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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 23 July 2018 to 6 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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 October 9th, 2017
Deviations:
no
Principles of method if other than guideline:
Additional information was taken from:
- MatTek Protocol: EpiOcularTM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals, for use with MatTek Corporation’s Reconstructed Human EpiOcularTM Model, 14. July 2014
- Stern M., Klausner M., Alvarado R., Renskers K., Dickens M., 1998. “Evaluation of the EpiOcular Tissue Model as an Alternative to the Draize Eye Irritation Test”. Toxicology in Vitro 12, 455-461
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of diazotised 3-amino-2-hydroxy-5-nitrobenzenesulphonic acid, coupled with 1,3-diaminobenzene and diazotised sodium 4-aminobenzenesulfonate, metallised with Basic Chromium (III) Sulphate
EC Number:
947-395-4
Molecular formula:
not applicable
IUPAC Name:
Reaction products of diazotised 3-amino-2-hydroxy-5-nitrobenzenesulphonic acid, coupled with 1,3-diaminobenzene and diazotised sodium 4-aminobenzenesulfonate, metallised with Basic Chromium (III) Sulphate
Test material form:
solid: particulate/powder

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: Main test: tissue 1 -51.2 mg (main test); tissue 2 - 50.1 mg. Additional test: tissue 1- 51.4 mg; tissue 2 - 49.5 mg

CONTROLS (POSITIVE, NEGATIVE)
- Amount(s) applied: 50 μl.
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours and 10 minutes (main test)
18 hours (additional test)
Number of animals or in vitro replicates:
two
Details on study design:
RhCE TISSUE USED
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 cul-tured 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.
- Day of delivery: 24. Jul. 2018
- Batch no.: 27060
- Additional Test on Coloured Test Items: Day of delivery:04. Sep. 2018; Batch no.:27066

CHEMICALS
- MTT solution: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (=MTT), which can be reduced to a blue formazan. A MTT stock solution of 5 mg/ml in DPBS buffer was prepared and stored in aliquots of 2 ml at – 20 ± 5 °C. 2 ml of the stock solution were thawed and diluted with 8 ml of assay medium (resulting in 1 mg/ml). This MTT-solution with the concentration of 1 mg/ml was used in the test. For the pre-test (testing the ability of direct MTT reduction), the stock solution was thawed and diluted with serum-free MEM directly before use. For the main test, the stock solution was thawed and diluted with assay medium directly before use.
- DPBS-Buffer: Dulbecco`s Phosphate Buffered Saline (DPBS) was used for the rinsing of the tissues. The buffer which was procured was used for rinsing the test item from the tissues. The buffer which was prepared was only used for preparing the MTT concentrate. Composition of the subset procured: KCl -0.2 g, KH2PO4-0.2 g, NaCl-8.0 g, Na2HPO4*7H2O-2.16 g, H2O-ad 1 litre. Composition of the subset prepared: KCl-0.2 g, KH2PO4-0.2 g, NaCl-8.0 g, Na2HPO4*2H2O-1.44 g, H2O-ad 1 litre.
- MEM with Phenol Red for Pre-Test: Serum-free MEM (Minimum Essential Medium)
- Assay Medium for Main Test: Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium)
- Isopropanol: p.A., 99.9 % used as extracting solvent for formazan.

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, 51.3 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 ≥ 95 % 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: 50.8 mg of the test item were 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 the mean OD for isopropanol, the mean OD of the test item solution was 0.12 (>0.08). 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 study, but no MTT assay was performed: instead of 300 µl MTT solution, 300 µl assay medium is 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 was ≤ 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 ≥ 95 % relative humidity for 1 hour (main test and additional test). 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 ≥ 95 % relative humidity for 16 hours (main test and additional test).
- Exposure 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 ≥ 95 % relative humidity for 29 minutes (main test and additional test). After that, 50 µl of the controls and a defined amount of the test item were applied in duplicate in one- 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 ≥ 95 % 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 (main test and additional test) 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 and 10 minutes (main test) and for 18 hours (addi-tional test) at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95 % 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 ≥ 95 % 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 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.
- Measurement: 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.

