Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September to 20 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 492 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
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:
July 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model
Version / remarks:
29 June 2015
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
German GLP Compliance Program (inspected on July 13-16, 2015 / Signed on September, 2015)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Appearance: pale brown solid, wax-like
Specific details on test material used for the study:
- Storage conditions: At room temperature, protected from moisture
(Dry area, unopened containers, optimum temp. 11-25 °C /
52-77 °F)

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 Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. This test guideline is applicable to solid and waxes, so is considered to be applicable to the test item.

CELL SYSTEM:
- EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (Ashland, MA01721, USA).
- Lot No.: 227005
- Keratinocyte strain: 4F1188
- Analysis for potential biological contaminants: none detected (HIV-1, Hep. B and Hep. C virus; Bacteria, yeast and other fungi)
- Tissue viability: 1.419, within the acceptance criteria (1.1-3.0)
- Barrier function: 15.97 min, within the acceptance criteria (12.2-37.5)
- Sterility: Sterile

- 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 of diameter).
- Transport: EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate.
- Storage: On day of 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 (nearly 17 hours).

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): Approximately 50 mg of the test item were tested topically on wetted duplicate EpiOcular™ tissues.
Duration of treatment / exposure:
6 hours
Observation period (in vivo):
NA
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
duplicate tissues
Details on study design:
- Details of the test procedure used: procedure for solids
- Doses of test chemical and control substances used: 50 mg
- Duration and temperature of:
exposure: 6 hours / 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
post-exposure immersion: 25 min / room temperature
post-exposure incubation period: 18 hours / 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
- Description of any modifications to the test procedure: none
- Test for direct MTT reduction: approximately 50 mg of the test item are added to 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was run concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT. Then the colour of the obtained solutions was evaluated.
- Assessment of Color Interference with the MTT endpoint: approximately 50 mg of the test item were added to 1.0 mL of water and to 2 mL isopropanol in a glass tube. 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, NSCliving): 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 were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Inserts were 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-labelled 6-well plate containing 2 ml isopropanol in each well so that no isopropanol was flowing into the insert. The corresponding negative and positive controls had to be treated identically without piercing. For this procedure it was necessary to seal the plates particularly thorough since a higher evaporation rate had to be expected due to the larger surface of wells in 6¬-well plates.
Extraction was performed for about 2 hours at room temperature on a shaker.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate(s).
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). 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 50% 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 freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) 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 labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is identified potentially requiring classification and labelling according to UN GHS (Category 2 of Category 1.
According to OECD guideline 492 a single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Data of 21 studies performed from July 2015 until end of end march 2017
Positive controls: Viability 31.59% (8.10% - 42.60%) / Absorption 0.48 (0.22- 0.64)
Negative controls: Absorption 1.50 (1.24 – 2.05)
- 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, Envigo CRS project No. 1729900
- Positive and negative control means and acceptance ranges based on historical data: yes
- 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:
Mean
Value:
95.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
31.8%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
The test item proved to be a MTT reducer in the MTT pre-test. An additional test with freeze-killed tissues was preformed to evaluate whether the test material is not binding to the tissue and leading to a false MTT reduction signal.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The negative control OD is > 0.8 and < 2.5 (values between 1.331 and 1.401).
- Acceptance criteria met for positive control: yes. The mean relative viability of the positive control is below 50% of the negative control viability (31.8%).
- Acceptable variability between tissue replicates: yes. The difference of viability between the two relating tissues of a single item is < 20% in the same run. This does also apply to the additional viable tissues (without MTT addition).

Any other information on results incl. tables

Table 7.3.2/1: Results after 6 hour treatment with the test item and the controls

Dose Group

OD

Well 1

OD
Well 2

Mean OD of 2 Wells

Mean OD of 2 Wells Blank corrected

Mean OD of 2 Tissue replicates

% Viability of individual Tissue replicates

Mean Viability (%)

Difference
Tissue1 - Tissue2

Corrected Viability Mean %

Blank

0.036

0.036

0.036

Negative Control Tissue 1

1.322

1.411

1.367

1.331

97.5

Negative Control Tissue 2

1.432

1.441

1.436

1.401

1.366

102.5

100.0

5.1

Positive Control Tissue 1

0.411

0.485

0.448

0.412

30.2

Positive Control Tissue 2

0.492

0.494

0.493

0.458

0.435

33.5

31.8

3.3

Test Item Tissue 1

1.264

1.385

1.325

1.289

94.4

Test Item Tissue 2

1.369

1.368

1.369

1.333

1.311

97.6

96.0

3.2

95.9

Negative Control Freeze-killed Tissue

Tissue 1

0.109

0.117

0.113

0.077

5.7

Negative Control Freeze-killed Tissue

Tissue 2

0.115

0.112

0.113

0.078

0.078

5.7

5.7

0.0

Test Item Add. Freeze-killed Tissue

Tissue 1

0.113

0.115

0.114

0.079

5.8

Test Item Add. Freeze-killed Tissue

Tissue 2

0.114

0.116

0.115

0.079

0.079

5.8

5.8

0.0

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study and with a percentage of tissue viability > 60%, the test item does not require classification for eye irritation or eye damage according to 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 proved to be an MTT reducer in the MTT pre-test. An additional test with freeze-killed tissues was preformed to determine a correction factor for calculating the true viability in the main experiment.

50 mg of the test item was applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

Treatment with the positive control induced a decrease below 50% compared with thenegative control value in the relative absorbance thus ensuring the validity of the test system.

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

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues.

The quality criteria required for acceptance of results in the test were satisfied. The tissue viability of the test item was 95.9% after the 6 -hour exposure period.

Under the experimental conditions of this study and with a percentage of tissue viability > 60%, the test item does not require classification for eye irritation or eye damage according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for eye irritation endpoint.