Graphene

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The followed OECD TG 439 is not validated for testing on nanomaterials.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test:
Histological analysis was carried out on hematoxylin/eosin stained RhE specimens after single exposure of few layer graphene (FLG) with the possibility to study dermal absorption.
- Short description of test conditions:
After material exposure, RhE specimens were collected, fixed and hematoxylin/eosin stained and analyzed under the Axio Scope A1, Axiocam 503, using the Zen2 software.
- Parameters analysed / observed: morphology of the skin model and presence of FLG above and within the epidermis

GLP compliance:

not specified

Remarks:

no information on GLP compliance available in this publication

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
FLG was exfoliated by ball milling of graphite

INFORMATION ON NANOMATERIALS
- Lateral dimension: 171 nm ± SD 147 nm (TEM)

- Lateral dimension distribution: 50–600 nm (TEM)
- No layers: 4
- C content %: 94.93 ± 0.28
- H content %: 0.55 ± 0.02
- N content %: 0.54 ± 0.02
- S content %: 0.32 ± 0.03
- O content %: <3.7
- weight loss at 600 °C: 6% (TGA)
- melamine races: 0.81%
- Fe content: 0.074 mg/L

Radiolabelling:

no

Type of coverage:

other: Semiocclusive: test item applicated as powder; occlusive: positive control and negative (vehicle) control in liquid (PBS)

Vehicle:

physiological saline

Duration of exposure:

42 min + 42 h post-treatment incubation

Doses:

- Testmaterial: Amount(s) applied (volume or weight with unit): 16 mg (32 mg/cm²)
- Negative / vehicle control: Amount(s) applied (volume or weight): not specified
- Positive control: Concentration (if solution): 5% w/v SDS or 5% w/v SDBS in PBS

Details on study design:

Test system was a human skin model of non-transformed (normal human) keratinocytes (SkinEthic™ Reconstructed Human Epidermis (RhE) model).
Exfoliated FLG (with melamine) was applicated in powdered form. Immediately after treatment (42 min exposure to FLG and/or positive controls followed by 42 h post-exposure), RhE specimens were collected from each insert and fixed in 10% (v/v) neutral buffered formaldehyde. RhE specimens were then dehydrated, embedded in paraffin and longitudinally sectioned to avoid any passive transport of GBMs deposited on the epidermis surface over the different layers. Sections were then stained with hematoxylin/eosin and analyzed under the Axio Scope A1 optical microscope (Zeiss, Milan, Italy) and microphotographs were collected through the Axiocam 503 color digital camera (Zeiss, Milan, Italy) using the Zen2 software, with a 40× magnification. Images were collected from 6 different samples.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: normal human keratinocytes
- Type of skin: SkinEthic™ Reconstructed Human Epidermis (RhE) non-transformed keratinocytes

Results

- Negative control: stratum corneum characteristically dense with high number of granules in the stratum granulosum; nuclei of proliferating keratinocytes of stratum spinosum and stratum basale typically rounded.

 

- FLG: no signs of epidermis irritation or other alterations, presence of aggregated FLG above the epidermis surfaces (small flat agglomerates). Increasing magnification small depots of FLG within the epidermis; depots were limited to the stratum corneum and were far smaller than the aggregates observable above the epidermis surfaces.

 

- FLG-SDS and FLG-SDBS: loss of the typical density of the stratum corneum, to a similar extent of that induced by the positive controls; morphological alterations, compatible with skin irritation, in the stratum corneum and stratum granulosum and fragmentation of nuclei of keratinocytes of the stratum spinosum and stratum basale; larger flat agglomerates of FLG-SDS and FLG-SDBS above the epidermis

 

- Positive controls SDS and SDB: dramatic morphological alterations typical of skin irritation, with loss of stratum corneum density and destruction and partial removal of the stratum corneum and stratum granulosum; fragmentation of nuclei of keratinocytes of the stratum spinosum and stratum basale.

 

Further results: CVD-graphene: absence of material on the epidermis surface, and no depots visible within the epidermis

Conclusions:

Histological analysis demonstrated the presence of small agglomerates of FLG above the epidermis. Small depots within the epidermis were observed but limited to the stratum corneum (these depots were far smaller than the aggregates observable above the epidermis).

Executive summary:

Study design

The test procedure of the study provided by this publication is well documented and meets generally accepted scientific principles, and is acceptable for assessment. In the study, histological analysis were carried out on hematoxylin/eosin stained RhE specimens after single exposure of few layer graphene (FLG) to study dermal absorption.

After material exposure, RhE specimens were collected, fixed and hematoxylin/eosin stained and analyzed under the Axio Scope A1, Axiocam 503, using the Zen2 software to observe morphological changes of the skin model and presence of FLG above and within the epidermis.

