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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
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
No Cytotoxicity or Genotoxicity of Graphene and Graphene Oxide in Murine Lung Epithelial FE1 Cells in Vitro
Author:
Bengtson S. et al.
Year:
2016
Bibliographic source:
Environmental and Molecular Mutagenesis 57:469-482 (2016)

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
DNA strand break levels were determined using the comet assay. PBS was used as negative control, 7.5, 15, 30 µm hydrogen peroxide as positive control. DNA strand breaks were quantified as comet tail length (TL) and % DNA in the tail (%DNA).
GLP compliance:
not specified
Remarks:
no information on GLP compliance available in this publication
Type of assay:
comet assay

Test material

Constituent 1
Reference substance name:
Reaction product of Graphite, acid-treated and potassium permanganate
IUPAC Name:
Reaction product of Graphite, acid-treated and potassium permanganate
Test material form:
solid: particulate/powder
Details on test material:
Graphene oxide in aqueous suspension (GO) was manufactured and supplied by Graphenea (San Sebastian, Spain). GO was synthesized by chemical exfoliation of graphite using a modified Hummer's method. Synthetic graphite was dispersed in concentrated sulphuric acid in an ice bath under magnetic stirring and potassium permanganate was slowly added to avoid overheating. The reaction was then heated at 35° C for 1 hr. The reaction is exothermic and to stop the reaction, water and later hydrogen peroxide was added and the reaction solution was transferred to an ice bath. The final solution was cleaned thoroughly with water followed by sonication to obtain GO. To remove non-exfoliated graphite, the inal solution of GO was sonicated (60 Hz) for 1 hr followed by centrifugation for 10 min (10,000 rpm).

Organic elemental composition Graphene oxide
C (wt%) 43.3 ± 1
C, H, N, O (wt%) 88.3
C (mmol/g)b 36.05 ± 1.44
H (mmol/g)b 21.73 ± 0.87
N (mmol/g)b 0.19 ± 0.01
O (mmol/g)b 26.56 ± 1.06
OH (mmol/g)c 26.56
COOH (mmol/g)c 13.28
OH (mmol/m2)d -
COOH (mmol/m2)d -
Inorganic elemental composition (oxide weight %)
SO3 0.02%
MnO 0.0023%
SiO2 0.04%
K2O -
CaO -
Cl 0.0081%
Al2O3 0.02%
Fe2O3 0.0011%
MoO3 -
ZnO -
CuO 0.0010%
NiO 0.0004%
Pd 0.0040%
Ru 0.0032%
Specific details on test material used for the study:
TABLE I. Characterization of GO:

Number of layers 2 - 3
Lateral size T EM (µm) 2 - 3
(Transmission electron microscopy)
Lateral size STEM (µm) ~1
(Scanning transmission electron microscopy)
Surface area B E T (m2/g) -
(Brunauer-Emmet-Teller)
Z-average DLS (nm)b 157
(dynamic light scattering)
PDIc 0.354
(Polydispersity Index)
Zeta potential (mV)d -39.3 ± 1.5
pH 7.02

b Mean hydrodynamic size (6 repeated measurements) in cell culture medium determined with dynamic light scattering.
c Polydispersity Index.
d Mean 6 SEM across 3 repeated measurements.

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
mammalian cell line, other: FE1 cells
Details on mammalian cell type (if applicable):
CELLS USED
The spontaneously immortalized murine pulmonary epithelial cell line (FE1), derived from the transgenic mouse strain 40.6 MUTA-Mouse [White et al., 2003] was used.

