Graphene

Endpoint summary

Description of key information

Skin irritation/corrosion

Key: In vivo, skin irritation, rabbit, OECD 404: not skin irritating (Cada, 2020)

Key: In vitro, skin irritation, RhE, OECD 439: not skin irritating (Fusco et al., 2020)

 

Eye irritation/corrosion

Key: In vitro, eye irritation, RhE (EpiOcular^TM), OECD 492: not eye irritating (Himmelsbach, 2021)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Dec 2019 to 28 Apr 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted July 28, 2015

Deviations:

yes

Remarks:

There were occasional procedural deviations that were minor and did not impact the integrity or outcome of the study.

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: 16–18 weeks at arrival (+ 7 days acclimation period before dosing)
- Weight at study initiation: 3.3 – 3.4 kg at start of treatment
- Housing: Animals were housed individually in stainless steel cages equipped with an automatic watering system.
- Diet: ad libitum (Envigo Teklad Certified Rabbit Diet (2031C))
- Water: ad libitum (Municipal tap water)
- Acclimation period: acclimation period of 7 days occurred between receipt of the animals and the start of dosing to accustom the animals to the laboratory environment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 10–15 air changes per hour
- Photoperiod (hrs dark / hrs light): a light dark cycle of 12 hours light/12 hours dark on most days, except during study protocol-designated procedures

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

physiological saline

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.5 + 0.01 g

VEHICLE
- Concentration: pre-wetted with 0.9% saline
- Lot/batch no.: 19E004

Duration of treatment / exposure:

4 hours

Observation period:

7 days

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 2 x 3 cm
- Type of wrap if used: gauze was taped to the skin on three sides with Elastikon, the dosing area was then closed with additional Elastikon. The pad was applied to skin and held in place with non-irritating tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the gauze was removed and the skin rinsed (not wiped) of any residual graphene with tap water, and patted dry
- Time after start of exposure: 4 hours ± 1 minute

OBSERVATION TIME POINTS
Prior to dosing, and approximately 1, 24, 48, and 72 hours (Day 4) postdose (after patch removal) as well as on Day 7

SCORING SYSTEM:
- Method of calculation: please refer to 'any other information on materials and methods'

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no skin irritation observed up to day 7

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no skin irritation observed up to day 7

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Dermal exposure to graphene had no impact on dermal scoring observations.
All time points, erythema and edema were scored as 0 at the dose site.

Other effects:

- Mortality: Dermal exposure to graphene had no impact on dermal scoring observations. All time points, erythema and edema were scored as 0 at the dose site.
- Clinical Observations: Dermal exposure to graphene had no impact on clinical observations. All animals were within normal limits at all observed time points.
- Body weights: Dermal exposure to graphene had no impact on body weights. All animals gained ~100 g body weight between Days -1 and 7.

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

Single dermal dosing of 0.5 g of graphene to male albino rabbits resulted in no skin irritation up to 7 days of observation.

Executive summary:

Study design

This study was conducted in compliance with the FIFRA GLP Standards of 40 CFR Part 160 and the generally applicable principles of the OECD, ENV/MC/CHEM (98) 17 as well as any applicable amendments. The study was conducted according to Guidelines OPPTS 870-2500 and OECD TG 404 (adopted July 28, 2015). The objective of this study was to determine potential dermal irritancy of graphene in rabbits. This information is utilized for the assessment and evaluation of the toxic characteristics of the test substance.

Graphene was administered dermally once to a 2 x 3 cm area to 3 albino male rabbits for 4 hours ± 1 minute on Day 1. Examined parameters were mortality and moribundity checks, clinical observations (including dermal scoring observations), and body weight measurements.

Results

Single dermal exposure to graphene had no effect on mortality, clinical observations, dermal scoring, or body weights. There were no erythema or edema observations at any time point following dermal exposure to Graphene, all animals were released from the study on Day 7.

Conclusion

Single dermal dosing of 0.5 g of graphene to male albino rabbits resulted in no skin irritation up to 7 days of observation.

