Registration Dossier

Administrative data

Description of key information

- Skin irritant Category 2 (OECD 439, K, Rel.1: positive ; OECD 431, K, Rel.1: negative)

- Eye irritant Not classified (OECD 492, GLP, K, Rel.1: negative)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study carried out between 09 February and 21 February 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 431 and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
French GLP Compliance Programme for chemical products (inspected on 30-31 January 2017 / signed on 27 April 2017)
Specific details on test material used for the study:
Test item was identified under the following code number: PH-17/0759
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not precised
Source strain:
other: Not applicable
Details on animal used as source of test system:
Not applicable
Justification for test system used:
Following the REACH bottom-up strategy, as the result of the OECD test guideline No. 439 was positive, the epiCS® Reconstructed Human Epidermis Model method was used to assess skin corrosion as recommended in the OECD test guideline No. 431.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
The test item was applied, as supplied, at the dose of 50 µL to 2 living and 4 killed Human skin model surfaces during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
50 µL of test item
- Concentration (if solution):
Not applicable: undiluted

VEHICLE
not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
50 µL of distilled water
- Concentration (if solution):
Not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
50 µL of potassium hydroxide 8N (8N KOH)
- Concentration (if solution): Not applicable
Duration of treatment / exposure:
3 min at room temperature or 1 hour at 37°C ± 1°C, 5% ± 1% CO2
Duration of post-treatment incubation (if applicable):
not applicable
Number of replicates:
2 tissues per condition
Triplicate measurement of OD570 for each tissue.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min
Value:
73.28
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The mean OD of negative control tissues after 3 minutes exposure was 0.691(between 0.233 and 0.933)
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour
Value:
101.43
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The mean OD of negative control tissues after 3 minutes exposure was 0.771 (between 0.233 and 0.933)
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Table 7.3.1/1: Individual and average values after 3 minutes exposure

 

Skin

OD

Mean OD / disc (#)

Mean OD / product

Viability %

Mean viability %

SD

Negative control

1

0.669

0.688

0.701

0.686

0.691

99.35

100.0

1.3

2

0.715

0.652

0.717

0.695

100.65

Positive control

3

0.025

0.025

0.024

0.025

0.042

3.62

6.08

4.9

4

0.059

0.058

0.059

0.059

8.54

Test item

13

0.418

0.403

0.415

0.412

0.506

59.67

73.28

27.2

14

0.603

0.600

0.596

0.600

86.89

#: mean of 3 values

OD: optical density

Table 7.3.1/2: Individual and average values after 1 hour exposure

 

Skin

OD

Mean OD / disc (#)

Mean OD / product

Viability %

Mean viability %

SD

Negative control

15

0.856

0.834

0.835

0.892

0.771

109.28

100.0

18.6

16

0.710

0.676

0.711

0.699

90.72

Positive control

17

0.004

0.004

0.004

0.004

0.003

0.52

0.39

0.3

18

0.002

0.002

0.002

0.002

0.26

Test item

27

0.661

0.627

0.626

0.638

0.782

82.80

101.43

37.2

28

0.961

0.907

0.906

0.925

120.05

#: mean of 3 values

OD: optical density

Table 7.3.1/3: Conclusion

Mean viability (%)

3 min exposure

1 hour exposure

Conclusion

Positive control

6.08

0.39

Corrosive sub-category 1B/1C

Test item

73.28

101.43

Non Corrosive

Interpretation of results:
other: non-corrosive
Conclusions:
In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item FENUGREEK ABSOLUTE does not have to be
classified in Category 1 “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.
Executive summary:

The aim of the study was to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®).

The test item FENUGREEK ABSOLUTE was applied as supplied, at the dose of 50 μL, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour. The application was

followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from

tissues. The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 431 dated 28 July 2015 and the method B.40bis of the Council regulation No. 440/2008.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 73.28% and 101.43% (considered as 100%) versus 6.03% and

0.39%, respectively; with the positive control item (potassium hydroxide 8N).

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item FENUGREEK ABSOLUTE does not have to be

classified in Category 1 “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Used as supplied
Test system:
human skin model
Source species:
other: reconstructed epidermises
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- 0.50 cm² reconstructed epidermis (Episkin SA, RHE/S/17) were received, and on the same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 1 mL of growth medium (Episkin SA) during 2 hours and 25 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of maintenance medium (Episkin SA).

