Extract obtained from powder of Theobroma cacao (Malvaceae) by co-extraction with ethanol and propylene glycol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-23 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 with a minor deviation: variability between tissue replicates of test item was >20%. Considering the results obtained, this deviation was considered as without impact on the conclusion of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Version / remarks:

adopted 28 July 2015

Deviations:

yes

Remarks:

variability between tissue replicates of test item was >20%

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

27 April 2017

Test material

Test material information

Constituent 1

Reference substance name:

Extract obtained from defatted powder of Theobroma cacao (Malvaceae) by extraction with ethanol

EC Number:

947-375-5

IUPAC Name:

Extract obtained from defatted powder of Theobroma cacao (Malvaceae) by extraction with ethanol

Test material form:

solid

Details on test material:

Name: cocoa oleoresine F9202
Batch No: K17 017-1
Appearance: dark brown sticky solid
CAS No: 84649-99-0
Production date: 17 January 2017
Expiry date: 16 January 2019
Storage condition of test material: room temperature

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: K17 017-1
- Date received: 21 February 2017
- Manufacturing date: 17 January 2017
- Expiration date: 16 January 2019
- Purity test date: January 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Used as supplied

FORM AS APPLIED IN THE TEST (if different from that of starting material): Test item was applied on a nylon mesh in order to cover the entire surface of the epidermis (corresponding to approximatively 50 mg).

Test animals / tissue source

Species:

human

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. 23772] were received on 21 March 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): ca. 50 mg
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

5 hours and 49 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)

Duration of post- treatment incubation (in vitro):

- Post-exposure immersion period: 25 minutes at room temperature
- Post-exposure incubation period: 18 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 MTT reduction with the test item:
The direct interaction of MTT with the test item was checked by adding approximatively 16 mg of the test item, previously applied on a nylon mesh, to 300 µL of a solution of MTT at 1 mg/mL.
- Test for the detection of the colouring potential of the test item:
The spectral properties at 570 nm of test item in isopropanol were checked by adding approximatively 16 mg of the test item, previously applied on a nylon mesh, to 1.5 mL of isopropanol (same conditions as in the main test).

MAIN TEST
- Pre-incubation of the tissues:
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 20 hours and 12 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 on a nylon mesh at an approximate dose of 50 mg, and then applied to the entire surface of 2 living RhCE tissue replicates during 5 hours and 49 minutes at standard culture conditions. In the same experimental conditions, a positive control (Methyl acetate), and a negative control (distilled water) were carried out. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 RhCE tissue replicates during 05 hours and 50 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. Residue of yellow coloration was noted on all RhCE after the rinse. This rinsing step was followed by a 25 minutes 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 an 18 hours 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 after extraction from tissues.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 2 hours and 55 minutes 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 02 hours at room temperature 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 at 570 nm was measured in triplicate samples of formazan extracts. The measured OD are proportional to the number of living cells. The measurement of OD was performed 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

Results

Irritation parameter:

other: % mean viability of the tissues

Run / experiment:

Main test

Value:

95.11

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:

PRELIMINARY TESTS
- Test for direct MTT reduction with the test item:
A yellow solution with test item at the bottom of the well was observed after 3 hours of incubation between 36.1°C and 37.0°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 yellow solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.025 which is less than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). Therefore the test item was considered not to interfere with the MTT assay and there was no need to add non-specific coloration controls to the study.

MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 95.11%, versus 31.07% in the positive control (Methyl acetate). Refer Tables 7.3.2/1 for more details.

ACCEPTANCE OF RESULTS:
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.

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	0.935	0.910	0.971	93.77	100	12.47
		0.920
		0.875
	2	1.023	1.031		106.23
		1.030
		1.039
Positive control	1	0.287	0.295	0.302	30.40	31.07	1.34
		0.294
		0.303
	2	0.311	0.308		31.74
		0.308
		0.304
Test item	1	0.804	0.806	0.923	83.05	95.11	24.11
		0.823
		0.791
	2	1.032	1.040		107.16
		0.994
		1.093

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

OD: optical density

 

Note

- As all extracts were diluted at 50% just before the OD measurement, the acceptability criteria for OD values should be in the range > 0.4 and < 1.25 for the negative control.

- The difference of viability between the two tissues of the test item group was 24.11% instead of ≤20% as initially scheduled. As the test item was clearly classified as no category, even when considering each replicate of the test item separately, this deviation was considered as without any impact of the conclusion and the viability of the study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions and in accordance with the Regulation (EC) No. 1272/2008, the test substance is identified as not requiring classification for eye irritation or serious eye damage; and corresponds to the UN GHS 'No category'. No hazard statement and the signal word are required.

Executive summary:

An in vitro eye irritation test using the Reconstructed Human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

The test substance was applied on a nylon mesh which was applied on the epidermis, in order to cover the entire surface of the epidermis (corresponding to approximatively 50 mg). The test substance was applied to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 5 hours and 49 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 25 minutes post-exposure immersion period at room temperature and an 18 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay.

In the preliminary tests, the test substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent tissue viability of the RhCE replicates treated with the test substance was 95.11%, versus 31.07% in the positive control (Methyl acetate). The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validated the experiment.

Under the test conditions and in accordance with the Regulation (EC) No. 1272/2008, the test substance is identified as not requiring classification for eye irritation or serious eye damage; and corresponds to the UN GHS 'No category'. No hazard statement and the signal word are required.