[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not applicable

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:

Differential cytotoxic effects of graphene and graphene oxide on skin keratinocytes

Author:

Pelin M. et al.

Year:

2017

Bibliographic source:

Sci. Rep. 7, 40572; doi: 10.1038/srep40572 (2017)

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this study the in vitro effects of GO on human skin HaCaT keratinocytes, a spontaneously immortalized non-tumor cell line, were evaluated.
GOs effect on mitochondrial activity of HaCaT cells was evaluated by the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) reduction assay.
GOs effects on HaCaT cells proliferation were evaluated by the sulforhodamine B (SRB) assay.
Cell membrane damages were evaluated by measuring Propidium iodide (PI) uptake (PI fluorescence inside the cells).

GLP compliance:

not specified

Remarks:

no information on GLP compliance available in this publication

Test material

Test material information

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: nanoform

Specific details on test material used for the study:

Summarized materials properties of GO see Table 1 "Any other information on materials and methods"
GO was prepared using the improved Hummer's method. A mixture of concentrated H2SO4/H3PO4 (180:20 mL), was added into a mixture of powdered graphite (1.5 g) and KMnO4 (1.8 g). Then, the resulting mixture was heated to 50 °C and stirred for 12 h. The reaction was then cooled to RT and poured in ice water (200 mL) with addition of H2O2 (0.5 mL, 30%). The mixture was filtered and washed with water. The resulting wet solid was re-dissolved in water (200 mL) and dialyzed until neutral pH and colorless aqueous solution was observed. The dialyzed suspension was centrifuged (4000 rpm, 1 h) in order to separate the graphite material. The supernatant was filtered and washed with ethyl ether, obtaining 2.6 g of brown solid.

In vitro test system

Test system:

other: human keratinocytes cell line HaCaT

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human keratinocytes

Details on animal used as source of test system:

not applicable

Justification for test system used:

Initially, the effects of GO on cell viability was evaluated by means of mitochondrial activity of HaCaT cells after different exposure times (24 up to 72 h) by the WST-8 assay. This assay, widely used to investigate mitochondrial damages of different GBMs on a wide range of cell models, was preferred to the MTT assay since the latter can generate a nonspecific signal due to a possible spontaneous reduction of the MTT reagent by GO, leading to false positive overestimation of cell viability.

Vehicle:

unchanged (no vehicle)

Details on test system:

Cell Culture:
The human skin HaCaT cell line was purchased from Cell Line Service (DKFZ, Eppelheim, Germany) and all cell culture reagents were from Euroclone (Milan, Italy). Cells were maintained in DMEM high glucose supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/mL penicillin and 0.1 mg/mL streptomycin. Cell cultures were maintained according to standard procedures in a humidified incubator at 37 °C with 5 % CO2, performing cell passages once a week. If not otherwise specified, for cytotoxicity experiments, cells were seeded in 96-wells plates at a density of 5 E3 cells/well. Experiments were carried out between passages 50 and 65.
Cells exposure to GO: For cytotoxicity assays, cells were exposed to GO (0.005 to 100 µg/mL) up to 72 h.

WST-8 reduction assay: GOs effect on mitochondrial activity of HaCaT cells was evaluated by the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) reduction assay. After exposure to GO, cells were washed three times with PBS (200 µL/well) and incubated for 4 h with fresh medium (100 µL/well) containing 10 µL of WST-8 reagent. Absorbance was subsequently read at 450 nm by an Automated Microplate Reader EL 311 s (Bio-Tek Instruments, Winooski, VT, USA). Data are reported as % of mitochondrial activity in cells exposed to GO with respect to untreated control cells.