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results
Irritation parameter:
other: % Tissue viability
Value:
72.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: the validity of the EpiOcularTM test was demonstrated in a proficiency study. For this purpose 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized. Therefore, the proficiency of the EpiOcularTM test was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: OD of negative control: 1.8 (main test), 2.0 (additional test), criterion 0.8> OD< 2.5 met.
- Acceptance criteria met for positive control: % mean relative viability of positive control 44.3 (main test), criterion % mean relative viability of positive control < 50 % of negative control met.
- Variation within replicates: main test 11.5 % (negative control), 5.6 % (positive control), 15.7 % (test item); additional test 0.3 % (negative control), 0.2 % (test item). criterion variation within replicates< 20 % met.
- Range of historical values if different from the ones specified in the test guideline: values for negative control and for positive control were within the range of historical data of the test facility

Any other information on results incl. tables

MEASURED VALUES MAIN TEST

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table.

Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate 1 2 3 4 5 6 7 8 Mean
Absorbance 0.035 0.036 0.036 0.036 0.035 0.036 0.036 0.039 0.036

The absorbance values of negative control, test item and positive control are given in the following table:

Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation Measurement Negative Control Positive Control Test material
Tissue 1 1 1.930 0.866 1.410
2 1.913 0.883 1.549
Tissue 2 1 1.721 0.772 1.216
2 1.712 0.779 1.184

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol

Mean Absorbance Negative Control, Positive Control and Test Item

Designation Negative control Positive control Test material
Mean – blank (Tissue 1) 1.886 0.839 1.444
Mean – blank (Tissue 2) 1.681 0.740 1.164

COMPARISON OF TISSUE VIABILITY

For the test item and the positive control, the following percentage values of tissue viability were calculated in comparison to the negative control:

% Viability Positive Control and Test Item

Designation Positive Control Test material
% Viability (Tissue 1) 47.0% 81.0%
% Viability (Tissue 2) 41.5% 65.3%
% Viability Mean 44.3% 73.1%

MEASURED VALUES OF THE ADDITIONAL TEST FOR COLOURED TEST ITEMS

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:

Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate 1 2 3 4 5 6 7 8 Mean
Absorbance 0.037 0.037 0.036 0.037 0.036 0.037 0.037 0.038 0.037

The absorbance values of negative control and test item are given in the following table:

Absorbance Values Negative Control and Test Item (OD at 570 nm)

Designation Measurement Negative Control Test material
Tissue 1 1 1.989 0.040
2 2.044 0.039
Tissue 2 1 2.025 0.043
2 1.997 0.044

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol

Mean Absorbance Negative Control and Test Item

Designation Negative control Test material
Mean – blank (Tissue 1) 1.980 0.003
Mean – blank (Tissue 2) 1.974 0.007

COMPARISON OF TISSUE VIABILITY (ADDITIONAL TEST FOR COLOURED TEST ITEMS)

For the test item, the following percentage values of mean tissue viability were calculated in comparison to the mean of the negative control:

% Tissue Viability

Designation Test material
% Viability (Tissue 1) 0.1 %
% Viability (Tissue 2) 0.3 %
% Viability Mean 0.2 %

COMPARISON OF TISSUE VIABILITY (CORRECTED)

The value of “% Viability (colourant control)” of the additional test was subtracted from “% Viability” of the main test.

% Tissue Viability

Designation Test material
% Viability mean main test 73.1 %
% Viability mean colourant control of additional test 0.2 %
% Viability corrected 72.9 %

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to the CLP Regulation (EC) no. 1272/2008
Conclusions:
After treatment with the test item, the mean value of relative tissue viability was 72.9 %.
Executive summary:

The eye hazard potential of the substance is evaluated in vitro in a Reconstructed human Cornea-like Epithelium (RhCE) model according to the OECD 492 Guideline. Before the main test the test item was assessed for its ability to directly reduce the vital dye MTT and also whether it has colour interference. As the test item showed intense coloring in the pre-test, there was the risk to influence the photometric measurement. Therefore, an additional test for intensely coloured test items was performed.

In the main test, the test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. 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 positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 44.3 % (< 50%). Validity criteria were met and therefore the test is considered as valid.

After treatment with the test item, the mean value of relative tissue viability was reduced to 72.9 %. This value is above the threshold for eye irritation potential (≤ 60 %).

Under the conditions of the test, the test material is considered non-eye irritant.