 

Results

The results demonstrated the presence of aggregated/agglomerated FLG above the epidermis surface, showing small flat aggregates/ agglomerates for FLG and larger ones for FLG-SDS and FLG-SDBS. This different behaviour may be related to the different physicochemical properties of these materials, FLG being characterized by an average lateral dimension far smaller than that of FLG-SDS and FLG-SDBS. FLG depots, far smaller than the aggregates/agglomerates observed above the epidermis surface, were observed only within the RhE stratum corneum (composed of completely keratinized keratinocytes). This result suggests that highly dispersed not-aggregated/ agglomerated materials could penetrate into the stratum corneum but could be not able to pass through the stratum granulosum down to the stratum spinosum and stratum basale.

As FLG (but not FLG-SDS and FLG-SDBS) is not able to deeply penetrate into the inner layers of the epidermis and, FLG is unable to directly interact with proliferating keratinocytes of the stratum basale.

 

Conclusion: Histological analysis demonstrated the presence of small agglomerates of FLG above the epidermis. Small depots within the epidermis were observed but limited to the stratum corneum (these depots were far smaller than the aggregates observable above the epidermis).

Data source

Reference

Reference Type:

publication

Title:

Skin irritation potential of graphene-based materials using a non-animal test

Author:

Fusco, L., Garrido, M., Martín, C., Sosa, S., Ponti, C., Centeno, A., Alonso, B., Zurutuza, A., Vázquez, E., Tubaro, A., Prato, M., Pelin, M.

Year:

2020

Bibliographic source:

Nanoscale, 12, 610-622

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

The followed OCED TG 439 is not validated for testing on nanomaterials. Therefore, additional tests were performed to address this matter.

Principles of method if other than guideline:

Note: the OECD TG 439 turned out to be applicable also for graphene-based material testing, since no interference with the methylthiazolyldiphenyl - tetrazolium bromide (MTT) reduction, used as a final readout, was found.

GLP compliance:

not specified

Remarks:

no information on GLP compliance available in this publication

Test material

Test material information

Constituent 1

Reference substance name:

carbon

EC Number:

801-282-5

Cas Number:

1034343-98-0

Molecular formula:

Cx

IUPAC Name:

carbon

Test material form:

solid: nanoform

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
few-layer graphene (FLG) was exfoliated by ball milling of graphite

INFORMATION ON NANOMATERIALS
- Lateral dimension: 171 nm ± SD 147 nm (TEM)

- Lateral dimension distribution: 50–600 nm (TEM)
- No layers: 4
- C content %: 94.93 ± 0.28
- H content %: 0.55 ± 0.02
- N content %: 0.54 ± 0.02
- S content %: 0.32 ± 0.03
- O content %: <3.7
- weight loss at 600 °C: 6% (TGA)
- melamine races: 0.81%
- Fe content: 0.074 mg/L

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: normal human keratinocytes

Vehicle:

physiological saline

Remarks:

vehicle for positive control

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
Room temperature
- Temperature of post-treatment incubation (if applicable):
37°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps:
25 times with 1 mL PBS
- Observable damage in the tissue due to washing:
not specified
- Modifications to validated SOP:
not specified
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:
Automated Microplate Reader EL 311s
- Wavelength:
570 nm
- Filter:
not specified
- Filter bandwidth:
not specified
NUMBER OF INDEPENDENT TESTING RUNS: three independent experiments (in triplicates)
PREDICTION MODEL / DECISION CRITERIA
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430: As a threshold given by OECD TG 439, viability ≤50% compared to negative control defines an irritant compound.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
16 mg (32 mg/cm²)

NEGATIVE / VEHICLE CONTROL
- Amount(s) applied (volume or weight):
not specified

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
not specified
- Concentration (if solution): 5% w/v SDS or 5% w/v SDBS in PBS

Duration of treatment / exposure:

42 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Test system

Type of coverage:

other: Semiocclusive: test item applicated as powder; occlusive: positive control and negative (vehicle) control in liquid (PBS)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

(16 mg FLG)

Run / experiment:

3 replicates/ experiment, three independent experiments

Value:

101

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

Positive control (5% SDS in PBS)

Run / experiment:

3 replicates/ experiment, three independent experiments

Value:

3

Remarks on result:

positive indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Remarks:

Positive control (5% SDBS in PBS)

Run / experiment:

3 replicates/ experiment, three independent experiments

Value:

2

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: no unspecific MTT reduction since the O.D. values recorded in FLG-exposed killed RhE were not significantly different from the negative controls (killed RhE not exposed to FLG) (preliminary set of experiments)
- Colour interference with MTT: no interference between MTT and FLG was found (preliminary set of experiments)

Any other information on results incl. tables

Results of histological analysis was carried out on hematoxylin/eosin stained RhE specimens:

- Negative control: the stratum corneum was dense, presence of a high number of granules in the stratum granulosum, rounded nuclei of proliferating keratinocytes of the stratum spinosum and stratum basale

- The positive controls SDS and SDBS: dramatic morphological alterations typical of skin irritation, with a loss of stratum corneum density as well as destruction and partial removal of the stratum corneum and stratum granulosum, fragmentation of nuclei of keratinocytes of the stratum spinosum and stratum basale.

