[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin irritation/corrosion

WoE: in vitro skin irritation (HaCaT cells, Pelin 2017, no guideline followed, Graphene oxides): very weak cytotoxicity on skin keratinocytes

WoE: in vivo skin inflammation after subcutaneous injection (mice, Yue 2012, no guideline followed, Graphene oxide): weak inflammatory response after subcutaneous injection

WoE: In vivo skin irritation (rabbit, Ema 2011, OECD Guideline 404, N-MWCNT): not skin irritating

WoE: In vivo skin irritation (rabbit, Ema 2011, OECD Guideline 404, MWCNT-7): not skin irritating

Eye irritation/corrosion

WoE: in vivo acute eye irritation (rabbit, Wu 2016, OECD Guideline 405, Graphene oxide): no eye irritation

WoE: in vitro eye irritation (RhCE and BCOP, Kolle 2016, OECD Guidelines 492 and 437, three MWCNT test materials): not eye irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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

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.

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

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)

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.

Reference 2

Endpoint:

skin irritation: in vivo

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:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: The inflammatory response in murine skin was analysed after subcutaneous injection of graphene oxide.
- Short description of test conditions: Mice were injected subcutaneously in the neck region with the 350 nm GO at series concentrations. Mice without any treatment were used as control. After 21 day post-injection, mice were sacrificed, and the neck tissues were harvested, fixed in 10% formaldehyde, and processed for histology with hematoxylin and eosin (H&E) stain.
- Parameters analysed / observed: Histology, migration of inflammatory cells in the skin

GLP compliance:

not specified

Remarks:

GLP compliance is not specified in this publication

Specific details on test material used for the study:

Atomic force microscopy characterization showed that GO was generally 350 nm in lateral dimension, and the average height was 3.9 nm.

Species:

mouse

Strain:

C57BL

Details on test animals or test system and environmental conditions:

C57BL/6 male mice (6-8 weeks) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products.

Type of coverage:

other: injected subcutaneously

Preparation of test site:

not specified

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

series concentrations

Duration of treatment / exposure:

single injection

Observation period:

21 days

Number of animals:

5

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

other: skin infiltration

Time point:

21 d

Remarks on result:

other: slight migration of inflammatory cells

Remarks:

score not applicable

Conclusions:

Subcutaneous injection of nanosized GO provoked only a slight skin infiltration of inflammatory cells in mice, showing a weak inflammatory response after subcutaneous injection of nanosized GO.

Executive summary:

The capacity of the test item graphene oxide to induce skin inflammation after subcutaneous injection was studied in mice. In this study, the immigration of inflammatory cells in the skin was studied after a single subcutaneous injection. For nanosized (250 nm), only a slight skin infiltration was found, whereas for microscaled (2 µm GO), a larger number of mononuclear cells (such as macrophages and lymphocytes) infiltrated subcutaneous adipose tissue, and lipid-filled vacuoles as well as tissue impairment appeared. Thus, this study shows that even after subcutaneous injection, nanosized GO induces only a weak inflammatory response in murine skin.

Reference 3

Endpoint:

skin irritation: in vivo

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:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

GLP compliance:

not specified

Remarks:

GLP compliance is not specified in this publication

Species:

rabbit

Strain:

other: Kbl:NZW

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kitayama Labes Co. Ltd. (Ina, Japan)
- Age at study initiation: 17 weeks
- Weight at study initiation: 2.7 - 3.6 kg
- Housing: individually in standard aluminum cages
- Diet: LRC4; Oriental Yeast Co. Ltd., Tokyo, Japan, ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 6 days prior to the start of the experiment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0-25.0 °C
- Humidity (%): 40-70%
- Air changes (per hr): ventilation of 15-21 air changes/h
- Photoperiod (hrs dark / hrs light): 12-h light (7:00-19:00)/dark (19:00-7:00) cycle

