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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 July 24th to 27th, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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:
July 28th, 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Reaction mass of dodecane-1-thiol and tridodecyl trithiophosphite
IUPAC Name:
Reaction mass of dodecane-1-thiol and tridodecyl trithiophosphite
Test material form:
liquid

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
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 cm^2.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: Negative control (Sterile demineralised water) and positive control (Methyl acetate)
Amount / concentration applied:
50 µl of the liquid test item was applied to each tissue.
Duration of treatment / exposure:
28 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
Duration of post- treatment incubation (in vitro):
120 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
Number of animals or in vitro replicates:
2 replicates for test item and 2 replicates for controls applied in 1 minute intervals.
Details on study design:
GENERAL PRINCIPLE OF THE STUDY:
Eye irritant materials are identified by their ability to produce a decrease in cell viability as determined. The cell viability is measured by dehydrogenase conversion of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison of untreated negative controls is used to predict the eye irritation potential.

PRE-TESTS:
-ASSESSMENT OF DIRECT REDUCTION of MTT
The test item was tested for the ability of direct MTT reduction. To test for this ability, 50 µl of the liquid 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 reagent plus 50 µl of H2O demin. was used as negative control. The colour of the MTT turned blue/purple, the test item is presumed to have reduced the MTT. The test item has shown the potential to reduce MTT. Considering that the same control was performed the skin irritation test according to OECD TG 439 in which the same principle based on MTT reduction was applied and considering that the negative result of skin-irritation test remained unchanged after the MTT correction, no further controls were performed for eye irritation test.
- ASSESSMENT OF COLOURED OR STAINING TEST ITEM
The test item is colourless. To assess, whether the test item will become coloured after contact with water or isopropanol, 50 µl of the liquid test item were added to 1 ml of sterile H2O demin. in a 6-well plate and incubated in the dark at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour. After incubation, no colour development was visible. Therefore, the main test was performed without colourant controls.

MAIN TEST:
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 30 minutes.
After overnight incubation, the tissues were pre-wetted with 20 µl DPBS buffer and then incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 minutes. After that, 50 µl of the controls and the test item were applied in duplicate in 1 minute intervals. This was done in such a fashion that the upper surface of the tissue was covered. 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 28 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
At the end of the 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 to and immersed in 5 ml of pre-warmed assay medium in a pre-labelled 12-well plate for 12 minutes post soak at room temperature.
After that, each insert was removed from the medium, the medium was decanted off the tissue and the insert was blotted on absorbent material and transferred into the respective well of a pre-labelled 6-well plate containing 1 ml assay medium. For post-treatment incubation, the tissues were incubated for 120 minutes 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 the empty 24-well-plate. Into each well, 2 ml isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was sealed and stored in the refrigerator overnight. On the next day, the plate was shaken for 2 hours at room temperature, protected from light.

MEASURAMENT
The inserts were pierced with an injection needle, taking care that all colour is extracted. The inserts were then discarded and 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 which was read in a plate spectrophotometer at 570 nm.

EVALUATION
The values of the 96-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®).
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results.

CALCULATION
% VIABILITY= [OD corrected test item or positive control/OD corrected mean negative control]*100 %

- RhCE tissue construct used, including batch number: 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 cm^2. EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Myln-ské Nivy 73, 82105 Bratislava, Slovakia (Batch number 23797).

-Doses of test chemical used: 50 μl for each replicate.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
Exposure: 28 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity;
Post-exposure: 120 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): based on the results of preliminary tests , test item has no colourant potential but it has the potential to reduce MTT. Considering that the same control was performed the skin irritation test according to OECD TG 439 in which the same principle based on MTT reduction was applied and considering that the negative result of skin-irritation test remained unchanged after the MTT correction, no further controls were performed for eye irritation test.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): two replicates.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): quantification of MTT formazan performed at 570 nm.

