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Toxicological information

Skin irritation / corrosion

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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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
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

Resultsopen allclose all
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%
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.