Ammonium glycyrrhizate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Principles of method if other than guideline:

AMES test was conducted in two independent experiments, plate incorporation test and preincubation test, each carried out without and with metabolic activation (a microsomal preparation derived from
Aroclor 1254-induced rat liver).
The genotypes of the test strains were confirmed as histidine and biotin requirement, (rfa-) deep rough character, UV-sensitivity, and ampicillin-resistance.

For the plate incorporation experiments, the test components were prepared as follows : 0.1 mL of Salmonella cell suspension containing approximately 1x108 viable cells in the late exponential or early stationary phase, 0.1 mL of MGL test solution, or positive control solvent or solvent alone, and 0.5 mL of S9 mix or phosphate buffer were added into 2 mL of soft agar containing 50 mM L-histidine HCl and 50 mM biotin held at 45°C.
The test components were mixed by vortexing the soft agar for 3 seconds at low speed and then poured onto a coded minimal glucose agar plate.

In the preincubation experiments, the test solution of Monoammonium Glycyrrhizinate was preincubated with the test strain solution containing approximately 1x108 viable cells and sterile buffer or the metabolic activation system for 20 minutes at 37°C prior to mixing the overlay agar and poured onto the surface of a coded minimal agar plate.
After 48 to 72 hours incubation of inverted plates in a dark 37°C incubator, the revertant colonies on the test plates and on the control plates were counted, and the presence of the background lawn on all plates was confirmed. Triplicate plates were examined at each concentration level and strain.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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

Specific details on test material used for the study:

The appearance and properties were a white yellowish-white odorless powder with mp=209°C. MGL was dissolved in 0.9% NaCl solution or dimethyl sulfoxide(DMSO).

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (post-mitochondrial fraction) was prepared from rats treated with Aroclor 1254, collected and stored in liquid nitrogen. S9 mix (5% S9) was freshly prepared on the day of the test.

Test concentrations with justification for top dose:

As no signs of cytotoxicity were noted at up to the top concentration of 5000µg/plate in the preliminary test without metabolic activation in test strain TA 100 employing a plate incorporation test, five concentrations separated by a half-log interval ranging from 100 to 5000µg/plate were employed in the independent experiments without and with metabolic activation.

Vehicle / solvent:

MGL was dissolved in 0.9% NaCl solution or dimethyl sulfoxide (DMSO).

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

methylmethanesulfonate

other: 2-amino-anthracene (with metabolic activation, for TA 98, TA 102, and TA 1537 strains)

Details on test system and experimental conditions:

AMES test was conducted in two independent experiments, plate incorporation test and preincubation test, each carried out without and with metabolic activation (a microsomal preparation derived from
Aroclor 1254-induced rat liver).
The genotypes of the test strains were confirmed as histidine and biotin requirement, (rfa-) deep rough character, UV-sensitivity, and ampicillin-resistance.

For the plate incorporation experiments, the test components were prepared as follows : 0.1 mL of Salmonella cell suspension containing approximately 1x108 viable cells in the late exponential or early stationary phase, 0.1 mL of MGL test solution, or positive control solvent or solvent alone, and 0.5 mL of S9 mix or phosphate buffer were added into 2 mL of soft agar containing 50 mM L-histidine HCl and 50 mM biotin held at 45°C.
The test components were mixed by vortexing the soft agar for 3 seconds at low speed and then poured onto a coded minimal glucose agar plate.

In the preincubation experiments, the test solution of MGL was preincubated with the test strain solution containing approximately 1x108 viable cells and sterile buffer or the metabolic activation system for 20 minutes at 37°C prior to mixing the overlay agar and poured onto the surface of a coded minimal agar plate.
After 48 to 72 hours incubation of inverted plates in a dark 37°C incubator, the revertant colonies on the test plates and on the control plates were counted, and the presence of the background lawn on all plates was confirmed. Triplicate plates were examined at each concentration level and strain.

Evaluation criteria:

Since the statistical evaluation of the results of the AMES test is still under discussion, and the range of spontaneous reversion frequencies of TA 98, 100, 102, 1535, and 1537 in our test circumstance are generally 20- 60, 100-200, 240-320, 10-35, and 3-20, respectively, the test item would be considered to show a positive response if the results were satisfied with the all following criteria : i) if the number of revertants would be significantly increased at p< 0.05 by U-test according to MANN and WHITNEY compared with the solvent control,
to at least 2-fold of the solvent control for TA 98, TA 100 and TA 102, and 3-
fold of the solvent control for TA 1535 and TA 1537 in both independent experiments, ii) if a significant concentration(log value) - related effect at p< 0.05 by Spearman’s rank correlation coefficient would be observed, and iii) if positive results would have to be reproducible and the histidine independence of the revertants would have to be confirmed by streaking random samples on histidine-free agar plates. Cytotoxicity was defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.

Statistics:

Since the statistical evaluation of the results of the AMES test is still under discussion, and the range of spontaneous reversion frequencies of TA 98, 100, 102, 1535, and 1537 in our test circumstance are generally 20- 60, 100-200, 240-320, 10-35, and 3-20, respectively, the test item would be considered to show a positive response if the results were satisfied with the all following criteria : i) if the number of revertants would be significantly increased at p< 0.05 by U-test according to MANN and WHITNEY compared with the solvent control,
to at least 2-fold of the solvent control for TA 98, TA 100 and TA 102, and 3-
fold of the solvent control for TA 1535 and TA 1537 in both independent experiments, ii) if a significant concentration(log value) - related effect at p< 0.05 by Spearman’s rank correlation coefficient would be observed, and iii) if positive results would have to be reproducible and the histidine independence of the revertants would have to be confirmed by streaking random samples on histidine-free agar plates. Cytotoxicity was defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.

Results and discussion

Test results

Key result

Species / strain:

other: All strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

other: Solvent control is used as negative control

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The effect of MGL on the bacterial reverse mutation activity using 5 Salmonella typhimurium stains TA 98, TA 100, TA 102, TA 1535 and TA 1537 has been addressed in two independent experiments, each carried out without and with metabolic activation by a rat post-mitochondorial fraction(S9 mix) from Aroclor 1254 induced animals. The first experiment was carried out as a plate incorporation and the second as a preincubation test.
In the result of the preliminary cytotoxicity test, no signs of cytotoxicity were noted in the plate incorporation test of TA 100 without metabolic activation at up to the top concentration of 5000 µg/plate. Hence, 5000 µg/ plate was chosen as the top concentration for the main study. While there were no signs of cytotoxicity noted in the plate incorporation test without and with metabolic activation and in the preincubation test with metabolic activation, cyototoxicity was observed in the preincubation test without metabolic activation at the top concentration of 5000 µg/plate in all test strains. On the other hand, no mutagenic effect was observed for MGL tested at up to 5000 µg/plate in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively. Consequently, under the present test conditions MGL tested at up to a cytotoxic concentration caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535, and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic
activation.

Under tests conditions of GLP study, results are considered scientifically valid to conclude on absence of mutagenic activity on prokaryotic cells.