6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

Adopted 18 June 2019

Deviations:

no

Principles of method if other than guideline:

This in vitro study was performed to assess the eye irritation potential of Macrolex Fluoreszenzrot 4B by means of the Human Cornea Model Test following.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione

EC Number:

244-320-0

EC Name:

6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione

Cas Number:

21295-57-8

Molecular formula:

C23H22N2O2

IUPAC Name:

10-(cyclohexylamino)-14-methyl-14-azatetracyclo[7.7.1.0²,⁷.0¹³,¹⁷]heptadeca-1(16),2,4,6,9,11,13(17)-heptaene-8,15-dione

Test material form:

solid

Details on test material:

Purity: 94.2%

Test animals / tissue source

Species:

other: in vitro test with human cornea cells

Strain:

other: three-dimensional human cornea model tissue model

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability:
In principle the EpiOcular™ eye irritation test (EIT) measures the ocular irritation potential of a test item by determination of cytotoxic effects on a reconstructed human cornea epithelium (RhCE) tissue model to discriminate chemicals not requiring classification for eye irritancy (UN GHS No Category) from those requiring classification. The EpiOcular™ EIT is not intended to differentiate between UN GHS Category 1 (serious eye damage) and UN GHS Category 2 (eye irritation). This differentiation would need to be addressed by another tier of a test strategy. As a BCOP has already been conducted and Category 1 can be excluded based on the result, this test can be used to determine, if the substance needs to be classified (Category 2) or not.
The EpiOcular tissue construct is a non-keratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air (surface 0.6 cm²). This allows the test item to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The experiment was carried out on the EpiOcular™ RhCE tissue construct (MatTek Corporation, Bratislava, Slovakia), Lot-No 30636.
The quality certificate of the test kits demonstrating its robustness is annexed to the report.
MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; MK-24-007-0055 (29 June 2015).

Test system

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): 50 mg (corresponds to according to Guideline a minimum of 83.3 mg/cm²)
- positive control: 50 µL
- negative control: 50 µL

Duration of treatment / exposure:

6 hours at standard culture conditions (5% CO2, 37°C, 95% humidity) followed by a post-soak immersion period of about 25 min in fresh medium

Duration of post- treatment incubation (in vitro):

18 hours (37°C, 5% CO2, 95% humidity)

Number of animals or in vitro replicates:

each 2 tissues for the test item, positive and negative controls

Details on study design:

- RhCE tissue construct used, including batch number: The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier.
RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.

- Doses of test chemical and control substances used: 50 mg of the solid test item, 50 µL negative control (deionized water) and positive control (neat methyl acetate), respectively, were applied to the EpiOcular™ tissue surface in duplicate.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods for solid test items: 37 ± 2°C; 6 hours exposure, 25 min. post-soak immersion, 18 hours post-treatment incubation

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): The optical pre-experiment (color interference pre-experiment) to investigate the test item’s color change potential in water or isopropanol led to a change in color. Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed purple color. Therefore, an additional test with freeze-killed tissues was necessary.
Since the OD of the test item in deionised water or isopropanol at 570 nm after blank correction was > 0.08 and the test item interfered with MTT, Non-Specific Killed Controls (NSKC) were necessary in the main experiment additionally.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): each 2 tissues for the test item, positive and negative controls

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: For interpretation of cell viability results the cut-off value distinguishing classified (irritant) from non-classified substances as given in OECD TG 492 was used:
- The test chemical is identified as not irritant and not requiring classification according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.
- The test chemical is identified as irritant and potentially requiring classification according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%.

- Positive and negative control means and acceptance ranges based on historical data: The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.8
- mean relative viability of the positive control (PC) is < 50 % (relative to negative control)
- the difference of viability between the two replicates is < 20 %.

Results and discussion

In vitro

Results

Irritation parameter:

other: mean percent tissue viability

Run / experiment:

mean of two tissue cultures

Value:

106.91

Vehicle controls validity:

not examined

Remarks:

no vehicle used

Negative controls validity:

valid

Remarks:

100 % final cell viability

Positive controls validity:

valid

Remarks:

33.63 % final cell viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

According to the conducted pre-check the test items were demonstrated to exert optical interferences directly affecting the test results that are not related to cytotoxic effects on tissue cells. Therefore killed controls and colour controls had to be conducted.

Colour of test item	MTT Reduction	Colour reaction in isopropanol	Colour reaction in water	Killed control test to be conducted	Colour control test to be conducted	Non specific Killed control test to be conducted
red	+	+	+	yes	yes	yes

+  positive reaction, -  negative reaction

As the final test item-treated tissue viability was > 60% relative to negative control, the test item was characterized as NOT having eye irritating properties (UN GHS No Category):

Compound	Final cell viability [%]	Category
Macrolex Fluoreszenzrot 4B	106.91	No category
Positive control	33.63	Cat. 1 / 2
Negative control	100.00	No category

All assay acceptance criteria were met.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

not irritant

Executive summary:

This in vitro study was performed to assess the eye irritation potential of Macrolex Fluoreszenzrot 4B by means of the Human Cornea Model Test following OECD TG 492. The test item proved to be an MTT reducer and to dye water and isopropanol in the color interference pre-experiment. The OD of the test item in isopropanol at 570 nm after blank correction was > 0.08. Therefore, additional tests with freeze-killed tissues, viable tissues and Non-specific Killed Control (NSKC) tissues (without MTT addition) had to be performed.

Each 50 mg of the solid test item or 50 µL of the negative control (deionised water) and of the positive control (methyl acetate), were applied to each duplicate tissue for 6 hours. Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

After treatment with the test item Macrolex Fluoreszenzrot 4B the mean relative viability value was 106.91% compared to the mean value of the negative control. This value is above the threshold for irritancy of ≤ 60%. Therefore, the test item does not need to be classified according UN GHS.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Macrolex Fluoreszenzrot 4B does not need to be classified for eye irritation according UN GHS.