- FLG: no signs of epidermis irritation or other alterations can be observed.

- Histological analysis demonstrated the presence of small flat agglomerates for FLG materials. Intriguingly, on increasing the magnification of the images, the presence of small depots of FLG was observed within the epidermis. These depots were limited to the stratum corneum and were far smaller than the aggregates observable above the epidermis surfaces.

 

Results of cytokine release (IL-1α , IL-6, IL-8) in RhE media after FLG exposure, evaluated by ELISA

- FLG did not induce a significant effect of IL-1α with respect to the negative control

- The positive controls SDS and SDBS significantly increased the release of IL-1α to 1478 pg/mL (increase of 2742 %; p < 0.001) and 645 pg/mL (increase of 1140 %; p < 0.001), respectively

- FGL and the positive controls SDS and SDBS did not induce a significant increase of IL-6 as compared to the negative control

- The positive controls SDS and SDBS significantly increased the release of IL-8 to 112 pg mL−1 (increase of 35%; p < 0.01) and 178 pg mL−1 (increase of 114%; p < 0.001), respectively

- FLG did not induce a significant release of IL-8 with respect to the negative control

 

Further results:

Considering that some of the FLG materials are obtained by exfoliation with surfactants, the effects induced by FLG-SDS and FLG-SDBS were evaluated (FLG exfoliated with 5% SDS or 5% SDBS):

- Significantly reduction of RhE viability: 85% (FLG-SDS) and 65% (FLG-SDBS). RhE viability reductions were significantly higher than that induced by FLG (−1%). Only FLG prepared with irritant surfactants (FLG-SDS and FLG-SDBS), reduced RhE viability at levels lower than those predicting skin irritation (≤50%), suggesting irritant properties.

- Histological analysis demonstrated the presence of aggregated material: Small flat agglomerates for FLG materials, appearing larger in FLG-SDS and FLG-SDBS. Loss of the typical density of the stratum corneum, to a similar extent of that induced by the positive controls, was observed in RhE exposed to FLG-SDS and FLG-SDBS.

- FLG-SDS and FLG-SDBS were able to significantly increase IL-1α, IL-6, IL-8

 

Further results of additional test item: disks of CVD-graphene monolayers (topically applied on RhE surfaces for the same exposure time):

- CVD-graphene did not reduce RhE viability at levels lower than the threshold given by OECD TG 439 (tissue viability ≤50%)

- No material (no depots or aggregates/agglomerates) on the epidermis surface or within the epidermis for CVD-graphene was found

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

In conclusion, the results demonstrate that few-layer graphene (FLG) (characterized by very low amounts of irritant surfactant residues) do not seem to induce skin irritation after a single acute exposure.

Executive summary:

Study design

This study was carried out with the aim to investigate the irritation potential of few-layer graphene (FLG) using the SkinEthic™ Reconstructed human Epidermis (RhE) a predictive in vitro 3D model, approved as an acute non-animal test for skin irritation according to OECD TG 439.

Even though not validated for nanomaterials, the OCED TG 439 turned out to be applicable also for GBM testing, since no interference with the methylthiazolyldiphenyl- tetrazolium bromide (MTT) reduction, used as a final readout, was found. Furthermore, direct epidermal exposure to powdered FLG mimics the actual human exposure, avoiding interference by the cell culture medium (protein corona formation). As negative (vehicle) and positive controls, tissues were exposed to phosphate buffered saline (PBS) or 5% w/v SDS, respectively. As an additional positive control, RhE was exposed also to 5% w/v SDBS.

Results

FLG did not reduce RhE viability at levels lower than those predicting skin irritation (≤50%), suggesting irritant properties. This result was further confirmed by measuring cytokine (IL-1α, IL-6 and IL-8) release by FLG-treated RhE and by histological analysis as additional readouts to implement the guideline. On the whole, these results demonstrate that FLG (prepared with the non-irritant exfoliation agents melamine) do not induce skin irritation after a single acute exposure.

Conclusion

The results demonstrate that FLG do not seem to induce skin irritation after a single acute exposure.