Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2017 - October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 492 without any deviation

Data source

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

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)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Appearance: very viscous liquid, brown to brown red
Substance type: UVCB (Natural Complex Substance)
CAS-No: 9000-24-2
EINECS-No: 232-532-6
Test substance storage: Dry area, unopened containers, optimum temp. 11-25°C
Purpose of testing: REACH
Date of expiration: 18 May 2020

Test animals / tissue source

Species:
other: Reconstructed human Cornea-like Epithelia
Details on test animals or tissues and environmental conditions:
- Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation]

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Concentration: Undiluted
Duration of treatment / exposure:
30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)
Duration of post- treatment incubation (in vitro):
- Post-exposure immersion period: 12 minutes at room temperature
- Post-exposure incubation period: 2 hours at standard culture conditions
Number of animals or in vitro replicates:
Test item, negative and positive controls were applied on duplicate tissues.
Details on study design:
MAIN TEST
- Pre-incubation of the tissues:
On the day of receipt, the tissues were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 20 hours and 10 minutes for the first run, 20 hours for the second run and 19 hours and 45 minutes for the third run, at standard culture conditions.
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
- Treatment and post-treatment incubation of the tissues:
The test item was applied, as supplied, at the dose of 50 μL, to 2 living PBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). In the same experimental conditions, a positive control (Methyl acetate), and a negative control (distilled water) were used. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 viable RhCE tissue replicates during 30 minutes at standard culture conditions.
After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS. The rinsed tissues were checked for any colouration and noted for comparable colour with the negative control. This rinsing step was followed by a 12-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. The RhCE constructs were then incubated for about a 2-hour post-exposure incubation (1 hour and 59 minutes for the first run,2 hours for the second and third run) at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.
- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD was proportional to the number of living cells.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 17 hours and 35 minutes for the first run, during 18 hours and 45 minutes for the second run and during 17 hours and 20 minutes for the third run, at 6±3°C in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2). The OD measurement of formazan extracts at 570 nm was measured in triplicate samples using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: % mean viability of the tissues
Run / experiment:
First run mean value
Value:
61.83
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: % mean viability of the tissues
Run / experiment:
Second run mean value
Value:
55.27
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
other: % mean viability of the tissues
Run / experiment:
Third run mean value
Value:
65.97
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:
MAIN TEST
MTT assay results:
- The mean percent tissue viability of the RhCE replicates treated with the test item was 61.83% versus 24.44% in the positive control (Methyl acetate).
A second test was run leading to mean percent tissue viability of 55.27% for the test item and 32.69% for the positive control. As no conclusion could be drawn from these 2 tests, a 3rd one was performed. This run led to mean percent tissue viability of 65.97% or the test item and 44.19% for the positive control.
Refer Tables 7.3.2/1 for more details.

Any other information on results incl. tables

Table 7.3.2/1: Main test - Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

Tissue

OD

Mean OD/disc (#)

Mean OD/product

Viability %

Mean viability %

Difference of viability %

Negative control

1

0.846

0.839

0.845

99.29

100.00

1.42

0.835

0.836

2

0.845

0.851

100.71

0.855

0.854

Positive control

1

0.227

0.214

0.207

25.33

24.44

1.78

0.211

0.205

2

0.202

0.199

23.55

0.198

0.198

Test item

1

0.507

0.511

0.523

60.47

61.83

2.72

0.517

0.509

2

0.537

0.534

63.20

0.540

0.526

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Second Main test - Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

Tissue

OD

Mean OD/disc (#)

Mean OD/product

Viability %

Mean viability %

Difference of viability %

Negative control

1

1.036

1.023

1.034

98.94

100.00

2.13

1.012

1.022

2

1.063

1.045

101.06

1.036

1.037

Positive control

1

0.357

0.357

0.338

34.53

32.69

3.68

0.360

0.353

2

0.394

0.319

30.85

0.352

0.210

Test item

1

0.600

0.592

0.572

57.25

55.27

3.97

0.588

0.589

2

0.557

0.551

53.29

0.544

0.558

Third Main test - Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

Tissue

OD

Mean OD/disc (#)

Mean OD/product

Viability %

Mean viability %

Difference of viability %

Negative control

1

0.829

0.849

0.861

98.61

100.00

2.79

0.861

0.857

2

0.889

0.873

101.39

0.909

0.822

Positive control

1

0.356

0.346

0.381

40.19

44.19

8.01

0.333

0.350

2

0.463

0.415

48.20

0.404

0.380

Test item

1

0.574

0.578

0.568

67.13

65.97

2.32

0.581

0.581

2

0.545

0.558

64.81

0.568

0.562

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Considering the mean viability of 61.02% obtained with the three runs performed, the test item does not require classification for eye irritation or serious eye damage according to Regulation EC No. 1272/2008.
Executive summary:

The aim of the study was to evaluate the eye hazard potential of the test item GALBANUM RDE SUPER INDE after topical administration on in vitro reconstructed human cornea-like epithelium tissues (EpiOcularTM tissue model).

The test item GALBANUM RDE SUPER INDE was applied, as supplied, at the dose of 50 μL, to 2 living PBS pre-treated RhCE (EpiOcularTM tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with PBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay. The experimental protocol was established in accordance with O.E.C.D. Test Guideline No. 492 adopted 28 July 2015.

The mean percent tissue viability of the RhCE replicates treated with the test item GALBANUM RDE SUPER INDE was 61.83 %, versus 24.44% in the positive control (Methyl acetate).

Results were borderline, insofar as mean percent tissue viability equal to 61.83 ± 2.72%, so a second test was performed under the same experimental conditions.

During the 2nd run, the mean corrected percent tissue viability of the RhCE replicates treated with the test item was 55.27%, versus 32.69% in the positive control (Methyl acetate).

As this result is discordant with the result of the first test, a third test was considered.

During the 3rd run, the mean corrected percent tissue viability of the RhCE replicates treated with the test item was 65.97%, versus 44.19% in the positive control (Methyl acetate).

Considering the mean viability of 61.02% obtained with the three runs performed, the test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.