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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
01 Feb 2018 to 26 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
08 May 2017

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek Corporation
Justification for test system used:
The test method is based on reconstructed human epidermis models, which closely mimic the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The procedure described under this test method allows the hazard identification of irritant substances in accordance with UN GHS Category 1 or Category 2, and is in line with the OECD 439 test guidance.
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C, 5% CO2 and 95% relative humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: untill all test item was removed

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3h
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The MTT conversion assay is a quantitative validated method, which is used to measure cell viability.
- Barrier function: The barrier function is assessed by determination of the concentration at which a marker substance reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50% (ET50) upon application of the marker substance at a specified, fixed concentration.
- Contamination: The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze-killed
- N. of replicates : 3
- Method of calculation used:
True viability = Viability of treated tissue – Interference from test chemical = OD tvt – OD kt
where OD kt = (mean OD tkt – mean OD ukt)
tvt = treated viable tissue kt = killed tissues
tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or equal to 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the mean relative tissue viability of three individual tissues exposed to the test substance is >50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30µL
- Concentration (if solution): undiluted D-PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30µL
- Concentration (if solution): 5% aqueous sodium dodecyl sulphate (SDS)
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
The measurements were made for three tissues per condition in 2 replicates.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main experiment
Value:
91.9
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:
- OTHER EFFECTS:
- Direct-MTT reduction: Yes
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Historical data of negative and positive controls 2014-2017

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values:

Negative control
Average OD(mean CV %): 1.776
Average Viability % :100
Viability [%]: 87.4–110.8
CV [%]: 1.1–11.8
Irritant (I) /Non-irritant (NI): NI
Number of unqualified experiments: 0

Positive control
Average OD(mean CV %): 0.085
Average Viability %: 5.1
Viability [%]: 1.3–10.2
CV [%]: 1.8–15.1
Irritant (I) /Non-irritant (NI): I
Number of unqualified experiments: 0

Applicant's summary and conclusion

Interpretation of results:
other: Not irritant
Remarks:
in accordance with Annex I of the CLP Regulation (1272/2008/EC).
Conclusions:
Based on the results of the in vitro skin irritation test for Ginger CO2-SE extract, the test substance does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The skin irritant properties of Ginger CO2 -SE extract were examined in a study performed according to OECD Guideline 439 (EpiDermTM model), under GLP.

Three tissues were used for each treatment and control group. The optical density (OD) was determined by using the MTT reduction assay.  30 mL undiluted Ginger CO2 -SE extract was applied to the model skin surface. D-PBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium.

The mean viability of cells exposed to Ginger CO2-SE extract was 91.9% of the negative controls (viability was corrected for direct MTT reduction). The mean optical density (OD) of 3 negative and positive control tissues were respectively 1.699 and 5.2% of the negative control. All acceptance criteria were fulfilled.

Based on the cell viability being >50% of the cut-off value, Ginger CO2-SE extract was considered to be non-cytotoxic and predicted to be non-irritant to skin.