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Toxicological information

Specific investigations: other studies

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Administrative data

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
other: not rated acc. to Klimisch
Rationale for reliability incl. deficiencies:
other: Any kind of reliability rating is not considered to be applicable, since the study is not conducted/reported according to a standardised guideline.

Data source

Reference
Reference Type:
publication
Title:
The protective role of Nrf2-Gadd45b against antimony-induced oxidative stress and apoptosis in HEK293 cells
Author:
Jiang, A. et al.
Year:
2016
Bibliographic source:
Toxicologiy Letters http://dx.doi.org/10.1016/j.toxlet.2016.05.016

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, the cytotoxicity of antimony trioxide to human embryonic kidney (HEK) 293 cells was investigated. Furthermore, the intracellular ROS level in HEK 293 cells was examined after treatment with antimony trioxide. The following concentrations were tested: 1 (cytotoxicity only), 2 (cytotoxicity only), 4 (cytotoxicity only), 8, 16 and 32 (cytotoxicity only) µM. A control was run concurrently.
GLP compliance:
not specified
Remarks:
not specified in this publication
Type of method:
in vitro
Endpoint addressed:
genetic toxicity

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Sigma Aldrich

Test animals

Species:
other: human embryonic kidney (HEK) 293 cells
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
NOTE: This is an in vitro study conducted with human embryonic kidney (HEK) 293 cells. Information about the cells used in this study is given below.

CELLS USED
- Source of cells: Shanghai Cell Bank of Type Culture Colllection of Chinese Academy of Sciences
- Methods for maintenance in cell culture: cells were routinely maintained in 1640 medium (Hyclone) with 10% fetal bovine serum and 100 U/mL penicillin-streptomycin at 37 °C in humidified atmosphere with 5% CO2.

Administration / exposure

Route of administration:
not specified
Vehicle:
not specified
Details on exposure:
please refer to the field "Details on study design" below.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Cytotoxicity assay: 24 hours
Frequency of treatment:
Cytotoxicity assay: once
Post exposure period:
Cytotoxicity assay: 4 hours
Doses / concentrationsopen allclose all
Dose / conc.:
1 other: µM
Remarks:
Cytotoxicity assay
Dose / conc.:
2 other: µM
Remarks:
Cytotoxicity assay
Dose / conc.:
4 other: µM
Remarks:
Cytotoxicity assay
Dose / conc.:
8 other: µM
Remarks:
Cytotoxicity and ROS assay
Dose / conc.:
16 other: µM
Remarks:
Cytotoxicity and ROS assay
Dose / conc.:
32 other: µM
Remarks:
Cytotoxicity assay
No. of animals per sex per dose:
not applicable
Control animals:
not specified
Details on study design:
CYTOTOXICITY ASSESSMENT:
MTT assay was carried out to examine cytotoxicity upon chemical treatment following the instructions from manufacturer (Roche). In brief, cells were seeded in 96-well plates and then treated with the test substance at different concentration. After treatment for 24 ours, 20 μL of MTT (5 mg/mL) was added into complete culture media and cells were cultured for additional 4 hours. Thereafter, the medium with MTT were aspirated, and 200 µL of DMSO was added into each well. Finally, 96-well plates were read at 490nm on a microplate reader (Thermo).
A control group was run concurrently.

INTRACELLULAR ROS LEVEL
Cells were cultured into 96-well pates and exposed to the test substance in accordance with the manufacturer’s instruction (Sigma-Aldrich). Dichlorofluorescein diacetate (DCF-DA) was added at a final concentration of 10 µM in the dark for 30 minutes before examination. Cells were then washed with PBS three times, and DCF fluorescence was then monitored using a microplate reader. The excitation and emission wavelength were 488 and 525 nm, respectively, and 0.1% H2O2 was used as a positive control to induce ROS in cells.
A control group was run concurrently.

STATISTICAL ANALYSIS:
The difference in data between two experimental groups were assessed using the two-tailed Student’s t-test or One-way ANOVA test. Data are presented as mean ± SD. P value <0.05 was considered statistically significant.

Examinations

Examinations:
please refer to the field "Details on study design" above.
Positive control:
Intracellular ROS level: 0.1 % H2O2

Results and discussion

Details on results:
CYTOTOXICITY ASSESSMENT:
Antimony trioxide exhibited a dose-dependent cytotoxicity against HEK293 cells for 24 hours (P < 0.05).

INTRACELLULAR ROS LEVEL:
Intracellular ROS content increased in a dose dependent manner with the peak reaching at 6 hours.

Please also refer to the field !Attached background material" below.

Applicant's summary and conclusion

Conclusions:
In this study, the results showed that antimony trioxide caused a dose-dependent cytotoxicity against HEK293 cells, and it caused an increased intracellular ROS content in a dose dependent manner with the peak reaching at 6 hours.