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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:
23 to 25 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 492 and in compliance with GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: 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) (adopted 28 July 2015)
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek in Vitro Life Science Laboratories, Mlynské Nivy 73, Bratislava, Slovakia, EpiOcular™ Eye Irritation Test (OCL-200-EIT), Protocol, 14 July 2014
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected from 2015-07-13 to 2015-07-16 / signed on 2015-09-14

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: clear colourless liquid
- Storage condition of test material: room temperature

Test animals / tissue source

Species:
other: human reconstructed cornea model (EpiOcular™)
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to A
nimal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. This test guideline is applicable to solids, liquids, semi-solids and waxes, so is considered to be applicable to the test item.

- Description of the cell system used:
CELL CULTURE:
- Source: EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (Ashland, MA01721, USA).
- Lot No.: 21572
- The EpiOcular™ tissue consists of normal, human-derived epidermal 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 which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ∅).
- Transport: EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate.
- Storage: The day after receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. An appropriate volume of EpiOcular™ Assay Medium was warmed to approximately 37 °C. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (18 hours).

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: Negative control: deionised water. Positive control: methyl acetate
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
Duration of treatment / exposure:
30 minutes
Observation period (in vivo):
NA
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
duplicate tissues
Details on study design:
- Details of the test procedure used: procedure for liquids
- RhCE tissue construct used, including batch number: EpiOcular™ kits (Lot No.: 21572)
- Doses of test chemical and control substances used: 50 μL
- Duration and temperature of:
exposure: 30 minutes / 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
post-exposure immersion: 11-13 min / room temperature
post-exposure incubation period: 120 minutes / 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
- Description of any modifications to the test procedure: none
- Test for direct MTT reduction: 50 μL of the test item are added to 1 mL of a 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 h. Untreated MTT solution was used as a control. Then the colour of the obtained solutions was evaluated.
- Assessment of Color Interference with the MTT endpoint: 50 μL of the test item are added to 2 mL of isopropanol and to 1 mL of water. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for one hour. The isopropanol mixture was left for 3 hours at room temperature.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT): 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 ml of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions and rinsed 3 times with DPBS afterwards.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570). No reference wavelength measurement was used.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The results are acceptable according to OECD TG 492, if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 60% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: not reported
- Complete supporting information for the specific RhCE tissue construct used: yes, attached to the study report
- Reference to historical data of the RhCE tissue construct: no
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: yes
- Positive and negative control means and acceptance ranges based on historical data: NA
- Acceptable variability between tissue replicates for positive and negative controls: yes
- Acceptable variability between tissue replicates for the test chemical: yes

Results and discussion

In vitro

Results
Irritation parameter:
other: Tissue viability
Run / experiment:
Main study
Value:
93.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
6.9%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour.

OTHER EFFECTS:
- Visible damage on test system: none reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The negative control OD is > 0.8 and < 2.5 (values between 1.568 and 1.653).
- Acceptance criteria met for positive control: yes. The mean relative viability of the positive control is below 50% of the negative control viability (6.9%).
- Acceptable variability between tissue replicates: yes. The difference of viability between the two relating tissues of a single item is < 20% (values between 0.5% to 7.0%) in the same run (for positive and negative control tissues and tissues of single test items).

Any other information on results incl. tables

Table 7.3.2/1: Results after treatment for 30 minutes

Dose Group

Absorbance (OD) of Tissue 1 and 2


Well 1

Absorbance (OD) of Tissue 1 and 2


Well 2

Mean Absorbance (OD) Tissue 1 and 2

Mean Absorbance (OD)* Tissue 1 and 2 minus Mean Blank

Mean Absorbance (OD) of
2 Tissues*

Rel. Aborbance [%] (Viability)
Tissue 1 and 2**

Absolute value of the Difference of the Rel. Absorbances [%]
Tissue 1 and 2

Rel. Absorbance

[% of Negative Control]** (Viability)

Negative Control

1.596

1.615

1.606

1.568

1.611

97.4

5.3

100.0

1.691

1.691

1.691

1.653

102.6

Positive Control

0.151

0.138

0.144

0.107

0.110

6.6

0.5

6.9

0.147

0.157

0.152

0.114

7.1

Test Item

1.480

1.491

1.485

1.448

1.504

89.9

7.0

93.4

1.459

1.738

1.598

1.561

96.9

Concerning acceptance criteria:

• The negative control OD is > 1.0 and < 2.6 (1.568 and 1.653).

• The mean relative viability of the positive control is below 60% of the negative control viability (6.9%).

•The difference of viability between the two relating tissues of a single item is < 20% (values between 0.5% to 7.0%) in the same run (for positive and negative control tissues and tissues of single test items).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
With a percentage of tissue viability > 60%, the test item does not require classification and labelling according to the Regulation (EC) No 1272/2008 (CLP) and to the GHS.
Executive summary:

A study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The study was conducted according to the OECD guideline No. 492 and in compliance with GLP.

The test item did not prove to be an MTT reducer in the MTT pre-test. Also its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

Each 50 µL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue.

After treatment with the negative control the absorbance values were well within the required acceptability criterion:

• mean OD > 1.0 and < 2.6 (MatTek criterion)

• mean OD > 0.8 and < 2.5 (OECD criterion)

thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 60% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system.

The difference of viability between the two relating tissues of a single test item was < 20% in the same run (for positive and negative control tissues and tissues of single test items).

Irritating effects were not observed following incubation with the test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (93.4%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess any eye irritating potential.

With a percentage of tissue viability > 60%, the test item does not require classification and labelling according to the Regulation (EC) No 1272/2008 (CLP) and to the GHS.