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Administrative data

Description of key information

Skin irritation (OECD TG 439): Not irritating

Eye irritation (OECD TG 437): Not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Feb 2018 - 29 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
2017
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
08 May 2017
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG, 49699 Lindern, Germany
- Characteristics of donor animals: age range of 6 to 12 months
- Storage, temperature and transport conditions of ocular tissue: Eyes were completely submerged in Hanks' Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL
- indication of any existing defects or lesions in ocular tissue samples: Only corneas from eyes free of defects were used.
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): 750 µL
- Concentration (if solution): undiluted

Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
Opacity: None
Corneal permeability: 90 +/- 5 min
Number of animals or in vitro replicates:
3 per condition
Details on study design:
SELECTION AND PREPARATION OF CORNEAS :
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.
The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas that had an opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
0.9% sodium chloride solution

POSITIVE CONTROL USED
1% NaOH solution in highly purified water

APPLICATION DOSE AND EXPOSURE TIME
750µL, 10 minutes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: only for corneal permeability measurement 90 +/- 5 min

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times, Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490, Tecan Sunrise visible light)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: decision criteria as indicated in the TG were used.
Irritation parameter:
in vitro irritation score
Run / experiment:
Cornea No. 7
Value:
-1.742
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Cornea No. 8
Value:
0.639
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Cornea No. 9
Value:
-1.487
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:
- Visible damage on test system: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

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

NaCl 0.9%: Opacity 0.062 (sd 0.934), Permeability 0.043 (sd 0.073), IVIS 0.579 (sd 1.097)
NaOH 1%: Opacity 72.67 (sd 20.908), Permeability 2.016 (sd 0.415), IVIS 102.903 (21.283)
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 eye 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 eye irritant properties of Ginger CO2-se extract were examined in a study performed according to OECD Guideline 437, under GLP. Three corneas were used for each treatment group (test item, negative control and positive control). The liquid test item Ginger CO2-se extractwas used undiluted. 0.9% NaCl solutionwas used as the negative control and 1% NaOH in water (highly purified water)as the positive control item. The test and control items were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was 10 minutes. The optical density (OD) was measured at a wavelength of 490 nm. The acceptance criteria of validity were fulfilled in this test. Following treatment with Ginger CO2-se extract a mean opacity and a mean permeability value of <0.01compared to the negative control were determined. The calculated IVIS was below the cut-off value of 3and consequently the test item is not classified as a severe irritant and is not considered corrosive.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin irritation (OECD TG 439)

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.

Eye irritation (OECD TG 437)

The eye irritant properties of Ginger CO2-se extract were examined in a study performed according to OECD Guideline 437, under GLP. Three corneas were used for each treatment group (test item, negative control and positive control). The liquid test item Ginger CO2-se extractwas used undiluted. 0.9% NaCl solution was used as the negative control and 1% NaOH in water (highly purified water) as the positive control item. The test and control items were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was 10 minutes. The optical density (OD) was measured at a wavelength of 490 nm. The acceptance criteria of validity were fulfilled in this test. Following treatment with Ginger CO2-se extract a mean opacity and a mean permeability value of <0.01 compared to the negative control were determined. The calculated IVIS was below the cut-off value of 3 and consequently the test item is not classified as a severe irritant and is not considered corrosive.

Justification for classification or non-classification

Based on the available information for Ginger CO2-se extract, the test substance does not need to be classified for skin or eye irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).