Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 -29 Apr 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
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)
Deviations:
yes
Remarks:
no historical data of positive and negative controls
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
human
Strain:
other: EpiOcular™

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: approx. 50 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
in duplicates for each treatment and contol group
Details on study design:
- RhCE tissue construct used, including batch number: EpiOcular™ (MatTek Corporation, Bratislava, Slovakia), Lot No.: 23707
- Tissue viability: The quality of the final product was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.918 ± 0.095 (acceptance criteria: 1.1 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 0.3% Triton X-100. The ET-50 value was determined to be 15.12 min (acceptance criteria: 12.2 - 37.5 min).
- Contamination: The cells used to produce the EpiOcular tissue were screened for the presence of viruses, bacteria, yeast and other fungi.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 6 hours exposure (37 °C), 25 min post-exposure immersion (room temperature), 18 hours post-exposure (37 °C)
- 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. Therefore, an additional test with viable tissues without MTT addition was not necessary. Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour. Therefore, an additional test with freeze-killed tissues was not necessary.
- Description of the method used to quantify MTT formazan: The absorbance at 570 nm of each well was measured with a plate reader (Versamax Molecular Devices, Ismaning, Germany). No reference wavelength measurement was used.
- 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.
- Acceptance criteria: The results are acceptable 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 and 3) the difference of viability between the two relating tissues of a single test substance is < 20% in the same run (for positive and negative control tissues and tissues of test substances).

Results and discussion

In vitro

Results
Irritation parameter:
other: % mean relative absorbance of 2 tissues
Run / experiment:
6 hours exposure
Value:
85.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.352 and 1.573) was in the range of > 0.8 and < 2.5.
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (13.0%).

The difference of viability between the two relating tissues of a single item is < 20% (values between 0.0% to 15.1%) in the same run (for positive and negative control tissues and tissues of single test items).

Any other information on results incl. tables

Table 2. Results after 6 hours incubation time

Test group

Mean absorbance*

 

 

Mean absorbance of 2 tissues*

Rel. absorbance (%)**

Absolute value of the difference of the rel. absorbance (%) Tissue 1 and 2

Rel. absorbance (% of negative control)

Tissue 1

Tissue 2

Tissue 1

Tissue 2

Negative control

1.352

1.573

1.463

92.4

107.6

15.1

100.0

Positive control

0.189

0.190

0.190

13.0

13.0

0.0

13.0

Test substance

1.332

1.159

1.245

91.1

79.2

 11.9

85.1

* Mean of two replicate wells after blank correction

** Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

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:
Under the conditions of the conducted test, the test substance is non irritating towards human-derived epidermal keratinocytes in the EpiOcular™ model.