Reference 2

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.

Reason / purpose for cross-reference:

reference to same study

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

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

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

Type of coverage:

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

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

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

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)

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 JUN 2021 to 20 SEPT 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

18 JUN 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: not applicable: normal, human-derived keratinocytes

Remarks:

Reconstructed human Cornea-like Epithelium

Details on test animals or tissues and environmental conditions:

- Justification of the test method (RhCE) and considerations regarding applicability:
The EpiOcularTM Eye Irritation Test (EIT) predicts the acute eye hazard potential of chemicals by measurement of tissue damage caused by cytotoxic effects in the reconstructed human cornea-like tissue model. It is utilized for the classification and labelling of chemicals concerning their eye hazard potential. The test system is applicable for substances like the test item, which is a solid material insoluble in water.
The EpiOcular™ EIT can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. Since minimal or no eye irritation /corrosion potential was expected the for test item, this system was used in the framework of a bottom-up approach.
- RhCE tissue or hCE cell construct used, including batch number:
Commercially available EpiOcularTM kit. The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm².
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 82105 Bratislava, Slovakia.
1. Main Test:
Designation of the kit: OCL-200-EIT
Batch no.: 34914
2. Additional Test Direct Reduction of MTT by the test item:
Designation of the kit: OCL-212-EIT
Batch no.: 30624

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Min test: 50.4 mg (tissue 1) and 50.5 mg (tissue 2). Additional test (MTT-reduction): 50.6 mg (tissue 1), 50.3 mg (tissue 2). The tissue surface is 0.6 cm².

