Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 June - 26 July 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:
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 09 Oct 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals / tissue source

Species:
human
Strain:
other: EpiOcular™
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo. The cells are not transformed or transfected with genes to induce an extended life span. The EpiOcular™ 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. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years and for a great variety of chemicals.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

The test was performed on a total of 2 tissues per dose group.
Duration of treatment / exposure:
6 h (37 ± 1 °C, 5 ± 1% CO2, 80 – 100 % relative humidity)
Duration of post- treatment incubation (in vitro):
post-exposure immersion: 25 min (room temperature)
post-treatment incubation: 18 h (37 ± 1 °C, 5 ± 1% CO2, 80 – 100 % relative humidity)
Number of animals or in vitro replicates:
duplicate tissues were investigated
Details on study design:
- RhCE tissue construct used, including batch number: EpiOcular™ tissues (OCL-200-EIT; MatTek, Lot No.: 27046)

MTT-REDUCING PRE-EXPERIMENT
To test for direct MTT reduction of the test item, 50.9 mg of the 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, 80 – 100 % relative humidity for 3 h. 1 mL of MTT solution plus 50 µL of demineralised water 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.

COLOURING POTENTIAL PRE-EXPERIMENT
49.6 mg of the test item were added to 2 mL isopropanol and incubated in 6-well plates on an orbital shaker for 2 h at room temperature. The test item was not completely soluble in isopropanol. Therefore, the test item solution was centrifuged for 30 s at 16,000g. Then, two 200 µL aliquots of the supernatant of the resulting solution and two 200 µL aliquots of neat isopropanol were transferred into a 96-well plate and the optical density (OD) was 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 0.16 (>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 test, but no MTT assay was performed. Instead of 300 µL MTT solution, 300 µL assay medium were used. The bound colour was extracted and the absorbance of the isopropanol extract was measured in the same way as in the MTT assay for coloured test items. As the colourant control result was ≤ 50% compared to the negative control, a data correction procedure could be performed.

EXPERIMENTAL PROCEDURE
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, 80 – 100% relative humidity for 1 h. 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, 80 – 100 % relative humidity for 16 h.
After overnight incubation, the tissues were pre-wetted with 20 µL Dulbecco`s Phosphate Buffered Saline (DPBS) buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1% CO2, 80 – 100 % relative humidity for 29 min. After that, 50 µL of the controls and 50 mg of the test item were applied in duplicate in one minute intervals. After dosing the last tissue, all plates were transferred into the incubator for 6 h at 37 ± 1 °C, 5 ± 1% CO2, 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 plates for 25 min 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 h at 37 ± 1 °C, 5 ± 1% CO2, 80 – 100 % relative humidity.
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 min at 37 ± 1 °C, 5 ± 1% CO2, 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 was flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 h 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.

PREDICTION MODEL
The ocular irritation potential of the test item was predicted from the relative mean tissue viabilities obtained after treatment compared to the negative control tissues concurrently treated with demineralised water. The test item is considered to be irritant to the eye if the relative tissue viability is less or equal to 60%. The test item is considered to be non-irritant if relative tissue viability is higher than 60%.

ACCEPTANCE CRITERIA
The test meets acceptance criteria if:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%.

CALCULATIONS
Note: The corrected optical density (OD) value of the negative control corresponds to 100% viability.

% viability = [ ODcorrected test item/positive control / ODcorrected negative control ] x 100

% viability (colourant control) = [ ODcorrected test item at colourant control / ODcorrected negative control ] x 100
NB: "% viability (colourant control)" refers to the negative control in the same experiment. THe value for "% viability (colourant control)" was subtracted from "% viability" of the main test.

Results and discussion

In vitro

Results
Irritation parameter:
other: mean relative viability (%)
Run / experiment:
6 h
Value:
103.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (main test: 2.0; additional test: 1.8) was in the range of > 0.8 and < 2.5.
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (main test: 38.2%).
- The difference of rel. absorbance between the two relating tissues of a single item is < 20% (main test: 1.6% for negative control, 5.0% for positive control, 9.7% for test item; additional test: 11.5% for negative control, 0% for test item).

Any other information on results incl. tables

Measured optical densities and calculated values are summarised in the a separate pdf document (OECD 492 results.pdf)

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
Conclusions:
After treatment with the test item, the mean value of relative tissue viability was 103.7 %. This value is well above the threshold for eye irritation potential (≤ 60%). The test item is, therefore, considered not to be irritating to eyes in the in vitro EpiOcular Eye Irritation Test (EIT).