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

other: olive oil

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
MWCNT-7 was moistened with a minimum amount of olive oil to prepare a paste for dermal application. The paste contained 2% MWCNT-7. This amount of MWCNT-7 was the maximum not leading to overlow when patched and the sample fully coated the skin. Olive oil was used to prepare the paste, because the olive oil was highly refined (neutral, denatured, and free of antioxidants) and widely used as the negative control in these expermiments.
An area of about 10 x 15 cm on the back of each rabbit was made free of fur using electric clippers and an electric shaver 24 h prior to testing. The MWCNT-7 paste (0.5 g) was evenly spread on a lint cloth (2.5 x 2.5 cm), applied to the skin, and covered with a gauze patch, which was held in place with non-irritating elastic bandage (Silkytex, Alcare Co. Ltd., Tokyo Japan).

Duration of treatment / exposure:

4 hours

Observation period:

Test sites were evaluated for signs of dermal irritation approximately 60 min, 24, 48, and 72 h after test substance removal.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure:
6.25 cm2 (2.5 x 2.5 cm) clipped area of skin
- Type of wrap if used:
gauze patch which was held in place with non-irritating elastic bandage

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure:
4 h

OBSERVATION TIME POINTS

60 min, 24 h, 48 h, and 72 h

SCORING SYSTEM:
Test sites were evaluated according to OECD TG 404.

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible within: 72 h

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritant / corrosive response data:

No clinical signs or changes in body weight gain were observed in any groups treated with MWCNT-7. No dermal responses, including erythema/eschar or edema, were found in rabbits treated with MWNT-7. The PII was calculated to be 0.

Other effects:

None

Conclusions:

No clinical signs or changes in body weight gain were observed. No dermal responses, including erythema/eschar or edema, were found in rabbits treated with MWNT-7. The PII was calculated to be 0.

Executive summary:

Acute dermal irritation was analyzed in a study performed according to method described in OECD guideline 404. New Zealand white rabbits received doses of 0.5 g of MWCNT-7-pastes containing 2% MWCNT-7 by application to a clipped area of the skin.

No clinical signs or changes in body weight gain were observed in any groups treated with MWCNT-7. No dermal responses, including erythema/eschar or edema, were found in rabbits treated with MWNT-7. The PII was calculated to be 0 in these group.

Hence based on the above findings MWCNT-7 test item was considered as non irritant to the skin of rabbits.

Reference 4

Endpoint:

skin irritation: in vivo

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:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

GLP compliance:

not specified

Remarks:

GLP compliance is not specified in this publication

Species:

rabbit

Strain:

other: Kbl:NZW

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kitayama Labes Co. Ltd. (Ina, Japan)
- Age at study initiation: 17 weeks
- Weight at study initiation: 2.7 - 3.6 kg
- Housing: individually in standard aluminum cages
- Diet: LRC4; Oriental Yeast Co. Ltd., Tokyo, Japan, ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 6 days prior to the start of the experiment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0-25.0 °C
- Humidity (%): 40-70%
- Air changes (per hr): ventilation of 15-21 air changes/h
- Photoperiod (hrs dark / hrs light): 12-h light (7:00-19:00)/dark (19:00-7:00) cycle

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

other: olive oil

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
N-MWCNT was moistened with a minimum amount of olive oil to prepare a paste for dermal application. The paste contained 1% N-MWCNTs. This amount of N-MWCNT was the maximum not leading to overlow when patched and the sample fully coated the skin. Olive oil was used to prepare the paste, because the olive oil was highly refined (neutral, denatured, and free of antioxidants) and widely used as the negative control in these expermiments.
An area of about 10 x 15 cm on the back of each rabbit was made free of fur using electric clippers and an electric shaver 24 h prior to testing. The N-MWCNT paste (0.5 g) was evenly spread on a lint cloth (2.5 x 2.5 cm), applied to the skin, and covered with a gauze patch, which was held in place with non-irritating elastic bandage (Silkytex, Alcare Co. Ltd., Tokyo Japan).

