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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2017 - March 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
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:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-isopropyl-4-methylbicyclo[3.1.0]hexan-3-one
EC Number:
208-912-2
EC Name:
1-isopropyl-4-methylbicyclo[3.1.0]hexan-3-one
Cas Number:
546-80-5
Molecular formula:
C10H16O
IUPAC Name:
(1S,4R,5R)-4-methyl-1-propan-2-ylbicyclo[3.1.0]hexan-3-one
Constituent 2
Chemical structure
Reference substance name:
DL-bornan-2-one
EC Number:
244-350-4
EC Name:
DL-bornan-2-one
Cas Number:
21368-68-3
Molecular formula:
C10H16O
IUPAC Name:
1,7,7-trimethylbicyclo[2.2.1]heptan-2-one
Constituent 3
Chemical structure
Reference substance name:
Cineole
EC Number:
207-431-5
EC Name:
Cineole
Cas Number:
470-82-6
Molecular formula:
C10H18O
IUPAC Name:
1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane
Constituent 4
Chemical structure
Reference substance name:
(1S, 4S, 5R)-4-Methyl-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-one
Cas Number:
471-15-8
Molecular formula:
C10H16O
IUPAC Name:
(1S, 4S, 5R)-4-Methyl-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-one
Constituent 5
Chemical structure
Reference substance name:
Camphene
EC Number:
201-234-8
EC Name:
Camphene
Cas Number:
79-92-5
Molecular formula:
C10H16
IUPAC Name:
2,2-diméthyl-3-méthylène-bicyclo[2,2,1]heptane
Constituent 6
Chemical structure
Reference substance name:
Humulene
EC Number:
229-816-7
EC Name:
Humulene
Cas Number:
6753-98-6
Molecular formula:
C15H24
IUPAC Name:
(1E,4E,8E)-2,6,6,9-tetramethylcycloundeca-1,4,8-triene
Constituent 7
Chemical structure
Reference substance name:
Pin-2(3)-ene
EC Number:
201-291-9
EC Name:
Pin-2(3)-ene
Cas Number:
80-56-8
Molecular formula:
C10H16
IUPAC Name:
2,6,6-trimethylbicyclo[3.1.1]hept-2-ene
Constituent 8
Chemical structure
Reference substance name:
Caryophyllene
EC Number:
201-746-1
EC Name:
Caryophyllene
Cas Number:
87-44-5
Molecular formula:
C15H24
IUPAC Name:
(1R,4E,9S)-4,11,11-trimethyl-8-methylidenebicyclo[7.2.0]undec-4-ene
Constituent 9
Chemical structure
Reference substance name:
(1S-endo)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol
EC Number:
207-353-1
EC Name:
(1S-endo)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol
Cas Number:
464-45-9
Molecular formula:
C10H18O
IUPAC Name:
endo-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol
Constituent 10
Chemical structure
Reference substance name:
Pin-2(10)-ene
EC Number:
204-872-5
EC Name:
Pin-2(10)-ene
Cas Number:
127-91-3
Molecular formula:
C10H16
IUPAC Name:
6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
Test material form:
liquid
Details on test material:
-Name: SAGE DALMATIAN OIL
-IUPAC Name: Essential oil of Salvia officinalis (Lamiaceae) obtained from leaves, flowers and stalks by steam distillation.
-CAS: 84082-79-1
-EINECS: 282-025-9
-Batch no.: BU201707
-Purity: 100 % wt – UVCB substance
-Appearance: pale to slightly yellow
-Expiry date: 03/07/2019
-Storage: Sample should be stored tightly closed, below ambient temperature, preferably under inert gas atmosphere in dark, lightproofed bottles.

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, batch No. 23767] were received on 21 February 2017.

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 µL
- Concentration (if solution): 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:
PRELIMINARY TESTS
- Test for direct interaction with MTT:
The direct interaction of MTT with the test item was checked by adding 16 µL of the test item to 300 µL of MTT solution at 1 mg/mL.
- Test for the detection of the colouring potential of the test item:
The spectral properties at 570 nm of the test item in isopropanol was checked by adding 16 µL of the test item to 1.5 mL of isopropanol.


MAIN TEST
- Pre-incubation of the tissues:
First run: On the day of receipt, the 12 tissues in their 24-well shipping container 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 19 hours and 45 minutes at standard culture conditions.
Second run: On the day of receipt, the 12 tissues in their 24-well shipping container 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 19 hours and 55 minutes 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 the entire surface of 2 living RhCE tissue replicates during 30 minutes at 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 a 2-hour post-exposure incubation 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 18 hours and 50 minutes 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
Run / experiment:
first run
Value:
60.34
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: mean % viability
Run / experiment:
second run
Value:
80.96
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
PRELIMINARY TESTS
- Test for direct interaction with MTT:
A yellow solution was observed after 3 hours of incubation between 36.4°C and 37.9°C, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
A colourless solution was obtained in isopropanol after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.002 which was less than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). Therefore the test item was considered to be not interfering with the MTT assay.

MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 60.34%, versus 24.44 % in the positive control (Methyl acetate), during the first run and 80.96% for the test item versus 18.36% for the positive control during the second rub. Results from the first run were borderline, insofar as mean percent tissue viability equal to 60.34 ± 8.01%, so a second test was performed under the same experimental conditions.
The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validate the experiment. 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: FIRST RUN

 

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

0.922

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

0.469

0.485

0.520

56.33

60.34

8.01

0.492

0.494

2

0.571

0.554

64.34

0.565

0.526

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

OD: optical density

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 SECOND RUN

 

Tissue

OD

Mean OD/disc (#)

Mean OD/product

Viability %

Mean viability %

Difference of viability %

Negative control

1

0.920

0.950

0.951

99.95

100.00

0.11

0.952

0.978

2

0.933

0.951

100.05

0.954

0.967

Positive control

1

0.023

0.025

0.175

2.63

31.46

31.46

0.026

0.027

2

0.318

0.324

34.09

0.321

0.334

Test item

1

0.715

0.750

0.770

78.91

80.96

4.10

0.749

0.787

2

0.786

0.789

83.01

0.787

0.795

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

OD: optical density

The difference of viability between the 2 tissues treated with the positive control was 31.46%, instead

of <20% at the maximum as initially scheduled.

Considering the results obtained (2 values < 50%) and the fact that this value remains in the range of historical negative control data, this deviation is considered as without impact on the conclusion of the study

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test substance was not identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).
Executive summary:

The test item was applied, as supplied, at the dose of 50 µL, to 2 living DPBS pre-treated RhCE (EpiOcularTMtissue model) during 30 minutes at, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS 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 withO.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 was 60.34%, versus 24.44% in the positive control (Methyl acetate).

Results were borderline, insofar as mean percent tissue viability equal to 60.34 ± 8.01%, so a second test was performed under the same experimental conditions.

 During the 2ndrun, the mean corrected percent tissue viability of the RhCE replicates treated with the test item was 80.96%, versus 18.36% in the positive control (Methyl acetate).

 

 In conclusion, under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.

No hazard statement and signal word are required.