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
1. Preparations: MTT concentrate was thawed and diluted with assay medium directly before use. The assay medium was warmed in the water bath to 37 ± 1°C. 6-well-plates were filled with 1 mL assay medium. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 1 hour. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated for 16 hours and 10 minutes (under the above mentioned conditions).
2. Exposure and Post-Treatment: After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and the tissues were incubated for 30 minutes (under the above mentioned conditions). After that, 50 μL of the controls and a defined amount of the test item were applied in duplicate in one-minute-intervals. All plates were transferred into the incubator for 6 hours (under the above mentioned conditions). At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature. After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity. After the post-treatment incubation, the MTT assay was performed.
3. MTT Assay and Extraction: A 24-well-plate was prepared with 300 μL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 1 mL isopropanol, taking care that no isopropanol was flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.
4. Measurement: The inserts were removed from the 6-well plate and discarded. 1 mL isopropanol was added into each well. Afterwards, the content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well-plate. The plate was read in a plate spectrophotometer at 570 nm. In addition, eight wells of the 96-well-plate were filled with 200 μL isopropanol each, serving as blank.
- Doses of test chemical and control substances used
50 mg test item and 50 μL of the controls per tissue
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
Exposure: at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity
Post-exposure immersion at room temperature (for 25 min)
Post-treatment incubation: at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity
- Justification for the use of a different negative control than ultrapure H2O (if applicable)
Sterile demineralised water was used
- Justification for the use of a different positive control than neat methyl acetate (if applicable)
Methyl acetate (C3H6O2, CAS No. 79-20-9) was used
- Description of any modifications to the test procedure
Inadvertently, isopropanol was pipetted onto each plate measured on the microtiter plate photometer. However, only 8 wells should be used as blank. Therefore, the OD values for isopropanol are only used from the first measured plate (main test). The deviation can be considered non-critical, since the values of the too much pipetted isopropanol are not relevant for the evaluation and have no influence on the result of the study.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
1. Assessment of Direct Reduction of MTT by the Test Item: As the test item is black, it was directly tested for the ability of direct MTT reduction, because it is obvious that the test item’s colour can interfere with the measurement of the MTT. Therefore, it was necessary to perform a functional test with freeze killed tissue that possess no metabolic activity but absorb and bind the test item like viable tissues. Freeze killed tissues were prepared by placing untreated tissues in a freezer (- 20 ± 5 °C) overnight. In addition to the normal test procedure described, the functional check employed two freeze-killed tissue treated with the MTT reducing test item and one untreated killed tissue that shows the small amount of MTT reduction due to residual NADH and associated enzymes within the killed tissue.
As the direct reduction of MTT by the test item was ≤ 50% of the negative control the net OD of the test item treated killed tissues was subtracted from the net OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
2. Assessment of Coloured or Staining Test Items: 49.6 mg of the test item were added to 2 mL isopropanol, incubated in 6-well plates on an orbital shaker for 2 hours at room temperature. Because the test item caused a turbidity in isopropanol due to insoluble solids, the respective solution was centrifuged (1 mL in Falcon tube for 30 seconds at 4500 rpm) and aliquots were taken from the supernatant.
Then, two 200 μL aliquots of the resulting solution and two 200 μL aliquots of neat isopropanol were transferred into a 96-well plate and measured with a plate reader at 570 nm.
After subtraction of the mean OD for isopropanol, the mean OD of the test item solution was 0.032 (≤ 0.08). Therefore, the main test was performed without colourant controls.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
Main test: 2 replicates
Additional test (MTT-Reduction) on freeze-killed tissues: 2 replicates
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
OD at 570 nm
- Description of the method used to quantify MTT formazan, if applicable
A 24-well-plate was prepared with 300 μL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 1 mL isopropanol, taking care that no isopropanol was flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.
The inserts were removed from the 6-well plate and discarded. 1 mL isopropanol was added into each well. Afterwards, the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well-plate. The plate was read in a plate spectrophotometer at 570 nm.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
If the cell viability is greater than 60 %, the substance is non eye irritant. If the tissue viability is equal or less than 60 %, the substance is at least eye irritant (cut-off point as specified for this test system in OECD test guideline 492).
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
The values for negative control and for positive control were within the range of historical data of the test facility.
Therefore, the experiment is considered valid.
- Complete supporting information for the specific RhCE tissue construct or hCE cells used
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 82105 Bratislava, Slovakia.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
All of the 15 proficiency chemicals were correctly categorized. Therefore, the proficiency of the EpiOcularTM test was demonstrated.
- Positive and negative control means and acceptance ranges based on historical data:
OD negative control: mean: 1.663 (range: 1.047 – 2.340), in study: 1.096
Relative Tissue Viability positive control: mean: 33.8% (range: 21.1 – 53.9%), in study: 30.9%
- Acceptable variability between tissue replicates for positive and negative controls
Variation within replicates: < 20%
- Acceptable variability between tissue replicates for the test chemical
Variation within replicates: < 20%

Irritation parameter:

percent tissue viability 

Remarks:

Tissue 1

Run / experiment:

Experiment 1/1

Value:

160.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent tissue viability 

Remarks:

Tissue 2

Run / experiment:

Experimen1/1

Value:

164.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Experiment 1/1

Value:

162.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

mean percent tissue viability 

Remarks:

corrected with freeze-killed tissue

Run / experiment:

Experiment 1/1

Value:

161.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: Range of historical values are within the range of the ones specified in the test guideline

Interpretation of results:

other: EU GHS criteris not met

Conclusions:

After treatment with the test item, the mean value of relative tissue viability was 162.6% and 161.6% after correction with the results on freeze-killed tissues, hence, the values are clearly above the threshold for eye irritation potential (≤ 60%).
Under the conditions of the test, the test item is considered non- eye irritant in the EpiOcularTM Eye Irritation Test.

Executive summary:

Study design

This key study was performed in order to evaluate the eye irritation hazard potential of the Graphene test material. The study was performed according to OECD TG 492 in a Reconstructed human Cornea-like Epithelium (RhCE) model (EpiOcular^TM Eye Irritation Test) and in compliance with GLP.

As the test item is black, it was directly tested on freeze killed controls in an additional test, because it was obvious, that the test item’s color could interfere with the measurement of MTT. Furthermore, the probability of an influence on the photometric measurement as well as a false negative result had to be excluded.