Duration of treatment / exposure:

4 hours

Observation period:

Test sites were evaluated for signs of dermal irritation approximately 60 min, 24, 48, and 72 h after test substance removal.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure:
6.25 cm2 (2.5 x 2.5 cm) clipped area of skin
- Type of wrap if used:
gauze patch which was held in place with non-irritating elastic bandage

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure:
4 h

OBSERVATION TIME POINTS

60 min, 24 h, 48 h, and 72 h

SCORING SYSTEM:
Test sites were evaluated according to OECD TG 404.

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

24/48/72 h

Score:

0.6

Reversibility:

fully reversible within: 72 h

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritant / corrosive response data:

In the group treated with N-MWCNTs, three rabbits exhibited very slight erythema, which was barely perceptible, 24 h after removal of the patches. In one rabbit, the erythema disappeared 48 h after the patch was removed. In two rabbits, the erythema disappeared 72 h after removal of the patches. The PII was calculated to be 0.6 in this group.

Other effects:

None

Conclusions:

In the group treated with N-MWCNTs, three rabbits exhibited very slight erythema, which was barely perceptible, 24 h after removal of the patches. The erythema disappeared after up to 72 h after removal of the patches. The PII was calculated to be 0.6 in this group.

Executive summary:

Acute dermal irritation was analyzed in a study performed according to method described in OECD guideline 404. New Zealand white rabbits received doses of 0.5 g of N-MWCNT-pastes containing 1% N-MWCNT by application to a clipped area of the skin. In the group treated with N-MWCNTs, three rabbits exhibited very slight erythema, which was barely perceptible, 24 h after removal of the patches. The erythema disappeared after up to 72 h after removal of the patches. The PII was calculated to be 0.6 in this group.

Hence based on the above findings N-MWCNT test item was considered as non irritant to the skin of rabbits.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

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:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

not specified

Remarks:

no information on GLP compliance available in this publication

Specific details on test material used for the study:

GO was prepared from graphite according to a modified Hummer's method, followed by probe sonication of GO dispersion for 4h (Yan et al., 2013a). AFM images showed that the size of GO nanosheets was ~120 nm, and their thickness were no more than 1.2 nm, which suggested that the prepared GO was single-layer nanosheets.
The estimated C/O ratio was ~2.4

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Third Military Medical University (30 Gaotanyan Street, Chongqing) and maintained in pathogen-free conditions
- Age at study initiation: 6 months

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

100 µL of GO (100 µg/mL)

Duration of treatment / exposure:

not specified

Observation period (in vivo):

1, 24, 48 and 72 h post GO treatment

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

3

Details on study design:

The acute eye irritation test was conducted according to OECD Guideline 405 (OECD TG 405, 2012). Three New Zealand White Rabbits (6 months of age) were checked and exhibited no abnormalities in the eyes. One drop of a topical ocular anesthetic (0.4% oxybuprocaine hydrochloride) was applied to each eye 5 min prior to GO application to minimize potential pain and distress. Subsequently, 100 µL of GO (100 pg/mL) were dripped into the conjunctival sac of the right eye after gently pulling the lower lid away from the eyeball. Both lids were then gently held together for approximately 1 s to prevent the loss of GO. The left eye remained untreated and served as the control. The responses, including corneal opacity, conjunctival redness, abnormality of the iris, and chemosis, were observed and graded according to OECD Guideline 405 (OECD TG 405, 2012) in rabbits at 1, 24, 48 and 72 h post GO treatment. The corneal epithelium was stained using fluorescein 72 h after GO treatment.
Twenty microliters and two microliters of 3% fluorescein was dripped into the conjunctival sac of the albino rabbits. The rabbits were examined using slit lamp with cobalt-blue light 2 min later, and photographs were obtained.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

no indication of irritation

Other effects:

no other effects available

Interpretation of results:

GHS criteria not met

Conclusions:

No indication of any eye irritation potential of graphene oxide in rabbits was found in a study conducted according to OECD 405.

