Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Sep - 01 Dec 2017
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 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:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test - Summary
Version / remarks:
22 July 2015
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
human
Strain:
other: Construct of normal human epidermal keratinocytes
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The EpiOcular™ human cell construct for eye irritation testing is a non-keratinized epithelium prepared from normal human keratinocytes (MatTek Corporation). It models the corneal epithelium with progressively stratified, but not cornified cells. Based on the "depth of injury model," the EpiOcular eye irritation test (EIT) is intended to differentiate those materials that are nonirritants (would not require a warning label in the European chemical classification systems) from those that would require labelling as either GHS eye irritant category 1 or 2.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live The EpiOcular™ human cell construct for eye irritation testing (OCL-200-EIT) (Lot No. 27015) was obtained from MatTek Corporation, and tested by MatTek for potential biological contaminants, sterility, barrier function and tissue viability, results for which were within acceptable ranges and indicated appropriate formation of the mucosal barrier and a viable basal cell layer.

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): 50 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 μL
- Lot/batch no.: RNBF7110

POSITIVE CONTROL
- Amount(s) applied: 50 μL
- Lot/batch no.: S6943111
Duration of treatment / exposure:
6 ± 0.25 h
Duration of post- treatment incubation (in vitro):
18 ± 0.25 h
Number of animals or in vitro replicates:
Duplicate tissues for each treatment, positive and negative control groups
Details on study design:
- Details of the test procedure used: EpiOcular™ was transferred to plates containing assay medium and incubated for 1 h, followed by transferring out the assay medium, adding fresh assay medium, and pre-incubating 16-24 hours at 37 °C, in a humidified atmosphere of 5% CO2. After pre-incubation, the tissues were pre-treated by wetting with DPBS and incubated at standard culture conditions for 30 ± 2 minutes. The tissues were treated with each dose group in duplicate, starting with the negative and positive control. The cultures were incubated for 6 ± 0.25 h at 37 °C, 5.0% CO2 / 95% air. After the exposure time, tissues were rinsed with DPBS to remove any residual test material. After rinsing, the tissues were immediately transferred to and immersed in assay medium for a 25 ± 2 minutes immersion incubation (post-soak). The tissues were then returned to assay medium, and post-incubated for an additional 18 ± 0.25 h at 37 °C, 5.0% CO2 / 95% air. Then the cultures were transferred to 0.3 mL/well of MTT reagent and incubated for 3 h ± 10 minutes. After incubation, the cultures were transferred to new tissue well-plates, and extracted in 2 mL of isopropanol, the extraction plates were sealed to inhibit isopropanol evaporation and carried out after storage overnight in the dark at 2 - 8 °C. Then the extracts were mixed to obtain homogeneous solutions, and duplicate volumes of 200 μL of each extraction solution were transferred to a 96-well plate and their optical density (ODs at 570± 30 nm) was recorded, while 200 μL of isopropanol was used as the blank.

- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200, OCL-212); Test Kit name: OCL-200 kit, OCL-212 kit (manufactured by MatTek Corporation); Lot number: 27015.

- Doses of test chemical and control substances used: test material: 50 mg of test substance; negative control: 50 μL of distilled water; positive control: 50 μL of methyl acetate.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Exposure: 6 ± 0.25 h at 37 °C, 5.0% CO2 / 95% air; Postexposure immersion: 25 ± 2 minutes at room temperature; Post-exposure incubation: 18 ± 0.25 h at 37 °C, 5.0% CO2 / 95% air.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: 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. The test substance was tested for direct MTT-reduction and results were negative.

- Number of tissue replicates used per test chemical and controls: Two replicates per treatment: test substance, positive and negative controls

- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): Wavelength of 570 ± 30 nm, measured on a spectrophotometer

- Decision criteria/Prediction model: 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 labeled irritant.

- Acceptable variability between tissue replicates for positive and negative controls: OD in the negative control substance group is > 0.8 and < 2.5. The cell viability in the positive control substance group is < 50%.

- Acceptable variability between tissue replicates for the test chemical: Differences of two tissue cell viabilities in each treatment group (test material, positive control, negative control) are < 20%.

Results and discussion

In vitro

Results
Irritation parameter:
other: % tissue viability
Run / experiment:
two tissue replicates per treatment
Value:
ca. 74.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
The mixture of 50 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT (non-specific reduction of MTT) equalled 0%.
The mixture of 50 mg test item per 1 mL A. dest. and per 2 mL isopropanol showed no colouring as compared to the solvent. Therefore, NSCliving (non-specific colour of additional viable tissues) equalled 0%.

ACCEPTANCE OF RESULTS:
The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.819%). The mean relative tissue viability (% negative control) of the positive control was < 50% (19.1%). The inter tissue difference of replicate tissues of all dose groups was < 20% (11.6%).

Any other information on results incl. tables

Table 2: Result of the Test Item Frits

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

1

2

1

2

OD570 values (blank-corrected)

1.818

1.737

0.312

0.365

1.430

1.213

1.847

1.704

0.328

0.356

1.426

1.230

Mean of the duplicates

1.833

1.720

0.320

0.360

1.428

1.221

Mean OD

1.776*

0.340

1.325

Mean SD OD

0.07

0.02

0.12

Tissue viability [%]

103.2

96.8

18.0

20.3

80.4

68.8

Relative tissue viability difference [%]***

6.3

2.3

11.6

Mean tissue viability [%]

100.0

19.1**

74.6

* Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

** mean relative tissue viability of the positive control is < 50%

*** relative tissue viability difference of replicate tissues is < 20%.

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:
In this study under the given conditions the test item showed no irritant effects to the eyes.