Ammonium glycyrrhizate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

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)

Test material

Test material information

1

Reference substance name:

Ammonium glycyrrhizate

EC Number:

258-887-7

EC Name:

Ammonium glycyrrhizate

Cas Number:

53956-04-0

Molecular formula:

C42H65NO16

IUPAC Name:

ammonium glycyrrhizate

In vitro test system

Test system:

isolated skin discs

Source species:

human

Cell type:

other: Reconstructed Human Epidermis

Cell source:

other: foreskin

Source strain:

other: not applicable

Vehicle:

water

Remarks:

distilled

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin SA, RHE
- Tissue batch number(s): 17-RHE-088
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 23/08/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 42 minutes after the test item application, the human epidermis were washed with 25 x 1 mL of DPBS (Dutscher - Batch No. 9130617). The rinsed tissues were checked for any coloration and noted to be
whitish, comparable to negative control tissues. They were incubated for a 42 hours post-treatment incubation-period in fresh medium at 37°C, 5% CO2.
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800
- Wavelength: 570 nm
- Filter: No filter was used for the measurement of OD
- Linear OD range of spectrophotometer: between 0 and 2.0

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE (from certificate of analysis)
- Viability: O.D. = 1.4
- Barrier function: 4.3 h
- Morphology: absence of significant histological abnormalities
- Contamination: the product was prepared and packaged using aseptic techniques
- Reproducibility: not specified

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg of test item on 0.50 cm2 human skin model equal to 32 mg/cm2
The test item was applied, as supplied, during 42 minutes at room temperature at the dose of 16 mg to the epidermal surface of 3 living Reconstructed Human epidermis previously moistened with 10 µL of distilled water.
42 minutes after the test item application, the human epidermis were washed with 25 x 1 mL of DPBS (Dutscher - Batch No. 9130617). The rinsed tissues were checked for any coloration and noted to be whitish, comparable to negative control tissues.

NEGATIVE CONTROL - DPBS
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution):

POSITIVE CONTROL - SDS
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 0.5 g in 10 mL equal to 50 g/L

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

They were incubated for a 42 hours post-treatment incubation-period in fresh medium at 37°C, 5% CO2. Then, the epidermis were put in contact with the MTT solution.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean viability based on 3 replicates

Value:

ca. 80.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

viability 1.2%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: The direct interaction of MTT with the test item was checked by adding 16 mg of the test item to 300 µL of solution of MTT at 1 mg/mL. A cloudy yellow solution was observed after 3 hours of incubation between 36.2°C and 37.4°C, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Coloration potential and spectral analysis of the test item:
In water, the coloration potential of the test item in water was checked by adding approximatively 16 mg of the test item to 300 µL of distilled water. A colorless solution with the test item at the bottom of the well was obtained after 3 hours of incubation between 36.2°C and 37.4°C, 5% CO2.
In isopropanol, the coloration potential of the test item in isopropanol was checked by adding approximatively 16 mg of the test item to 1.5 mL of isopropanol. A colorless solution was obtained after 2 hours of incubation at room temperature.
No significant coloration appeared. Therefore the test item will not interfere with the MTT assay.

DEMONSTRATION OF TECHNICAL PROFICIENCY: a summary of proficiency chemicals testing has been provided in appendix of the report between 2009 and 2016, results observed are in line with those waited.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes; in line with historical control
- Acceptance criteria met for positive control: yes; in line with historical control
- Acceptance criteria met for variability between replicate measurements: yes; SD = 4.5 below 18

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under test conditions of OECD 439 (guideline of 28 july 2015) GLP study, test item is not considered as skin irritant with respect to CLP regulation.