Registration Dossier

Administrative data

Description of key information

Skin irritation in vitro (OECD TG 439): Not Irritant (WoE based on Ginger CO2-SE Extract)

Skin irritation in vitro (OECD TG 439): Irritant (WoE based on Ginger Hot Flavor CO2-TO Extract)

Skin irritation in vitro (OECD TG 431): Not corrosive (WoE based on Ginger Hot Flavor CO2-TO Extract)

Eye irritation in vitro (OECD TG 437): Not Irritant (WoE based on Ginger CO2-SE Extract)

Eye irritation in vitro (OECD TG 437): Not Irritant (WoE based on Ginger Hot Flavor CO2-TO Extract)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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
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:
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
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 for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood
- Temperature of post-treatment incubation (if applicable): 37°C, 5% CO2 and 95% relative humidity

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:
0
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: skin 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 Hot Flavor CO2-TO extract, the test substance needs 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 Hot Flavor CO2-TO 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 test material 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 the test material was 0.0% 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 Hot Flavor CO2-TO extract was considered to be cytotoxic and predicted to be irritant to skin.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10 Apr 2018 - 18 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
July 29, 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
07 July 2016
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.
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
At 37°C for the for 1 hr
At room temperature for 3 minutes
- Temperature of post-treatment incubation (if applicable):
37°C

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
- 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)

CONTROL TISSUES USED IN CASE OF MTT COLOUR INTERFERENCE
- N. of replicates 3
- Non-specific colour control (NCcc): The NCcc was performed concurrently per exposure time due to the inherent biological variability of living tissues. The true tissue viability was calculated as the percent tissue viability obtained with living tissues exposed to the interfering test item and incubated with MTT solution minus the percent non-specific colour obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT, run concurrently to the test being corrected (% Color Corrected Viability).

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- the test item is considered to be corrosive to skin and classified as category 1 (or optional category 1A ), if the viability after 3 minutes exposure is less than 50%;
- the test item is considered to be corrosive to skin and classified as sub-category 1B-and-1C, if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%;
- the test item is considered to be non-corrosive to skin, if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): 8 N KOH
Duration of treatment / exposure:
3 minutes or 1 hour
Duration of post-treatment incubation (if applicable):
3 hours induction with MTT
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
105.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure
Value:
116.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes, but additional test with freeze-killed tissues was performed
- Colour interference with MTT: yes, but non-specific colour control included

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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 if different from the ones specified in the test guideline: refer to table in any other information section.

Historical data of negative and positive controls
(
Most recent background data from GLP studies of the years 2015 - 2017 (n = 14).

Material

Average OD

(mean% difference ±SD)

Average viability [%] (mean% difference ±SD)

Range

Range

No. of

unqualified experi-ments

Viability [%]

% difference

Short time incubation – 3‑min

Negative control

(Non-Corrosive)

1.624

(2.5±2.4)

100

(2.5±2.4)

93.7–106.3

0.14–8.6

0#1

8 N KOH

(Corrosive)

0.126

(8.8±7.4)

7.6

(8.8±7.4)

2.0–15.6

<0.01–23.7

0

Long time incubation – 60‑min

Negative control

(Non-Corrosive)

1.650

(4.0±5.0)

100

(4.0±5.0)

85.6–114.4

0.13–18.3

0#1

8 N KOH

(Corrosive)

0.090

(5.7±9.8)

5.9

(5.7±9.8)

2.0–12.6

0.30–38.4

0#2

OD: Optical density. Viability for negative control is set = 100%

SD: Standard deviation

CV: Coefficient of variation

#1       Unqualified results =      if the mean OD of the NC tissues is < 0.8 or > 2.8

                                      if difference in viability for duplicate tissues > 30%

#2       Unqualified results =      8 N KOH: viability > 15% (1-hour exposure)

Interpretation of results:
other: not corrosive
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 Hot Flavor CO2-TO extract, the test substance does not need to be classified for skin corrosion in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The corrosivity to the skin of Ginger Hot Flavor CO2-TO extract was examined in a study performed according to OECD Guideline 431 (EpiDermTM model), under GLP.

Two tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. Ginger Hot Flavor CO2 -TO extract was applied topically as supplied (liquid). Sterile deionised water was used as the negative control. 8 N KOH was used as the positive reference item. The test item and the reference items were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.

In comparison to the negative controls, the mean viability of cells exposed to the test item (% color corrected viability) was 105.9% after a 3-minute exposure period and 116.9% after a 1 hour exposure and hence, the 3-minute and the 1-hour exposure values were above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of ≥50% and ≥15%, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean optical density (OD) of the negative control of 2 tissues was 1.504 (3 minute exposure) or 1.619 (1-hour exposure) and was well within the acceptable range of ≥ 0.8 to ≤ 2.8. The viability of cells treated with the positive reference item 8 N KOH were 8.6% (3 minute exposure) and 4.3% (1-hour exposure) of the negative control and, hence well below the 15% cut-off value at the 1-hour exposure.

The difference of viability between the two tissue replicates (at 20 - 100% viability) was below the limit of acceptance of 30%. Hence, all acceptance criteria were fulfilled.