Executive summary:

An in vivo eye irritation test was performed according to OECD Guideline 405 (OECD TG 405 2012) in New Zealand white rabbits by scoring the lesions of conjunctiva, cornea and iris at specific intervals after exposure of the ocular surface to the test item graphene oxide (GO). The eyes of the rabbits were examined at 1, 24, 48, and 72 h after dripping 100 µg/mL GO into the conjunctival sac. No rabbits exhibited corneal opacity, conjunctival redness, abnormality of the iris, or chemosis at any time point after the instillation of GO. A corneal fluorescein staining assay was used to assess GO toxicity to corneal epithelium. There was no impaired corneal epithelium 72 h post-GO treatment. This result indicated that GO did not induce any acute eye irritation or corrosion in rabbits.

Reference 2

Endpoint:

eye 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:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar 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)

GLP compliance:

not specified

Remarks:

no information on GLP compliance available in this publication

Specific details on test material used for the study:

see table 1 in "Any other information on materials and methods"

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
Justification for the Selection of the Test System
This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years.
Justification for the Selection of the Test Method
This in vitro method is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS “No Category”) without further testing within a tiered testing strategy from those requiring classification and labelling (UN GHS categories 1 and 2). Therefore, it can be used for regulatory purposes as an initial step in the bottom-up approach or as one of the last steps in a top-down approach to test eye irritation/corrosion potential. It is not intended to differentiate between UN GHS “Category 1” (serious eye damage) and UN GHS “Category 2” (eye irritation) which would require additional testing. Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the MTT dye to formazan conversion by the EpiOcular™ tissue construct after topical exposure to the test substance.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The test was carried out with the EpiOcular™ reconstructed human cornea-like epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 46 mg of MWCNT (NM-400) were applied directly atop the EpiOcular™ tissue

Duration of treatment / exposure:

90 min

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

duplicates

Details on study design:

The EpiOcular™-EIT was performed in two variants. The MWCNT (NM-400) was submitted to a protocol variant, i.e. as described by the supplier MatTek and Harbell et al. The test protocol variant differ in respect to the exposure and post-exposure immersion periods laid down for solid test materials.

In the main tests, two tissues were treated with either the test material. On the day of arrival in the laboratory, the EpiOcular™ tissues were transferred to sterile 6-well plates with 1 mL DMEM and pre-conditioned at standard culture conditions (37 °C, 5 % CO2, 90-95 % humidity) in the incubator for 16-24 h. After pre-incubation, the tissues were pre-treated with 20 µL PBS and further incubated at standard culture conditions for 30 min. Using a sharp spoon or pipette, the dry-powder or liquid test items, respectively, were applied to cover the entire tissue surface. Control tissues were concurrently exposed to 50 µL highly de-ionized water (NC) or methyl acetate (PC). After test material application, the tissues were placed into the incubator for the following exposure periods: 90 min.
To remove the test materials, the tissues were washed with sterile PBS and immediately immersed into 12-well plates, pre-filled with 5 mL pre-warmed medium per well to remove test material residuals. After 12 min each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL pre-warmed medium per well (post-exposure immersion). Subsequently, the tissues were incubated at standard culture conditions (post-exposure incubation) for 18 h. During the post-exposure immersion and incubation periods, weak cytotoxic effects might reverse, and more pronounced effects might increase.
Upon completion of the respective post-exposure periods, the assay medium was replaced by 0.3 mL MTT solution. After incubating the tissues for 3 h, the tissues were washed with PBS to terminate the MTT incubation. The produced formazan was extracted by incubating the tissues in isopropanol at room temperature overnight or on a plate shaker for at least 2 h. The optical density of the formazan extracts was determined spectrophotometrically at a wavelength of 570 nm (OD570). For each microtitre plate, blank values were established from 4 wells filled with isopropanol.

Calculation of mean relative tissue viability
Tissue OD570 values were calculated by subtracting the mean blank value of the respective microtitre plate from the measured tissue OD570 value, and mean OD570 values were calculated for the two tissues of each treatment group. The quotient of the mean OD570 values of the test material-treated tissues and those of the NC (i.e. the mean relative tissue viability) was determined to evaluate whether or not a test material is an irritant:
• Mean relative tissue viabilities <60 % indicated 'irritancy to the eye'
• Mean relative tissue viabilities >60 % indicated 'no irritancy to the eye'.

