Fenugreek, ext.

Skin irritation / corrosion

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Fenugreek, ext.

EC Number:

283-415-1

EC Name:

Fenugreek, ext.

Cas Number:

84625-40-1

Molecular formula:

Not applicable (UVCB)

IUPAC Name:

Absolute of Trigonella foenum graecum L. (Leguminosae) obtained from seeds by organic solvent treatment and subsequent ethanol extraction

Test material form:

liquid: viscous

Remarks:

Brown-yellow viscous liquid

Details on test material:

Test item: Fenugreek Absolute
Batch No.: 2709379
Identity: 100 % UVCB Substance
Appearance: Brown-yellow viscous liquid
Storage conditions: Ambient temperature (10 °C to 30 °C), in original aluminium bottle, dark
Expiry date: October 24, 2018

Specific details on test material used for the study:

Test item was identified under the following code number: PH-17/0759

In vitro test system

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.

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.