Reaction products of diazotized Dodecyl Aniline coupled with 8-acetylamine-1-hydroxynaphthalen-3,6-disulphonic Acid, sodium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 11. Sep. 2017 to 19. Oct. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

disodium 3-[(1E)-2-(4-dodecylphenyl)diazen-1-yl]-5-acetamido-4-hydroxynaphthalene-2,7-disulfonate

EC Number:

946-819-5

Molecular formula:

Not applicable for UVCB substance

IUPAC Name:

disodium 3-[(1E)-2-(4-dodecylphenyl)diazen-1-yl]-5-acetamido-4-hydroxynaphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

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.

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Main Test: 52.4 mg (Tissue 1) and 52.4 mg (Tissue 2)
- Additional Test: 51.4 mg (Tissue 1) and 50.9 mg (Tissue 2)

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:

RhCE TISSUE CONSTRUCT USED, INCLUDING BATCH NUMBER
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Main Test:
Designation of the kit: OCL-200-EIT
Batch no.: 27006
Additional Test:
Designation of the kit: OCL-200-EIT
Batch no.: 27009
- Details of the test procedure used

PRE-TESTS
- Assessment of Direct Reduction of MTT by the Test Item
The test item was tested for the ability of direct MTT reduction. To test for this ability, 49.0 mg of the solid test item were added to 1 mL of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 and 80 – 100 % relative humidity for 3 hours. 1 mL of MTT solution plus 50 µL of H2O demin. was used as negative control. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.
- Assessment of Coloured or Staining Test Items
50.2 mg of the test item was added to 2 mL isopropanol, incubated in 6-well plates on an orbital shaker for 3 hours at room temperature. 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 OD for isopropanol, the OD of the test item solution was >0.08 Therefore the test item was possibly interacting with the photometrical measurement and an additional test on colourant controls was performed. 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 solution, 300 µL assay medium was used. The bound colour was 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 was performed.

MAIN TEST
- Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use. The assay medium was warmed in the water bath to 37 ± 1°C. 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-min- 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 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 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 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 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.

DESCRIPTION OF EVALUATION CRITERIA
Eye irritation was assessed using the following criteria (source: MatTek Corporation):
> 60 % Viability, Non eye irritant, No GHS category
≤ 60 % Viability, Serious eye damage/for eye irritant, GHS category 1 or 2

REFERENCE TO HISTORICAL POSITIVE AND NEGATIVE CONTROL RESULTS
Parameter Optical Density Negative Control (Demineralised H2O) Relative Tissue Viability Positive Control (Methyl acetate)
Exposition time 6 hours 6 hours
Mean 1.555 33.4 %
Standard deviation 0.266 8.0 %
Range 1.047 – 2.340 21.1 – 53.9 %

-Acceptance Criteria
1. Mean OD of the tissue replicates treated with the negative control should be > 0.8 and < 2.5
2. Mean viability of the tissue replicates exposed for 6 hours with the positive control, expressed as % of the negative control, should be < 50%
3. The difference of viability between two tissue replicates should be less than 20%.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

% viability

Run / experiment:

corrected mean

Value:

36.6

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Remarks:

% viability

Run / experiment:

mean (main test)

Value:

39

Results 3

Irritation parameter:

in vitro irritation score

Remarks:

% viability

Run / experiment:

mean (colourant control)

Value:

2.4

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
-OD of negative control: 1.6
-% mean relative viability of positive control: 42.2 %
-Variation within replicate: 0.2 % (negative control), 3.5 % (positive control), 4.0 % (test item).
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: classified either Category 1 or Category 2 according to the CLP Regulation (EC 1272/2008)

Conclusions:

Under the conditions of the test system, the test item is considered 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, an in vitro study 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. As the test item is intensely coloured, an additional test for colour interference was performed to exclude wrong photometrical measurement values due to intense colour of the test item. 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. 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 < 2.5, OD was 1.6. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 42.2 % (< 50 %). Variation within tissue replicates was acceptable (< 20 %). After treatment with the test item, the mean value of relative tissue viability was 36.6 %. This value is below the threshold for eye irritation potential (≤ 60 %). The EpiOcularTM Eye Irritation Test 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). Thus, the test item is considered either eye irritant or inducing serious eye damage in the EpiOcularTM Eye Irritation Test, under the test conditions.