Extract obtained from the shell 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:

27 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

9 October 2017

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

948-055-8

Molecular formula:

not applicable as it is a UVCB

IUPAC Name:

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

Test material form:

liquid: viscous

Details on test material:

- Physical appearance: dark brown to black viscous liquid
- Storage conditions: in refrigerator (2-8°C) protected from light

Specific details on test material used for the study:

- Stability at higher temperatures: stable

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse Vitelco, 's-Hertogenbosch, The Netherlands.
- Storage, temperature and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected an transported in physiological saline in a suitable container under cooled conditions.
- The eyes were checked for unacceptable defects and those exhibiting defects were discarded.
- Preparation for use: isolated corneas were stored in a petri dish with cMEM medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum, they were mounted in a corneal holder of which the compartments were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C. After the incubation, opacity determinations were performed and corneas that had an initial opacity reading of >7 were not used.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: sufficient amount for complete coverage of the cornea (due to the viscous nature of the test material)

NEGATIVE CONTROL: Physiological saline
- Amount applied: 750 µL

POSITIVE CONTROL: Ethanol
- Amount applied: 750 µL

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ±10 minutes in cMEM for opacity measurements and subsequently 90 ±5 minutes in sodium-fluorescein for permeability determinations

Number of animals or in vitro replicates:

3 for the negative control, the positive control and the treatment group each.

Details on study design:

TREATMENT METHOD: The medium from the anterior compartment was removed and 750 µl of either the negative control, the positive control or an amount of the test item to completely cover the cornea was introduced onto the epithelium of the cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.


REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). OD490 values of less than 1.500 were used in the permeability calculation.
- Other: possible pH effects of the test substance on the corneas were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA (see table 1):
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces IVIS >55 is defined as a corrosive or severe irritant (UN GHS: category 1);
For a test substance that induces an IVIS >3 and ≤55, no prediction on irritant potency can be made.

ACCEPTABILITY CRITERIA:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3 replicates

Value:

4.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean IVIS: 74

Other effects / acceptance of results:

- The corneas treated with the test item showed opacity values between 1.6 and 3.4, permeability values were ranging from 0.118 to 0.163 and IVIS were 3.5, 3.6 and 5.8 (n=3). Since the IVIS were > 3 ≤ 55, no prediction on the irritancy can be made.

OTHER EFFECTS:
- The corneas were translucent after 10 minutes treatment with the test item.
- No pH effect of the test item was observed on the rinsing medium.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, results were within the historical range (opacity and permeability values were between 0.7 and 1.2 and between -0.001 and 0.013, respectively)
- Acceptance criteria met for positive control: yes, results were within historical range (IVIS ranging from 56-103). Corneas were turbid after 10 minutes of treatment.

Any other information on results incl. tables

Table 2 Overview of results per replicate

Treatment	Final Opacity^b	Final OD490^b	In vitro Irritancy Score^a
Negative control	1.2	-0.001	1.2
	0.7	0.013	0.9
	0.8	0.002	0.8
Positive control	16	2.615	56
	21	5.439	103
	15	3.195	63
Test item	1.6	0.134	3.6
	3.4	0.163	5.8
	2.2	0.118	3.5

^a  In vitro irritancy score (IVIS) = opacity value + (15 x OD₄₉₀value).

^b  Positive control and test item are corrected for the negative control.

Table 3 Historical Control Data for the BCOP Studies

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-2.9 – 3.0	-0.034 – 0.100	-2.8 – 3.0	28.0 – 110.9
Mean	0.18	0.00	0.23	55.28
SD	1.10	0.01	1.13	15.14
n	113	113	113	88

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Feb 2015 to Feb 2018.

Applicant's summary and conclusion

Interpretation of results:

other: no prediction can be made based on this study.

Conclusions:

In the Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, Cocoa Shell Extract (Pot) induced a mean IVIS of 4.3. Based on this result, no prediction for eye irritation can be made and no conclusion for classification according to GHS and Regulation (EC) 1272/2008 can be drawn.