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

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 March 2018 - 20 April 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

31 May 2008

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:

The test item is stable at higher temperatures.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 28326
- Surface: 0.6 cm^2
- Pretreatment: The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 ml DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC.

INTERFERENCE WITH THE MTT ENDPOINT:
- Test for color interference by the test item: 50 μL test item was added to 0.3 mL Milli-Q water and the mixture was incubated for approx. 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue/purple color change was observed.
- Test for reduction of MTT by the test item: 50 μL test item was added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approx. 1 hour at 37.0 ± 1.0°C. At the end of the exposure time it was checked if a blue/purple color change was observed or a blue/purple precipitate was observed.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 35.7-36.2°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: after exposure and after incubation tissues were washed with phosphate buffered saline (once)
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Amount of MTT-medium: 300 μL
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2 per exposure duration + 2 for the negative control and the positive control each

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Freeze killed tissues: The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 mL DMEM. Further use of killed tissues was similar to living tissues.
- N. of replicates : 2 treated with test item and 2 non-treated
- Method of calculation used: nonspecific MTT reduction (NSMTT) was calculated as the difference between the mean OD of the untreated freeze-killed tissues and test item treated freeze-killed tissues expressed as percentage of the mean of the negative control tissues.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one experiment per exposure period (two in total) with 3 independent OD570 measurements per replicate.

ACCEPTABILITY CRITERIA
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
- The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
- The %NSC should be ≤ 30% relative to the negative control OD.
- The non-specific MTT reduction should be ≤ 30% relative to the negative control OD.

DECISION CRITERIA (see table 1 in 'any other information on materials and methods')
A test item is considered corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
- In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non-corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
- In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL
- The test item was applied directly, undiluted, on top of the tissues.

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL (8N KOH)

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours in MTT medium

Number of replicates:

2 tissues per test item per exposure time (4 tissues in total)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

85

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability: 14%

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

57

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability: 6.8%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes
- Colour interference with MTT: yes

- The non-specific reduction of MTT by the test item was 5.3% and 17% of the negative control tissues after 3 minutes and 1 hour respectively.
- The color interference by the test item was 0.18% and 0.31% of the negative control tissues after 3 minutes and 1 hour respectively.
- The nonspecific color in freeze-killed tissues by the test item was 0.31% and 0.17% of the negative control tissues after 3 minutes and 1 hour respectively.

DEMONSTRATION OF TECHNICAL PROFICIENCY: the results of the positive control data were within the historical data range and thereby showing the test system functioned properly (see table 2).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the two tissues of the negative control was within the laboratory historical control data range (i.e., 1.929 for the 3-minute exposure period and 2.077 for the 1-hour exposure period).
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following 1-hour exposure to the positive control was <15 % (i.e., 6.8%).
- Acceptance criteria met for variability between replicate measurements: yes, the Coefficient of Variation (CV) between tissue replicates was ≤ 30% (i.e., ≤ 17%)

For individual OD measurements, see table 3 in 'any other information on results'.

Any other information on results incl. tables

Table 2 Historical data for skin corrosion studies

	Negative control		Positive control
	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (OD570)	1-hour treatment (OD570)
Range	1.258 – 2.615	1.371 – 2.371	0.0172 – 0.56	0.046 – 0.339
Mean	1.80	1.82	0.19	0.14
SD	0.26	0.22	0.09	0.05
n	111	110	106	103

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 November 2014 to November 2017.

Table 3 Individual OD Measurements at 570 nm

	3-minute application (OD570)                      A              B		1-hour application (OD570)                      A              B
Negative control OD570measurement 1 OD570measurement 2 OD570measurement 3
	2.0311	1.9918	2.2718		1.9492
	1.9092	1.9584	2.3495		1.9074
	1.9917	1.9408	2.3106		1.9249
The test item OD570measurement 1 OD570measurement 2 OD570measurement 3
	1.7698	1.8343	1.6028	1.5923
	1.7056	1.7980	1.5710	1.5642
	1.7650	1.8599	1.6033	1.5313
The test item on freeze killed tissue OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.2532	0.2989	0.5294	0.4981
	0.2486	0.2889	0.5124	0.4746
	0.2498	0.2900	0.5100	0.4762
Non - treated freeze killed tissue OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.1769	0.1656	0.1559	0.1324
	0.1751	0.1631	0.1518	0.1264
	0.1760	0.1630	0.1522	0.1277
NSC living OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.0453	0.0455	0.0476	0.0484
	0.0446	0.0452	0.0483	0.0498
	0.0451	0.0460	0.0476	0.0479
NSC dead OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.0525	0.0437	0.0473	0.0428
	0.0521	0.0429	0.0484	0.0439
	0.0518	0.0444	0.0470	0.0428
Positive control OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.3143	0.3388	0.1836	0.1838
	0.3015	0.3280	0.1882	0.1798
	0.3060	0.3367	0.1873	0.1773

OD = Optical density

Duplicate exposures are indicated by A and B.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Not classified according to Regulation (EC) 1272/2008

Conclusions:

In the in vitro skin corrosion test, Cocoa Shell Extract (Pot) was found to be not corrosive to the skin (mean tissue viability of 85% and 57% after a 3-minute and a 1-hour exposure period, respectively). The test item is not classified according to GHS and according to Regulation (EC) 1272/2008.