Sulforhodamine B (SRB) assay: GOs effects on HaCaT cells proliferation were evaluated by the sulforhodamine B assay. After exposure to GO, cells were washed three times with PBS (200 µL/well), fixed with 50% (v/v) trichloroacetic acid for 1 h at 4 °C and stained for 30 min with 0.4% SRB in 1% (v/v) acetic acid. After washings with 1% (v/v) acetic acid, the protein-bound dye was dissolved in 10 mM TRIZMA base solution and the absorbance was read by an Automated Microplate Reader EL 311 s (Bio-Tek Instruments, Winooski, VT, USA) at 570 nm. Data are reported as % of cell proliferation after GOs exposure with respect to untreated control cells.

Propidium iodide (PI) uptake: Cell membrane damages were evaluated by measuring PI fluorescence inside the cells. Briefly, ater exposure to GO, cells were washed three times with PBS and then exposed to 3.0 E-6 M PI in PBS for 30 min at 37 °C. As a positive control, 0.1% (vol/vol) Triton-X in PBS were added. Fluorescence intensity was read by a Fluorocount Microplate Fluorometer (Packard, Germany) with excitation wavelength of 485 nm and emission wavelength of 590 nm. Each sample was subsequently permeabilized with 0.1% Triton-X for 30 min to measure total fluorescence (index of total cell content). Data are reported as % of PI with respect to positive control cells, after normalization on cell content.

Statistical analysis:
Results are presented as mean ± SE from at least three independent experiments performed in triplicate. Non-linear regression of concentration-effect data was performed using GraphPad Prism version 4.00 for computing the concentration giving the 50% of the effect (EC50). Statistical differences among EC50 values were evaluated by Student t-test (significant differences, p < 0.05), data obtained by comparison of different GBMs were analyzed by a two-way ANOVA analysis followed by Bonferroni's post-test (PrismGraphPad, Inc.; San Diego, CA, USA) while data obtained by long-term analysis were analyzed by a one-way ANOVA analysis followed by Bonferroni's post-test (PrismGraphPad, Inc.; San Diego, CA, USA) and significant differences were considered at p < 0.05.

Control samples:

yes, concurrent no treatment

yes, concurrent positive control

Amount/concentration applied:

Applied concentrations: 0.005 to 100 µg/mL

Duration of treatment / exposure:

WST-8 assay: up to 72 h,
Sulforhodamine B (SRB) assay: up to 72 h,
Propidium iodide (PI) uptake: up to 72 h,

Duration of post-treatment incubation (if applicable):

none

Number of replicates:

not specified

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: mitochondrial activity, cell proliferation, cell membrane damage

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: slight effects on mitochondrial activity, cell proliferation and membrane integrity after prolonged exposure to high test concentrations (up to 100 µg/mL)

Applicant's summary and conclusion

Conclusions:

Weakly cytotoxic effects of graphene oxide on HaCaT keratinocytes were found after prolonged exposure (72 h) to high concentrations of graphene oxide.

Executive summary:

In this in vitro study, a potential cytotoxic effect of the test item graphene oxide on human epidermal keratinocytes (cell line HaCaT) was studied. The effect of graphene oxide (lateral dimension: 622 +/- 581 nm) on cell viability was evaluated by means of mitochondrial activity of HaCaT cells after different exposure times (24 up to 72 h) by the WST-8 reduction assay, the effect on cell proliferation was evaluated by the sulforhodamine B assay and cell membrane damages were evaluated by measuring the uptake of propidium iodide.

Low cytotoxic effects were observed in the WST-8 assay (mitochondrial activity), and cell proliferation was not modulated after exposure to up to 100 µg/mL graphene oxide for up to 48 h, whereas a prolonged exposure of 72 h slightly reduced cell proliferation by up to 15%. The EC50 value estimated for the PI uptake assay was 23.5 µg/mL.

Similar experiments were conducted in parallel for two additional graphene oxide test items with similar C/O ratios slightly larger sizes (lateral dimensions: 845 +/- 427 nm; 979 +/- 498 nm) found a very slight increase in effects on keratinocytes for these slightly larger test items.

These results suggest that only high concentrations and long exposure times to graphene oxide could impair mitochondrial activity associated with plasma membrane damage in keratinocytes, suggesting very low cytotoxic effects of graphene oxide in the skin.