Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-09-12 - 2017-12-22 (experimentla phase: 2017-09-25 - 2017-09-28)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP

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:
OECD Guideline for the Testing of Chemicals, Part 492, adopted 28. Jul. 2015, “Re-constructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage”
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany

Test material

Constituent 1
Reference substance name:
Yucca schidigera, ext.
EC Number:
607-033-5
Cas Number:
223749-05-1
IUPAC Name:
Yucca schidigera, ext.
Constituent 2
Chemical structure
Reference substance name:
Maltodextrin
EC Number:
232-940-4
EC Name:
Maltodextrin
Cas Number:
9050-36-6
IUPAC Name:
D-glucose
Test material form:
solid: particulate/powder
Remarks:
light tan powder
Details on test material:
Storage: Room Temperature: (20 ± 5°C)
Specific details on test material used for the study:
The test item was stored in the test facility in a closed vessel at room temperature (20±5°C).

Test animals / tissue source

Species:
human
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The EpiOcularTM Eye Irritation Test (EIT) predicts the acute ocular irritation potential of chemicals by measurement of its irreversible tissue damage caused by cytotoxic effects in the human cornea model. Within a testing strategy, the EpiOcularTM EIT is used as a re-placement of the Draize Eye Irritation Test.
It is utilized for the classification and labelling of chemicals concerning their eye irritation potential. The EpiOcularTM EIT is intended to differentiate substances that are “not eye irritant” from those that require labelling as either GHS category 1 or 2 for serious eye damage resp. eye irritation potential.
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 per-centage reduction of cell viability in comparison of untreated negative controls is used to predict the eye irritation potential.
The test was performed according to the protocol provided from MatTek Corporation.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
Specification
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.
Origin
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Day of delivery: 26. Sep. 2017
Batch no.: 27006

Test system

Vehicle:
unchanged (no vehicle)
Remarks:
tissues were pre-wetted with 20 µL DPBS buffer
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50.6 mg or 50.1 mg
Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
25 minutes post soak + 18 hours post-treatment incubation
Number of animals or in vitro replicates:
2 repilcates measured 2 times
Details on study design:
- Details of the test procedure used
The EpiOcularTM Eye Irritation Test (EIT) predicts the acute ocular irritation potential of chemicals by measurement of its irreversible tissue damage caused by cytotoxic effects in the human cornea model. Within a testing strategy, the EpiOcularTM EIT is used as a replacement of the Draize Eye Irritation Test.
It is utilized for the classification and labelling of chemicals concerning their eye irritation potential. The EpiOcularTM EIT is intended to differentiate substances that are “not eye irritant” from those that require labelling as either GHS category 1 or 2 for serious eye damage resp. eye irritation potential.
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.
The test was performed according to the protocol provided from MatTek Corporation.

- RhCE tissue construct used, including batch number
Specification: 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.
Origin: EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Day of delivery: 26. Sep. 2017
Batch no.: 27006

- Doses of test chemical and control substances used
Negative Control: Sterile demineralised water, prepared by the laboratory using an ion exchanger and membrane filtration through sterile filters, batch no.: 20170815.
Positive Control: Methyl acetate (C3H6O2, CAS No. 79-20-9), procured from MatTek, batch no.: 032817ISA.
50 µL of the controls and a defined amount of the test item were applied:
Tissue 1: 50.6 mg
Tissue 2: 50.1 mg

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
All 6-well-plates were pre-incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours.
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.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
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, 50.0 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 colour of the MTT turned blue/purple, the test item is presumed to have reduced the MTT.
The test item has shown its ability to reduce MTT and was classified as irritant in the main test. Therefore, it was not necessary to perform a functional test with freeze killed tissues.
Assessment of Coloured or Staining Test Items
The test item is colourless. To assess, whether the test item will become coloured after contact with water or isopropanol, 47.8 mg of the solid 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. For solid test items, moreover, 49.9 mg of test item were added to 2 mL isopropanol and incubated in a 6-well plate for 3 hours at room temperature.
After incubation, no colour development was visible. Therefore, the main test was performed without colourant controls.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
Each 2 tissues.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
The plate was read in a plate spectrophotometer at 570 nm.

- Description of the method used to quantify MTT formazan
MTT Reagent: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (=MTT), which can be reduced to a blue formazan, prepared by the laboratory.
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.
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.
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.
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.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Eye irritation is assessed using the criteria given in the following table (source: MatTek Corporation):
% Viability Assessment GHS classification
> 60 % Non eye irritant No GHS category for eye irritation
≤ 60 % Serious eye damage/eye irritant GHS category 1 or 2

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
Values for negative control and for positive control were within the range of historical data of the test facility.

- Positive and negative control means and acceptance ranges based on historical data
Values for negative control and for positive control were within the range of historical data of the test facility

- Acceptable variability between tissue replicates for positive and negative controls / Acceptable variability between tissue replicates for the test chemical
Criterion Demanded Found
OD of negative control > 0.8 and < 2.5 1.6
% mean relative viability of positive control < 50% of negative control 42.2%
Variation within replicates < 20% 0.2% (negative control)
3.5% (positive control)
0.9% (test item)

Results and discussion

In vitro

Results
Irritation parameter:
other: % viability
Run / experiment:
mean
Value:
4.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
either eye irritant or inducing serious eye damage
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not stated

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: no

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
either eye irritant or inducing serious eye damage
Conclusions:
The study was conducted under GLP according to OECD guideline 492 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritating potential of the test substance to the eye in vitro.
After treatment with the test item, the mean value of relative tissue viability was reduced to 4.6 %. This value is below the threshold for eye irritation potential (≤ 60%).
Under the conditions of the test system, Yucca extract is considered either eye irritant or inducing serious eye damage in the EpiOcularTM Eye Irritation Test, but no distinct classification according to EU GHS can be made. 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). For exact classification, further testing with other suitable test methods would need to be performed. Taking into account that the neat substance was not corrosive to skin, a classification as Eye Irr. Cat. 2 is considered possible.
Executive summary:

One valid experiment was performed according to OECD TG 492 under GLP.

The test item Yucca extract 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 two tissue replicates.

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 4.6 %.

This value is below the threshold for eye irritation potential (≤ 60%).

According to the OECD Guideline 492, 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). For these purposes, further testing with other suitable test methods is required.

Under the conditions of the test, Yucca extract is considered either eye irritant or inducing serious eye damage in the EpiOcularTM Eye Irritation Test.