In the main test, the test item was applied to a three-dimensional human cornea tissue model for an exposure time of 6 hours. The solid test item was applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control.

 

Results

Overall, one valid main experiment with an additional test was performed.

The result of the additional test showed that MTT reduction by the test item did influence the measurement and a correction of the result of the main test was necessary. The result of the valid main test was corrected with the result of the additional test on freeze-killed tissues and the corrected viability showed that MTT reduction by the test item itself did not influence the result of the study.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.8, OD was 1.096. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 30.9% (< 50%). The variation within tissue replicates of the controls and the test item was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 162.6% and 161.6% after correction with the results on freeze-killed tissues. This value is above the threshold for eye irritation potential (≤ 60%). Test items above the threshold are considered non-eye irritant.

 

Conclusion

Under the conditions of the test, the test item is considered non- eye irritant in the EpiOcularTM Eye Irritation Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation /corrosion

The available key in vivo study was performed according to Guidelines OPPTS 870 -2500 and OECD TG 404 (adopted July 28, 2015) and in accordance with GLP. The study was conducted in compliance with the FIFRA GLP Standards of 40 CFR Part 160 and the generally applicable principles of the OECD, ENV/MC/CHEM (98) 17, as well as any applicable amendments. 

The objective of this study was to determine potential dermal irritancy of graphene in rabbits. This information is utilized for the assessment and evaluation of the toxic characteristics of the test substance.

Graphene was administered dermally once to a 2 x 3 cm area to 3 albino male rabbits for 4 hours ± 1 minute on Day 1. Examined parameters were mortality and moribundity checks, clinical observations (including dermal scoring observations), and body weight measurements.

Single dermal exposure to graphene had no effect on mortality, clinical observations, dermal scoring, or body weights. There were no erythema or edema observations at any time point following dermal exposure to Graphene, all animals were released from the study on Day 7.

In conclusion, single dermal dosing of 0.5 g of graphene to male albino rabbits resulted in no skin irritation up to 7 days of observation.

 

Comparable results were obtained by in vitro studies. The available key in vitro 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 (Fusco et al., 2020).

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.

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.

In conclusion, the results demonstrate that FLG do not seem to induce skin irritation after a single acute exposure.

 

In a supporting study, investigating the cytotoxic effect of FLG on human epidermal keratinocytes (HaCaT), the observations lead to the conclusion that FLG exert only very weak cytotoxicity on skin keratinocytes (Pelin et al., 2017)

 

Eye irritation:

This key in vitro study was performed in order to evaluate the eye irritation hazard potential of the Graphene test material. The study was performed according to OECD TG 492 in a Reconstructed human Cornea-like Epithelium (RhCE) model (EpiOcular^TMEye Irritation Test) and in compliance with GLP.

As the test item is black, it was directly tested on freeze killed controls in an additional test, because it was obvious, that the test item’s color could interfere with the measurement of MTT. Furthermore, the probability of an influence on the photometric measurement as well as a false negative result had to be excluded.

In the main test, the test item was applied to a three-dimensional human cornea tissue model for an exposure time of 6 hours. The solid test item was applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control.

 

Overall, one valid main experiment with an additional test was performed.

The result of the additional test showed that MTT reduction by the test item did influence the measurement and a correction of the result of the main test was necessary. The result of the valid main test was corrected with the result of the additional test on freeze-killed tissues and the corrected viability showed that MTT reduction by the test item itself did not influence the result of the study.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.8, OD was 1.096. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 30.9% (< 50%). The variation within tissue replicates of the controls and the test item was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 162.6% and 161.6% after correction with the results on freeze-killed tissues. This value is above the threshold for eye irritation potential (≤ 60%). Test items above the threshold are considered non-eye irritant.

Under the conditions of the test, the test item is considered non- eye irritant in the EpiOcularTM Eye Irritation Test.

Justification for classification or non-classification

Based on the available data, the registered substance does not require classification and labelling for skin or eye irritation according to Regulation (EC) No 1272/2008.