TREATMENT
- The test item was applied as supplied, at the approximate dose of 16 µL, on the epidermal surface of 3 living human skin models during 42 minutes at room temperature.
- In the same experimental conditions, a positive control (5% SDS) and a negative control (DPBS) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment. To ensure a good contact with the epidermises, during all the treatment period, the test item was recovered with a nylon mesh provided by Episkin SA.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, the human epidermises were washed with 25 x 1 mL of DPBS. The rinsed tissues were checked for any coloration and noted to be slightly brown instead of being whitish as for the coloration of the negative control tissues. Residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse. They were incubated for a 42 hours and 05 minutes post-treatment incubation period in fresh medium at 37°C, 5% CO2. Then, the epidermises were put in contact with the MTT solution.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The cell viability was quantified by the measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD were proportional to the number of living cells.
- The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was measured by determining the OD (Optical Density) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).
- The OD of MTT extract was measured in triplicate. The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

VIABILITY CALCULATION:
- The results were expressed as a viability percentage compared with the negative control: viability % = (mean OD test item / mean OD negative control) * 100
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table 7.3.1/1.

PREDICTION MODEL / DECISION CRITERIA
The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:
- The test item is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is >50%.
- The test item is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is "non-corrosive". In accordance with Regulation EC No. 1272/2008, the test item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.
- The test item is considered to be irritant or corrosive to skin if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤50% and in absence of information on a skin corrosion test. In accordance with the Regulation (CE) No.1272/2008 and in absence of information on a skin corrosion test, the item has to be classified in Category 2 "Irritant" or in Category 1 "Corrosive". The corresponding hazard statement is respectively, "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger".
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 µL
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
42 minutes at room temperature
Duration of post-treatment incubation (if applicable):
42 hours post-incubation period at 37°C, 5% CO2
Number of replicates:
3 living human skin models
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
main test (duration 42 minutes
Value:
16.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The mean percent viability of the treated tissues was 16.1%, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate ).

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

 

Skin

 

OD

Mean OD / disc(#)

Mean OD / product

Viability

%

Meanviability

%

SD

viability

 

Conclusion

 

 

0.973

 

 

 

 

 

1.002

 

 

 

 

 

100.0

 

 

 

 

5.3

 

 

1

1.074

1.047

104.5

 

 

1.095

 

 

Negative

 

2

0.938

1.047

1.064

 

1.016

 

101.4

control

 

 

3

0.873

0.965

0.991

 

0.943

 

94.1

 

 

0.009

 

 

 

 

 

0.010

 

 

 

 

 

1.0

 

 

 

 

0.1

 

 

 

 

Irritant

 

4

0.011

0.010

1.0

 

 

0.011

 

 

Positive

 

5

0.010

0.012

0.012

 

0.011

 

1.1

control

 

 

6

0.010

0.010

0.010

 

0.010

 

1.0

 

 

0.145

 

 

 

 

 

0.162

 

 

 

 

 

16.1

 

 

 

 

0.8

 

 

 

 

Irritant

 

19

0.162

0.156

15.6

 

 

0.163

 

 

Test item

 

20

0.159

0.169

0.174

 

0.167

 

16.7

PH-17/0759

 

 

21

0.976

0.973

1.028

 

0.992*

 

-

#: mean of 3 values (triplicate of the same extract) OD: optical density

*: aberrant value, not taken into account for the calculation of cell viability.

 

Acceptability criteria:

·     SD ≤18%

·     Negative control: OD value of the 3 replicates in the range ≥ 0.8 and ≤3.0.

The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range ≥ 0.4 and ≤1.5 for the negative control.

 

Acceptability criteria:

The standard deviation between the viability of the three tissues treated with the test item was 47.9% instead of18% at the maximum as initially scheduled.

 Reason:

One epidermis presented with a mean OD at 0.992 (aberrant value, not taken into account for the calculation of cell viability) versus 0.156 and 0.167 for the two other epidermises.

 Considering the results obtained, this deviation is considered as without impact on the conclusion of the study.

Note

If the viability obtained for the test item is greater than 50%, the test item has to be considered as non-irritant.

If the viability obtained for the test item is less than or equal to 50% and the result of skin corrosion test is non-corrosive, the test item has to be considered as irritant.

 If the viability obtained for the test item is less than or equal to 50% and in absence of information on skin corrosion, the test item has to be considered as corrosive or irritant.

Interpretation of results:
other: Category 2 (irritating to skin) or Category 1 (corrosive) based on GHS criteria
Conclusions:
Under the test conditions and in accordance with Regulation EC No. 1272/2008 and in absence of information on skin corrosion, the test item has to be classified in Category 2 "Irritating to skin" or in Category 1 "Corrosive". The hazard statement "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger" are required.
Executive summary:

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE®model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.

 

The test item was applied as supplied, at the dose of 16 μL, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of PBS and a 42 hours post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

 

The mean percent viability of the treated tissues was16.1 %, versus 1.0 % in the positive control (5% Sodium Dodecyl Sulfate).