Under the present test conditions Ginger Hot Flavor CO2 -TO extract tested at two exposure periods of 3 minutes or 1 hour was non-cytotoxic and, hence, predicted to be non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
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 an irritant and is not considered corrosive.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
1.306
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:
2.079
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:
1.914
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 Hot Flavor CO2-TO 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 Hot Flavor CO2-TO 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 was 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 of 1.806 ± 0.387 and a mean permeability value of <0.01 compared to the negative control were determined. The substance had an IVIS value of 1.766 ± 0.407, which was below the cut-off value of 3 and consequently the test item is not classified as a severe eye irritant and is not considered eye corrosive.

Additional information

No study information is available regarding the irritation/corrosion of the substance Ginger oil CO2-Total Extract. However, there is sufficient weight of evidence information available from two independent sources to provide appropriate evidence to fulfil the information requirement. Therefore, in line with section 1.2 of Annex XI in regulation (EC) No 1907/2006, a weight of evidence (WoE) approach was used.

In this WoE approach the results from OECD TG 439 and 437, GLP studies, performed on two qualities of ginger oil extracts (Ginger CO2-SE extract and Ginger oil Hot Flavor CO2-TO extract) were used in order to fulfil the irritation/corrosion endpoint for Ginger oil CO2-Total Extract. These two qualities of ginger oil constitute to the volatile and the non-volatile fraction of the target UVCB (Ginger oil CO2-Total Extract). The two fractions combined cover the constituents present in Ginger oil CO2-Total Extract, albeit in higher concentration ranges in both fractions. By assessing the study results of both fractions in a WoE approach, there is adequate and reliable information available to assess if Ginger oil CO2-Total Extract has or has not a particular dangerous property.

Ginger CO2-SE Extract - 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.

Ginger oil Hot Flavor CO2-TO - skin irritation OECD TG 439

The skin irritant properties of Hot Flavor CO2-TO 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 test material 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 the test material was 0.0% 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, Hot Flavor CO2-TO Extract was considered to be cytotoxic and predicted to be irritant to skin.

Ginger oil Hot Flavor CO2-TO - skin corrosion OECD TG 431

The corrosivity to the skin of Hot Flavor CO2-TO Extract was examined in a study performed according to OECD Guideline 431 (EpiDermTM model), under GLP.

Two tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. Ginger Hot Flavor CO2-to extract was applied topically as supplied (liquid). Sterile deionised water was used as the negative control. 8 N KOH was used as the positive reference item. The test item and the reference items were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.

In comparison to the negative controls, the mean viability of cells exposed to the test item (% color corrected viability) was 105.9% after a 3-minute exposure period and 116.9% after a 1 hour exposure and hence, the 3-minute and the 1-hour exposure values were above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of ≥50% and ≥15%, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean optical density (OD) of the negative control of 2 tissues was 1.504 (3 minute exposure) or 1.619 (1-hour exposure) and was well within the acceptable range of ≥ 0.8 to ≤ 2.8. The viability of cells treated with the positive reference item 8 N KOH were 8.6% (3 minute exposure) and 4.3% (1-hour exposure) of the negative control and, hence well below the 15% cut-off value at the 1-hour exposure.

The difference of viability between the two tissue replicates (at 20 - 100% viability) was below the limit of acceptance of 30%. Hence, all acceptance criteria were fulfilled. Under the present test conditions Ginger Hot Flavor CO2-to extract tested at two exposure periods of 3 minutes or 1 hour was non-cytotoxic and, hence, predicted to be non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.

Ginger CO2 -TO Extract - eye irritation OECD TG 437

The eye irritant properties of Ginger Hot Flavor CO2-TO 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 was 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 of 1.806 ± 0.387 and a mean permeability value of <0.01 compared to the negative control were determined. The substance had an IVIS value of 1.766 ± 0.407, which was below the cut-off value of 3 and consequently the test item is not classified as a eye irritant and is not considered eye corrosive.

Ginger oil Hot Flavor CO2 -SE - 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 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.

WoE discussion - Ginger oil CO2-Total Extract

For skin, a non-irritant effect was observed for Ginger CO2-SE extract and an irritant effect for Ginger oil Hot Flavor CO2-TO extract. As the two fractions combined cover the constituents present in Ginger oil CO2-Total Extract, it can be concluded that the non-volatile Hot Flavor CO2-TO fraction which typically represents 42.22% of the Ginger oil CO2-Total Extract can give rise to skin irritation.

These WoE results are considered appropriate evidence to fulfil the information requirement, and justify the classification of Ginger oil CO2-Total Extract as a Category 2 skin irritant, according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

For eye, no irritant effect was observed for Ginger CO2-SE extract or for Ginger oil Hot Flavor CO2-TO extract. As the two fractions combined cover the constituents present in Ginger oil CO2-Total Extract, it can be concluded that Ginger oil CO2-Total Extract does not give rise to eye irritation. These WoE results are considered appropriate evidence to fulfil the information requirement.  Ginger oil CO2-Total Extract does not need to be classified for eye irritation according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Justification for classification or non-classification

Based on the available information, Ginger oil CO2-Total Extract needs to be classified for skin irritation (Skin Irrit. 2 / H315) but not for eye irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).