MEDIA USED
FE1 cells were cultured in an incubator (37°C, 5% CO2) in cell culture medium (DMEM/ F.-121 GlutaMAX, Life Technologies, 31331-028) supplied by 2% heat-inactivated Fetal Bovine Serum (Gibco, 10106-169), 1% Penicillin (10,000 IU/ml) Streptomycin (10,000 pg/ml, Biological Industries, 949¬208), and 0.001% Epidermal growth factor (Sigma E4127).
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
0, 5, 10, 25, 50, 100, 200 µg/ml
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
other:
Remarks:
As positive control, FE1 cells exposed to 7.5, 15, and 30 µM hydrogen peroxide
Details on test system and experimental conditions:
DNA strand break levels were determined using the comet assay.
As positive control, FE1 cells exposed to 7.5, 15, and 30 µM hydrogen peroxide for 30 min were included and showed statistically signiicantly increased DNA strand break levels compared to PBS exposed cells. Levels of DNA strand breaks were determined in FE1-cells after exposure to GO (5-200 µg/ml) following 3 hr and 24 hr of exposure.
In brief, 10 µl cell suspensions of FE1 cells, preserved in 17% DMSO + 83% fetal bovine serum, were embedded in agarose gel on 20-well Trevigen comet slides (Gaithersburg, MD). Slides were placed in lysis buffer overnight at 4°C. The next day, the slides were placed in alkaline solution for 30 min prior to alkaline electrophoresis (25 min, 1.15 V/cm, and 294 mA) with circulation (70 ml/min). After electrophoresis, slides were neutralized for 10 min. The slides were stained with SYBR Green for 30 min. FE1 cells exposed for 30 min at 4°C to PBS or 7.5, 15, 30 µm hydrogen peroxide were included as negative and positive control, respectively. Analysis and scoring of DNA strand breaks was performed with IMSTAR path-inder system (IMSTAR, Paris, France). DNA strand breaks were quantified as comet tail length (TL) and %DNA in the tail (%DNA).
Evaluation criteria:
For statistical analysis comet tail length (TL) and % DNA in the tail (% DNA) were normalized to the mean control level (0 µg/ml) for the respective experiment.
Statistics:
All statistical analyses were performed in R (v3.10) and Rstudio (v 0.98.1091). The statistical analyses were performed with One Way Analysis of Variance (ANOVA) and presented as mean +/- standard error of the means (SEM). Data were separated by individual particle and dose was set as categorical variable. In case of signiicant main effect of dose (signiicance level; 0.05), a pairwise comparison across doses was performed with Tukey's honest significant difference (HSD) test with adjusted correction (significant level; 0.05, confidence interval; 0.95).

Results and discussion

Test results
Key result
Species / strain:
mammalian cell line, other: FE1 cells
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
The levels of DNA strand breaks were presented as TL and % DNA. As positive control, FE1 cells exposed to 7.5, 15, and 30 µM hydrogen peroxide for 30 min were included and showed statistically significantly increased DNA strand break levels compared to PBS exposed cells. Levels of DNA strand breaks were determined in FE1-cells after exposure to GO (5-200 µg/ml) following 3 hr and 24 hr of exposure. Exposure had no effect on DNA strand break levels at doses up to 200 µg/ml at either time point.

Applicant's summary and conclusion

Conclusions:
The few layered GO was genotoxic to FE1 murine lung epithelial cells at concentrations up to 200 µg/ml.
Executive summary:

This study analyzed the genotoxic effects of graphene oxide on FE1 cells ( murine pulmonary epithelial cell line).

The levels of DNA strand breaks were assessed with the comet assay and presented as TL and % DNA. As positive control, FE1 cells exposed to 7.5, 15, and 30 µM hydrogen peroxide for 30 min were included and showed statistically significantly increased DNA strand break levels compared to PBS exposed cells. Levels of DNA strand breaks were determined in FE1-cells after exposure to GO (5-200 µg/ml) following 3 hr and 24 hr of exposure to reflect both transient and prolonged genotoxicity. Exposure had no effect on DNA strand break levels at doses up to 200 µg/ml at either time point.

The result of this study is supported by an in vivo cytogenicity test of Bengtson et al. (2017) where no dose-dependent increase in DNA breaks was observed for graphene oxide after pulmonary exposure of Graphene oxide in mice, therefore the test substance graphene oxide is not considered as genotoxic.