 

Under the test conditions and in accordance with Regulation EC No. 1272/2008 and in absence of information on skin corrosion, the test item has to be classified in Category 2 "Irritating to skin" or in Category 1 "Corrosive". The hazard statement "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger" are required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
January - March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Species:
other: Reconstructed human Cornea-like Epithelia
Details on test animals or tissues and environmental conditions:
- Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation]
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Concentration: Undiluted 50 µL
Duration of treatment / exposure:
30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)
Duration of post- treatment incubation (in vitro):
- Post-exposure immersion period: 12 minutes at room temperature
- Post-exposure incubation period: 2 hours at standard culture conditions
Number of animals or in vitro replicates:
Test item, negative and positive controls were applied on duplicate tissues.
Details on study design:
MAIN TEST
- Pre-incubation of the tissues:
On the day of receipt, the tissues in their well shipping container were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 20 hours and 34 minutes at standard culture conditions.
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
- Treatment and post-treatment incubation of the tissues:
The test item was applied, as supplied, at the dose of 50 μL, to 2 living PBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). In the same experimental conditions, a positive control (Methyl acetate), and a negative control (distilled water) were used. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 viable RhCE tissue replicates during 30 minutes at standard culture conditions.
After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS. The rinsed tissues were checked for any colouration and noted for comparable colour with the negative control. This rinsing step was followed by a 12-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. The RhCE constructs were then incubated for a 2-hour post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.
- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD was proportional to the number of living cells.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 17 hours and 40 minutes at 6±3°C in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2). The OD measurement of formazan extracts at 570 nm was measured in triplicate samples using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
Irritation parameter:
other: % tissue viability
Value:
96.49
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The mean percent tissue viability of the RhCE replicates treated with test item was 96.49% versus 17.72% in the positive control (Methyl acetate).

Table 7.3.2/1: Main test - Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

Tissue

OD

Mean OD/disc (#)

Mean OD/product

Viability %

Mean viability %

Difference of viability %

Negative control

1

1.128

1.054

1.041

101.25

100.00

2.50

1.062

0.971

2

1.018

1.028

98.75

1.054

1.012

Positive control

3

0.204

0.193

0.185

18.54

17.72

1.63

0.184

0.189

4

0.172

0.176

16.91

0.175

0.180

Test item

9

1.178

1.061

1.005

101.92

96.49

10.85

1.025

0.979

10

0.986

0.948

91.07

0.953

0.904

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions adopted and in accordance with Regulation EC No. 1272/2008, test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.


Executive summary:

Test item was applied, as supplied, at the dose of 50 µL, to 2 living DPBS pre-treated RhCE (EpiOcularTMtissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

The experimental protocol was established in accordance with O.E.C.D. Test Guideline No. 492 adopted 09 October 2017.

 

The mean percent tissue viability of the RhCE replicates treated with test item was 96.49% versus 17.72% in the positive control (Methyl acetate).

 

 In conclusion, under the experimental conditions adopted and in accordance with Regulation EC No. 1272/2008, test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.

No hazard statement andthe signal word are required.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE®model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.

 

The test item was applied as supplied, at the dose of 16 μL, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of PBS and a 42 hours post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

 

The mean percent viability of the treated tissues was16.1 %, versus 1.0 % in the positive control (5% Sodium Dodecyl Sulfate).

 

Skin corrosion

The test item FENUGREEK ABSOLUTE was applied as supplied, at the dose of 50 μL, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour. The application was

followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from

tissues. The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 431 dated 28 July 2015 and the method B.40bis of the Council regulation No. 440/2008.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 73.28% and 101.43% (considered as 100%) versus 6.03% and

0.39%, respectively; with the positive control item (potassium hydroxide 8N).

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item FENUGREEK ABSOLUTE does not have to be

classified in Category 1 “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.

Skin irritation/corrosion conclusion: the substance is therefore classified as skin irritation 2( H315) as it has been shown to be irritant but not

Eye irritation:

Test item was applied, as supplied, at the dose of 50 µL, to 2 living DPBS pre-treated RhCE (EpiOcularTMtissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 2 hours post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay.

The experimental protocol was established in accordance withO.E.C.D. Test Guideline No. 492 adopted 09 October 2017.

 

 The mean percent tissue viability of the RhCE replicates treated with test item was96.49%versus 17.72% in the positive control (Methyl acetate).

 

 In conclusion, under the experimental conditions adopted and in accordance with Regulation EC No. 1272/2008, test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.

No hazard statement andthe signal word are required.

Justification for classification or non-classification