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Diss Factsheets

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to 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
GLP compliance:
yes (incl. QA statement)

Test material

impurity 1
Reference substance name:
Unknown impurities
Unknown impurities
impurity 2
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Dihydrogen oxide
Constituent 1
Chemical structure
Reference substance name:
pentasodium 4-amino-3-(2-{4-[2-(2,4-disulfonatophenyl)diazen-1-yl]-3-methylphenyl}diazen-1-yl)-5-hydroxy-6-[2-(4-nitro-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate
Cas Number:
Molecular formula:
pentasodium 4-amino-3-(2-{4-[2-(2,4-disulfonatophenyl)diazen-1-yl]-3-methylphenyl}diazen-1-yl)-5-hydroxy-6-[2-(4-nitro-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate
Test material form:
solid: particulate/powder
Details on test material:
Batch XMEF 1476

Test animals / tissue source

other: in vitro test with human cornea cells
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:
Eye irritation is generally defined as "the production of reversible changes in the eye". The potential for chemical induced eye irritation is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. It was usually determined in vivo in the Draize rabbit eye irritation test as described in OECD guideline 405. In a pre-validation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential.
A limitation of the Test Guideline OECD 492 is that it does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B), as defined by UN GHS. For these purposes further testing with other suitable test methods is required.
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.
The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazol 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.
The technical proficiency of the test system according to OECD Guideline 492 guideline recommended proficiency substances was demonstrated.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- 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

Irritation parameter:
other: mean percent tissue viability
Run / experiment:
mean of two tissue cultures
Vehicle controls validity:
not examined
no vehicle used
Negative controls validity:
100 % final cell viability
Positive controls validity:
5.30 % final cell viability
Remarks on result:
no indication of irritation
Other effects / 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
 black + + +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):


 Final cell viability


 Test item 88.77 No category
 Positive control 5.30 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
not irritant
Executive summary:

This in vitro study was performed to assess the eye irritation potential of the registered substance 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 the mean relative viability value was 88.77% 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, the registered substance does not need to be classified for eye irritation according UN GHS.