Ammonium glycyrrhizate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

yes

Remarks:

The stock solution 100mM was prepared using the percentage of purity equal to 98.7% instead of 99.6%. The maximal concentration tested was therefore 100.9mM instead of 100mM, however, this difference does not affect the study result.

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

According to the column 2 of of the appendix VII of the Regulation EC 1907/2006, in vivo studies are not required if at least one or two in vitro study has been carried out and allow a classification for the substance.

Details on the study design:

The test item was prepared at 100 mM in water and the positive control was prepared at 100 mM in acetonitrile.
They were incubated in excess with the peptides at 1:10 and 1:50 ratio for cysteine and lysine peptides respectively.
Each sample was tested 3 times from 3 independent solutions.

*Preparation of the test item and positive control

Test item
According to the study plan, solubility of the test item in Acetonitrile was checked before the main test.
The test item being non soluble at 100 mM in acetonitrile, other suitable diluents have been tested given the order defined in the study plan. The chosen solvent was water.

The weight of test item was calculated according to the following formula:
"Weight (g) =" "0.01 × Vf × MW" /"Purity"
where MW = 840 g/mol and purity = 98.7%.
255 mg was pre-weighted in a glass vial in order to prepare, right for use, 3 ml of a limpid 100 mM solution.

Positive control
The volume of the positive control was calculated according to the following formula:
"Volume (ml) =" "0.01 × fV × MW" /"Purity × ¿"
MW= 132.16 g/mol
Purity = 99.1%
Density ¿ = 1.048
fV (final volume) = 3 ml
38.2 µl of the positive control were distributed in a 5 ml glass vial in order to prepare, right before use, 3 ml of a limpid 100 mM solution.
* Preparation of the peptide solution

Peptide solutions were prepared at 0.667 mM:
• 13.9 mg of cysteine peptide were pre-weighted then dissolved, right before the incubation, in 27.8 ml of phosphate buffer (100 mM; pH 7.5 ± 0.05).
• 14.5 mg of lysine peptide were pre-weighted then dissolved, right before the incubation, in 28 ml of ammonium acetate buffer (100 mM; pH 10.2 ± 0.05).

* Preparation of the sample for the test

Samples were dissolved immediately before use (100 mM positive control solution was kept for the 2 runs). Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively. All the replicates were prepared with the same peptide stock solutions.
The vials were capped and mixed carefully avoiding bubbling, then placed in the HPLC system sampler at 25°C ¿ 2.5°C. HPLC analysis started 24 hours ± 2 hours after addition of peptides.
Immediately upon addition of the test item solution to the peptide solution, just prior the beginning of the HPLC analysis and at the end of the analysis, samples vials were checked.

* Preparation of calibration curve

A calibration curve was generated for cysteine and lysine peptides. Peptides standards were prepared in 20% acetonitrile in buffer solution (phosphate buffer for cysteine peptide and ammonium acetate for lysine peptide).
Six standard solutions between 0.534 mM and 0.0167 mM (dilution factor 2) were prepared from the stock solution (0.667 mM) by serial dilution. The dilution buffer was also included as blank in the standard calibration curve.

* Preparation and manipulation of the HPLC system
The HPLC system was used according to the current working instruction IL MAT 24.
According to the guideline, the set-up parameters were adjusted to guarantee an appropriate elution and integration of the cysteine and lysine peptides.
Proficiency substances recommended in the OECD guideline were performed in these conditions. Results are shown at the end of the report.
The HPLC column was installed and equilibrated at 30°C with 50% of phase A and 50% of phase B, for at least 20 minutes before use. The gradient (cf. § 7.8) was performed at least one time before to use the column.

* HPLC analysis
A complete sequence was performed for each sample.
The column was re-equilibrated to initial conditions (90% Phase A and 10% of phase B) at least 4 minutes between each injection.

* Sequence of the analysis

The analysis was programmed according to the following principles:
• The reference controls B were placed at the beginning and at the end of the analysis (3 repetitions).
• The reference controls C were placed at the beginning of each repetition.
• The standards of the calibration curve and the reference controls A were placed in order to be analyzed progressively throughout the sequence.

Lysine and cysteine analysis were conducted on separate day and test item was freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample starts 22 to 26 hours after the test item was mixed with the peptide solution. The HPLC analysis time was less than 30 hours

Key result

Parameter:

other: Depletion in lysine peptide (%)

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

(55.31%)

Key result

Parameter:

other: depletion in cysteine peptide (%)

Value:

5.66

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

(73.06%)

Interpretation of results:

other: Based on a part of integrated approach, these results support a non-classification.

Conclusions:

The sensitivity of the test item is determined by calculating the mean percentage of depletion of Lysine and Cysteine.
The test item, GLYCYRRHIZATE MONOAMMONIACAL, shows mean depletion of 0% for Lysine and 5.66% for Cysteine, i.e. an overall average of 2.83% reflecting no or minimal reactivity and therefore a negative prediction of DPRA.

Based on OECD 442C, GLP study, results are considered scientifically valid to be used as part of an integrated approach to support a non classification.

Please refer to endpoint summary for conclusion.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

yes

Remarks:

The stock solution dilution was prepared 4X in treatment medium-4% DMSO instead of 100X. This has no impact on the study result because the test item was tested in the expected final condition (i.e 2000 µM as maximal concentration).

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

According to the column 2 of of the appendix VII of the Regulation EC 1907/2006, in vivo studies are not required if at least one or two in vitro study has been carried out and allow a classification for the substance.

Specific details on test material used for the study:

Storage conditions: Darkness + room temperature + hygroscopic
Color : White
Physical state at 20°C : Solid
Purity : 98.7%
Homogeneity : Yes
Molecular weight : 840 g/mol
pH : 4.2-4.8 (solution at 1% in ethanol 50%)

Details on the study design:

Cells suspension are adjusted to a density of 8.104 cells/ml in seeding medium.
125 µl of the cell suspension at 8.104 cells/ml (i.e. 104 cells per well) were distributed in three white plates for the induction measurement and two transparent plates to assess the cytotoxicity. The seeded plates were incubated 24 hours ± 1 hour at 37°C, 5% CO2.

The second day, in the 5 seeded plates, the medium was aspirated and replaced with 150 µl of treatment medium. Then the 4 X plate was replicated 5 times: 50 µl from the 4 X plate were placed in each of the three white plates and in the two transparent plates. The plates (1 X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37°C, 5% CO2).

After 48 hours, the luciferase activity was measured with an integration time of 2 seconds.

Viability was assessed measuring absorbance at 540 nm.

Positive control results:

Positive control : cinnamaldehyde
Mean Imax : 3.94

Key result

Parameter:

other: Mean Imax

Value:

1.28

Vehicle controls validity:

valid

Negative controls validity:

other: negative control is also vehicle control

Positive controls validity:

valid

Interpretation of results:

other: Based on a part of integrated approach, these results support a non-classification.

Conclusions:

Based on OECD 442D, GLP study, results are considered scientifically valid to be used as part of an integrated approach to support a non classification.

Please refer to endpoint summary for conclusion.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

Skin sensitisation

According to the column 2 of the appendix VII of the Regulation EC 1907/2006, in vivo studies are not required if at least one or two in vitro study has been carried out and allow a classification for the substance. Therefore, one in chemico study DPRA assay and one in vitro study Keratinosens assay address have been conducted according to OECD 442 C and 442 D, respectively. Studies were carried out on GLYCYRRHIZATE MONOAMMONIACAL and both of them provided negative results.

Based on a part of integrated approach, these results support a non-classification, so, it can be concluded GLYCYRRHIZATE MONOAMMONIACAL is not a skin sensitiser.