- Description of the method used to quantify MTT formazan: spectrophotometer.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
% Viability > 60 % : Non eye irritant: No GHS category for eye irritation
% Viability: ≤ 60 %: Eye irritant: GHS category 1 or 2

- ACCEPTANCE CRITERIA:
OD of negative control: > 0.8 and < 2.5
Tissue viability of positive control: < 50 % (relative to negative control)
Variation within two tissue replicates of test item and controls: < 20 %

Results and discussion

In vitro

Results
Irritation parameter:
other: Viability %
Run / experiment:
Mean of 2 tissue replicates
Value:
ca. 87.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
RESULTS OF MAIN TEST:
After 28 minutes of exposure followed by product washing and a post incubation of 120 minutes a residual cell viability (mean of two replicates) of 87.5 % has been quantified for test item.

RESULTS OF PRELIMINARY TESTS:
In the preliminary test potential MTT reduction by the test item was observed. Normally, an additional test (quantitative) on MTT reduction should be performed. Considering that the same control was performed the skin irritation test according to OECD TG 439 in which the same principle based on MTT reduction was applied and considering that the negative result of skin-irritation test remained unchanged after the MTT correction, no further controls were performed for eye irritation test.
In the preliminary test to assess coloured test item, no colour development was visible; therefore, the main test was performed without colourant controls.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The mean viability for negative and positive controls was in the range of historical data.

ACCEPTANCE OF RESULTS:
OD of negative control: > 0.8 and < 2.5: fulfilled (1.5)
Tissue viability of positive control: < 50% (relative to negative control): fulfilled (34.1%)
Variation within two tissue replicates of test item and controls: < 20%: fulfilled (3.2 % for negative control; 4.4 % for positive control and 7.1% for test item).

Any other information on results incl. tables

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

Designation

Measurement

Negative Control

Positive Control

TEST ITEM

Tissue 1 

1

1.584

0.593

1.319

2

1.588

0.588

1.315

Tissue 2 

1

1.552

0.524

1.395

2

1.523

0.523

1.455

Mean Absorbance Negative Control, Positive Control and Test Item (Note Mean Blank = 0.036)

Designation

Negative Control

Positive Control

 TEST ITEM

Mean – blank

(Tissue 1)

1.550

0.555

1.281

Mean – blank

(Tissue 2)

1.502

0.488

1.389

% Viability Positive Control and Test Item

Designation

Positive Control

 TEST ITEM

% Viability (Tissue 1)

36.3 %

83.9 %

% Viability (Tissue 2)

31.9 %

91.0 %

% Viability Mean

34.1 %

87.5 %

Historical data

Parameter

Optical Density

Negative Control

Relative Viability %

Positive Control

 

Demineralised H2O

Methyl acetate

Exposition time

28 minutes

Mean

1.851

31.3 %

Standard deviation

0.283

8.2 %

Range

1.253 – 2.437

12.4 – 57.2 %

Study17030701G891

1.526

34.1%

Applicant's summary and conclusion

Interpretation of results:
other: not classified as eye irritant, according to the CLP Regulation (EC n.1272/2008).
Conclusions:
The test item is identified as not irritant to the eyes according to the CLP Regulation (EC n.1272/2008).
Executive summary:

The substance was tested for its eye irritation potential with an in vitro test according to OECD TG 492 Reconstructed human Cornea-like Epithelium (RhCE) test method.

The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 28 minutes. 50 µl of the liquid test 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. 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 1.5. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 34.1 % (< 50 %). Variation within tissue replicates was acceptable (< 20%).

In a pre-test, potential MTT reduction by the test item was observed. Normally, an additional test (quantitative) on MTT reduction should be performed. Considering that the same control was performed the skin irritation test according to OECD TG 439 in which the same principle based on MTT reduction was applied and considering that the negative result of skin-irritation test remained unchanged after the MTT correction, no further controls were performed for eye irritation test.

After treatment with the test item, the mean value of relative tissue viability was 87.5 %. This value is well above the threshold for eye irritation potential (~ 60%).

Based on the results obtained, the substance is classified as not irritant to the eyes.

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