study, no classification for germ cell mutagenicity was proposed by RAC.


See RAC opinion CLH-O-0000006810-77-01/F adopted 11 June 2020 for detailed explanation

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on all available information the substance is devoid of any relevant genotoxic potency.