Acceptance criteria: In case one of the following acceptance criteria (AC) as described in the OECD TG 492 was not met, repetition of the EpiOcular™-EIT was considered.
• AC for the NC: The OD570 of the NC reflects the laboratory-specific tissue viability under the specific conditions of the assay. It was considered acceptable if the mean OD570 of the NC was > 0.8 and < 2.5 and the historical in-house mean at the respective time of testing was met (OD570 of NC for liquids / solids: 1.490 ± 0.106 / 1.361 ± 0.138).
• AC for the PC: In-house, the PC methyl acetate usually elicits relative tissue viabilities of approx. 25 % (historical in-house means at the time of testing in accordance wit: OD570 of PC for liquids/solids: 0.388 ± 0.098/0.318 ± 0.119). In addition to these historical means, all relative tissue viability values < 50 % were considered acceptable.
• AC for tissue variability: The relative inter-tissue variability (ITV %) between the two tissues of a treatment group was considered acceptable if it was <20 %.

Irritation parameter:

other: tissue viability [%]

Run / experiment:

mean

Value:

92

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: not stated

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: yes

Conclusions:

The test item Multi-Walled Carbon Nanotubes (NM-400) did not elicit eye irritation in the EpiOcular™-EIT assay.

Executive summary:

In the present study according to OECD TG 492 the eye irritating / damaging potential of MWCNT (NM-400) was analysed. Since irritant substances are cytotoxic to the corneal epithelium after a short time exposure the cytotoxic effects of the test item on EpiOcular™ RhCE tissue construct, a reconstituted three-dimensional human corneal epithelium model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 90 min exposure period and 18 h post-treatment period and compared to those of the concurrent negative control. The mean relative tissue viability (% negative control) was  > 60 % (92 %).

The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritating potential of the test substance to the eye in vitro.

The test item Multi-Walled Carbon Nanotubes (NM-400) does not reveale eye irritation potential in the EpiOcular™-EIT under the chosen test conditions (i.e. indicating likelihood of 'neither Category 1 nor 2'). In the same study, two further multi-walled carbon nanotube test materials (NM-401, NM-402) were studied. As can be seen in the table, these two did als not provoke a significant response in this test. In addition, this publication reports results of another in vitro eye irritation test, the Bovine Corneal Opacity and Permeability (BCOP) test according to OECD TG 437, for these three MWCNT test materials. In this test, the three MWCNT materials NM-400 (750 mg applied), NM-401 (48 mg applied), and NM-402 (750 mg applied) yielded in vitro irritation scores (IVIS) of -4.8 +/- 2.3, -5.3 +/- 0.4, and -3.9 +/- 0.7, respectively.

Taken together, these studies give no indication for an eye irritation potential of carbon-based MWCNTs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Skin irritation / corrosion:

No skin irritation potential is expected for the test item graphene oxide. In non-guideline studies on epidermal keratinocytes (Pelin et al., 2017) and in mice, dosed with nanosized graphene oxide via subcutaneous injection, very weak responses to graphene oxide were found. However, as the epidermal barrier was circumvented in this in vivo study, this exposure route is considered as worst-case scenario which clearly overestimates the bioavailability of graphene oxide in the skin, as nanomaterials usually do not penetrate the skin as recently summarized e.g. by the ECETOC Nano Task Force (Landsiedel et al., 2017). The assumption of a neglectable skin irritation potential of graphene oxide is further supported by in vivo studies on related carbon-based nanomaterials: Multiwalled carbon nanotubes (MWCNT) were found not skin irritating in rabbits (OECD TG 404, Ema et al., 2011, in line with Murthy et al., 2011)), singlewalled carbon nanotubes (SWCNT) were found not skin irritating in rabbits (OECD TG 404, Ema et al., 2011), and fullerene C60 were also not skin irritation in rabbits (OECD TG 404, Ema et al., 2013). In conclusion, classification of graphene oxide as skin irritant according to Regulation (EC) No 1272/2008 is not warranted.

 

Eye irritation:

No eye irritation potential is expected for the test item graphene oxide.