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Diss Factsheets

Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11. Sep. 2017 - 28.Sep. 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:
OECD Guideline for the Testing of Chemicals, Part 492, adopted 28. Jul. 2015, “Reconstructed
Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage”/ Protocol provided by MatTek Corporation
Deviations:
no
Principles of method if other than guideline:
Additional information was taken from:
- EpiOcular TM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals” by MatTek Corporation, document no. MK-24-007- 0055, dated 14. Jul. 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
Reference substance name:
Myo-Inositol, hexakis(dihydrogen phosphate), sodium salt
EC Number:
238-242-6
EC Name:
Myo-Inositol, hexakis(dihydrogen phosphate), sodium salt
Cas Number:
14306-25-3
Molecular formula:
C6H18O24P6.xNa
IUPAC Name:
Esters of sodium hydrogen phosphate with myo-Inositol, plant-derived (old name: myo-Inositol, hexakis(dihydrogen phosphate), sodium salt)
Test material form:
solid: bulk
Details on test material:
- Appearance: white solid powder
- Homogeneity: homogenous
- Density: 2.0401 ± 0.0009 g/cm3 at 20 ± 0.2 °C
- Moisture content: 0.1 - 10% (water is an inherent constituent of the UVCB substance)

Test animals / tissue source

Species:
human
Strain:
other: Keratinocyte strain 4F1188
Details on test animals or tissues and environmental conditions:
- Commercial Name: EpiOcular™ kit.
- Supplier: MatTek in Vitro Life Science Laboratories, Slovakia
- Designation of the kit: OCL-200-EIT
- Batch: 27006

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
The following amounts were applied to the tissues:
Tissue 1: 50.1 mg test item
Tissue 2: 52.3 mg test item
Controls: 50µl each
Duration of treatment / exposure:
Incubation for 6 hours at 37 ± 1 °C, 5 ± 1.0 % CO2 and 80-100% rel. humidity.
Duration of post- treatment incubation (in vitro):
For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
Number of animals or in vitro replicates:
The solid test item was applied to two tissue replicates.
Details on study design:
Pre - Tests
1. 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,
47.7 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.
2. 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 minutes. For solid test items, moreover, 49.3 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.
MAIN TEST
1. 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.
2. 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.
3. MTT Assay and exrtaction
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 3 hours 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.
4 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-wellplate.
Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a plate
spectrophotometer at 570 nm.
DECISION CRITERIA
Viability > 60 % Non eye irritant - No GHS category for eye irritation
Viability ≤ 60 % Eye damage / Eye irritant -GHS category 1 or 2

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: Viability
Run / experiment:
Tissue 1
Value:
67.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Viability
Run / experiment:
Tissue 2
Value:
66.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Validity criteria and results are stated below:
Validity
Criterion Demanded Found
OD of negative control > 0.8 and < 2.5 1.6
% mean relative viability < 50% of negative control 42.2%
of positive control
Variation within replicates < 20% 0.2% (negative control)
3.5% (positive control)
1.0% (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:
GHS criteria not met
Conclusions:
After treatment with the test item, the mean value of relative tissue viability was 66.9 %. This value is above the threshold for eye irritation potential (≤ 60%).
Under the conditions of the test the substance is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.
Executive summary:

The test item 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 66.9 %. This value is above the threshold for eye irritation potential (≤ 60%). Under the conditions of the test, the test item is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.

- Justification of the test method and considerations regarding applicability

This in vitro study will be performed in order to evaluate the eye hazard potential of the substance.

The EpiOcular™ 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 reconstructed human cornea-like tissue model. Within a testing strategy, the EpiOcular™ EIT is used as a replacement of the in vivo Draize Eye Irritation Test. It is utilized for the classification and labelling of chemicals concerning their eye irritation potential. The EpiOcular™ EIT can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. A limitation of this guideline is that it neither allows discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B). For these purposes, further testing with other suitable test methods is required