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Diss Factsheets

Toxicological information

Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
24 April, 2017
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
study conducted on the analogue substance; the read across justification is detailed in section 13. The Reliability of the Source Study is 1.
Justification for type of information:
The complete read-across justification is detailed in section 13.

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:
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Acid Brown 107 - Similar Substance 01
Acid Brown 107 - Similar Substance 01
Test material form:
solid: particulate/powder

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
Commercially available EpiOcularTM kit: the 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 cm^2.

EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
I) Main Test: OCL-200-EIT (Designation of the kit), batch no.: 23777
II) Additional Test MTT-Reduction: OCL-200-EIT (Designation of the kit), batch no.: 23719
The tissues were stored in the freezer until use.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
-Tissue 1: 54.3 mg (Main test), 51.4 mg (Additional Test MTT reduction), 50.1 mg (Additional Test Coloured test item)
-Tissue 2: 49.4 mg (Main test), 49.8 mg(Additional Test MTT reduction), 53.3 mg (Additional Test Coloured test item)
Duration of treatment / exposure:
6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 –100 % relative humidity
Duration of post- treatment incubation (in vitro):
18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity
Number of animals or in vitro replicates:
2 tissue replicates
Details on study design:
-Assessment of Direct Reduction of MTT by the Test Item
As the test item is dark brown, it was directly tested on freeze killed controls without any further test, because it is obvious that the test item’s colour can interfere with the measurement of the MTT product.
Freeze killed tissues were prepared by placing untreated tissues in freezer (–20 ± 5 °C) overnight. Once killed, the tissue may be stored indefinitely in the freezer.
In addition to the main test described, this additional test employed two freeze-killed tis-sues treated with the test item and one killed tissue treated with negative control (demin. water), to show the small amount of MTT reduction due to residual NADH and associated enzymes within the killed tissue. The test procedure was identical with the main test, but killed tissues were used additionally.
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.
-Assessment of Coloured or Staining Test Items
The test item was directly tested on colourant controls without any further pre-tests, be-cause it was obvious that the test item can interfere with the MTT product.
The additional test was performed in order to evaluate the amount of colour bound to the tissues. The test item was applied to two additional tissues (= colourant controls) and the test was performed in the same way as described for the main test, but no MTT assay was performed: instead of 300 µL MTT reagent, 300 µL medium was used. The bound colour is extracted and the absorbance of the isopropanol extract was measured in the same fashion as in the MTT assay for coloured test items (without piercing the tissues).
As the colourant control result is ≤ 50 % of the viable negative control, a data correction procedure will be performed.

On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the MTT solvent directly before use. The assay medium was warmed in the water bath to 37 ± 1°C.
The 6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. 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 80 – 100 % 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 at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours.
-Exposition and Post-Treatment
After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 minutes.
After that, 50 µL of the controls and a defined amount of the test item were applied in duplicate in 1- minute- intervals. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts were removed from the plates in 1-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 80 – 100 % relative humidity. After the post-treatment incubation, the MTT Assay was performed.
-MTT Assay and Extraction
A 24-well-plate was prepared with 300 µL freshly prepared MTT-reagent 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 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is 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. 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. Eight wells with 200 µL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.

The values of the 96-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®) (all calculations were performed with unrounded values. Therefore, re-calculation with the rounded values given in the tables may lead to slightly different results).
I) Calculation
-Calculation of mean OD of the blank isopropanol (ODBlk)
-Subtraction of mean ODBlk of each value of the same experiment (corrected values)
-Calculation of mean OD of the two replicates for each tissue
-Calculation of mean OD of the two relating tissues for controls and test item
(Note: Corrected OD value of negative control corresponds to 100 % viability)
To calculate the relative absorbance, the following equation was used:
% Viability = [OD corrected test item/OD corrected mean negative control]x100 %
II) Data Correction with Additional Tests
-Test with Freeze-killed Tissues
% Viability (freeze-killed) = [OD corrected test item (freeze killed)/OD corrected mean negative control]x100 %
-Test of Coloured or Staining Test Items
% Viability (colourant-control): [OD corrected test item (colourant control)/OD corrected mean negative control]x100 %

The experiment is considered as valid if:
-optical density OD of the negative control is > 0.8 and < 2.5
-mean relative viability of positive control is < 50 % of negative control viability
-the difference in % viability (compared with the negative control) of any two replicates is < 20 %

The means of negative control and positive control of all performed experiments were stated and compared with the values which were found in this study.

Results and discussion

In vitro

Irritation parameter:
other: % viability mean (corrected)
Negative controls validity:
Positive controls validity:
Remarks on result:
other: The correction values of both additional tests of “%Viability Mean” (MTT-Reduction) and “%Viability Mean” (Coloured Test Item) subtracted from the “%Viability Mean” of the main experiment
Other effects / acceptance of results:
As the “% Viability (freeze-killed)” result is ≤ 50 % of the viable negative control in the main test, the data correction procedure was performed. The value of“% Viability (freeze-killed)” was subtracted from “% Viability” of the main test.
As the colourant control result is ≤ 50 % of the viable negative control, the data correction procedure was performed. The value of “% Viability (colourant control)” was subtracted from “% Viability” of the main test.

All validity criteria were met. The criterion for optical density of the negative control was fulfilled: The OD value was 1.5 (> 0.8 and < 2.5). The positive control induced a decrease in the relative absorbance as compared to the negative control to 42.0%. Variation within the replicates was acceptable (< 20%).
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.

Applicant's summary and conclusion

Interpretation of results:
other: either eye irritant or inducing serious eye damage
The test item is considered as either eye irritant or inducing serious eye damage in the EpiOcularTM Eye Irritation Test.
Executive summary:

In order to evaluate the potential of the test item to evoke eye irritation in a Reconstructed human Cornea-like Epithelium (RhCE) model in an in vitro study, the EpiOcularTM Eye Irritation Test was performed, according to the OECD Guideline 492 (2015). The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. The solid test item was applied to each tissue. 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. As the test item colour is very dark, additional tests for data correction were performed to exclude colour interference during the photometric measurement.

After treatment with the test item, the mean value of tissue viability was 3.0 %. All the validity criteria were met. This value is below the threshold for eye irritation potential (≤ 60 %). Under the conditions of the test, the test item is considered either an eye irritant or inducing serious eye damage in the EpiOcularTM Eye Irritation Test.