Graphene

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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)

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

Test animals / tissue source

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

Test system

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%

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Applicant's summary and conclusion

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.