Extract from the seeds of Trigonella foenum-graecum (Fabaceae) obtained by extraction with polar solvents

Endpoint summary

Description of key information

Not skin and eye irritating (not classified).

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:

27 November 2019 - 20 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification/Synonym FENUGREEK EXTRACT
Substance type UVCB substance
EC No. 950-727-0
Storage conditions room temperature, protected from light

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

not specified

Details on animal used as source of test system:

Multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and a multilayered stratum corneum

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
EPISKIN™ - 0.38 cm2
- Quality control for skin discs: Histology scoring, magnitude of viability and barrier function (IC50 determination).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C, 5 %CO2
- Temperature of post-treatment incubation: 37°C, 5 %CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: 25mL of sterile D-PBS

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength: 595 nm

Study Acceptability Criteria
The assay was considered valid if the following criteria were met:
– Blank controls: mean OD value < 0.1.
– Negative controls: mean OD value ≥ 0.6 and ≤ 1.5, SD of % viability ≤ 18.
– Positive controls: mean viability expressed as percentage of the negative control
≤ 40% and SD of % viability ≤ 18.
– Test item: SD of % viability ≤ 18

Criteria Classification
Mean relative viability ≤ 50% UN GHS Category 2 or 1
Mean relative viability > 50% UN GHS No Category (for member states that do not adopt optional Category 3)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 ± 2mg

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied: 20 µL
- Concentration: 5% D-PBS

POSITIVE CONTROL
- Amount(s) applied: 20 µL
- Concentration: 5% (w/v) SDS

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

Recovery 42 ± 1 hour
MTT staining 3 hours

Number of replicates:

Live tissue 3 per conditions
Killed tissue at least 2 per conditions

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Live Tissue

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Killed Tissue

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Study Acceptability Criteria
The assay was considered valid if the following criteria were met:
– Blank controls: mean OD value < 0.1.
– Negative controls: mean OD value ≥ 0.6 and ≤ 1.5, SD of % viability ≤ 18.
– Positive controls: mean viability expressed as percentage of the negative control
≤ 40% and SD of % viability ≤ 18.
– Test item: SD of % viability ≤ 18

Criteria Classification
Mean relative viability ≤ 50% UN GHS Category 2 or 1
Mean relative viability > 50% UN GHS No Category (for member states that do not adopt optional Category 3)

Interpretation of results:

GHS criteria not met

Conclusions:

The mean cell viability of the test item treated tissues, after the blank subtraction, was 94%. Based on the results obtained, the test item is classified as non-irritant to the skin (UN GHS No Category).

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:

25 November 2019 - 11 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification/Synonym FENUGREEK EXTRACT
Substance type UVCB substance
EC No. 950-727-0
Recommended storage Ambient (18-20°C) and protected from temperatures below 4°C; dry (< 70% rh);
conditions to be stored under dark conditions

Species:

chicken

Strain:

other: Ross 308

Details on test animals or tissues and environmental conditions:

Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens. The heads were transported at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3ºC to 20.2ºC) during the transport. After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).

Amount / concentration applied:

Used as such, whole surface of eye covered

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

3 samples
3 positive controls (acetid acid)
1 negative control ( NaCl solution)

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
A Drop of fluorescein solution was applied onto the cornea surface and rinsed off with isotonic saline. Checking of the integrity of the cornea, removing of the eyeball. Unnecessary material was removed from the eye. Eye was fixed with steel clamp in vertical position.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Changes in thickness were not observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment.

NUMBER OF REPLICATES
3 samples

NEGATIVE CONTROL USED
1 negative control ( 30 µL saline solution)

POSITIVE CONTROL USED
3 positive controls (30 µL 10 % w/w acetid acid)

APPLICATION DOSE AND EXPOSURE TIME
undiluted
10 seconds

OBSERVATION PERIOD
30, 75, 120, 180 and 240 minutes

REMOVAL OF TEST SUBSTANCE
Cornea surface was rinsed thoroughly with saline solution at ambient temperature.

Irritation parameter:

percent corneal swelling

Run / experiment:

75 min

Value:

2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

240 min

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Value:

0.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Value:

0.2

Other effects / acceptance of results:

Tables of ICE classification, see tables attached below.

Interpretation of results:

GHS criteria not met

Conclusions:

The overall ICE score was 3xI. According to the guideline OECD 438, Test Substance is categorized as “No Category”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)