Ammonium glycyrrhizate

Eye irritation

Administrative data

Endpoint:

eye 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 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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 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

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The 0.60 cm2 Reconstructed human Cornea-like Epithelia were received on 17 october 2017. The same day, 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 filyer. They were places in 6 wells culture plate with which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 19 hours and 20 minutes at standard culture conditions.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours and 15 minutes

Number of animals or in vitro replicates:

2 replicates (RhCE)

Details on study design:

The test item was applied on a nylon mesh at an approximate dose of 50mg to the entire surface of 2 living RhCE tissues replicate during 6 hours at standard culture conditions.

In the same experimental conditions, a positive control (Methyl acetate) and a negative control (distilled water) were carried out. The controls were applied, as supplied, at the dose of 50µL, to the surface of 2 RhCE tissue replicates during 6 hours 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 coloration and noted to be of comparable colour with the negative control treated tissues (whitish). The rinsing step was followed by a 25-minutes-post-exposure immersion period at room temperature in 5mL of fresh medium to remove any test item absorbed into the tissue.

The RhCE constructs were then incubated for an 18 hours and 15 minutes post-exposure incubation at standard culture conditions in 1mL of fresh medium at 37 °C, 5% CO2.

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 ad appearance of clear cytotoxic effects. The RhCE tissue viability was measured by enzymatic covnersion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues. The RhCE constructs were places in 300µL of a MTT solution at 1.0 mg/ml for 3 hours and 55 minutes at standard culture conditions. The precipitated blue formazan products was then extracted from the tissues by placing each insert in 2mL of isopropanol during 2 hours and 08 minutes at room temperature in the dark.
The concentration of formazan was measured by determining the optical density at 570 nm, just after dilution of the extraction in isopropanol (1:2)
The optical density was measured in triplicate samples of formazan extracts. The measured optical density are proportional to the number of living cells. The measurment was performed using the ELx800 absorbance microplate reader.

Results and discussion

In vitro

Results

Irritation parameter:

other: Viability %

Value:

12.32

Negative controls validity:

valid

Remarks:

100.00%

Positive controls validity:

valid

Remarks:

19.83%

Applicant's summary and conclusion

Interpretation of results:

other: Based on a part of integrated approach, these results support a classification in Category 2 or Category 1 of GHS.

Conclusions:

Based on OECD 492 (2015), GLP study, results are considered scientifically valid to be used as part of an integrated approach to support a classification in Category 2 or Category 1 according to GHS and CLP regulation.

Refer to endpoint summary for final conclusion.