O-6-deoxy-α-L-mannopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl (2α,3β,4α)-2,3,23-trihydroxyurs-12-en-28-oate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25/05/2011 - 17/06/2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

O-6-deoxy-α-L-mannopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl (2α,3β,4α)-2,3,23-trihydroxyurs-12-en-28-oate

EC Number:

240-851-7

EC Name:

O-6-deoxy-α-L-mannopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl (2α,3β,4α)-2,3,23-trihydroxyurs-12-en-28-oate

Cas Number:

16830-15-2

Molecular formula:

C48H78O19

IUPAC Name:

[6-[[3,4-dihydroxy-6-(hydroxymethyl)-5-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxymethyl]-3,4,5-trihydroxyoxan-2-yl] 10,11-dihydroxy-9-(hydroxymethyl)-1,2,6a,6b,9,12a-hexamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylate

Test material form:

solid

Details on test material:

Name: ASIATICOSIDE
IDEA code: ID-11/01446
Batch number: 337 B
Aspect: Powder
Color: White
Storage conditions: Room temperature
Test item nature: Cosmetic ingredient
Expiry date: 05/11/2012

Method

Target gene:

His D, His G, His C

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The choice of strains is made according to OCDE Guideline. It is five strains of Salmonella typhimurium LT2.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The solubility test did not show any insolubility of the test item. Therefore, the maximal concentration retained is 5000 μg/plate.
According to OCDE guideline, 5 concentrations of the test item have been studied with approximately half log interval, according to OCDE Guidelines. These doses (rounded to higher value) used for the preliminary cytotoxicity test are therefore the following: 5000, 1600, 500, 160 and 50 μg/plate.
As the preliminary experiment didn't reveal cytotoxicity of the test item, this range of concentrations has been conserved for the test 1.
According to the results obtained in the test 1, the Study Director decided to maintain range of concentrations for Test 2.
Each test dilution and each reference item are tested on 3 Petri plate.

Vehicle / solvent:

The most commonly used solvent are deionized water for the analyze and the dimethylsulfoxide (DMSO), or any appropriate solvent compatible with the test system and the test item or other solvent can be used at the request of the Sponsor if they are known or if it has been demonstrated that they are not cytotoxic nor genotoxic. The compatibility with the test item is therefore under the Sponsor responsability.
A preliminary dissolution of the test item is prepared in DMSO (DMSO final concentration 3.85%).
The other tested solutions will be obtained by serial dilution from this stock solution. These solutions are prepared extemporaneously each day of manipulation.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: amino-2-anthracène

Details on test system and experimental conditions:

Media and growth conditions:
For each experiment, the test strains cultures are prepared in nutrient broth from frozen stocks and incubated at 37°C+/-1°C on shaken platter to allow the culture to grow up to the late exponential or early stationary phase of growth (approximately 10^8-10^9 cells/ml). The optical density of each culture will be used to check the cell density. Each strain of the test system is tested in triplicate with and without metabolic activation system.
Microbial suspension is put in contact with the test item or positive controls, mixed with top agar and poured over minimal agar medium plate. After, solidification, plates are incubated at 37°C +/- 1 °C during 48 to 72 hours. Positive and negative controls are included in the experiment.

Evaluation criteria:

Acceptance criteria of data:
The test is considered valid if the following criteria are fulfilled:
-the sterility tests are conform
-the mean negative controls are within the historical data
-the solvent used (negative control) must not show genotoxic activity
-the revertants rate obtained for the positive controls must be in agreement with the historical data
-the positive controls must show a revertants number equal at least to the double of the spontaneous rate of reversion for TA98, TA100 and TA102 and the triple of the spontaneous rate of reversion for TA1535 and TA1537
-no more than 5% of the plates of the test are lost through contamination or any other unforeseen event
-at least 3 concentrations are available for mutagenicity assessment

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Applicant's summary and conclusion

Conclusions:

Based on the result of this study, the test item, ID-11/01446 was found to be non mutagenic and non pro-mutagenic under the test conditions.

Executive summary:

Study summary

The ability of the test item supplied by PHYCHER BIODEVELOPPEMENT, to induce mutation was assessed using the bacterial reverse mutation test (Ames test). The test was performed on five Salmonella typhimurium strains.

 

The test item dilutions were prepared in DMSO.

A preliminary cytotoxicity test was performed on S.typhimurium TA100 strain.

The test is performed at the concentrations 5000, 1600, 500, 160, 50 μg/plate, with and without S9.

 

The preliminary study did not shown any cytotoxicity of the test item, therefore this concentration range was used for the genotoxicity test 1.

According to the result obtained for the test 1, the study director decided to use the same dilution range for the test 2.

 

The revertant analyse show that:

-no cytotoxic effect was observed

-no concentration of the test item showed ratio R higher or equal at least to the double of the spontaneous rate of reversion for TA98, TA100 and TA102 and to the triple of the spontaneous rate of reversion for TA1535 and TA153, with and without metabolic activation.

-no dose response was observed, whatever the test system or conditions of the test.

-however, signs of precipitate were observed during test 2 on concentration 5000 μg/plate without metabolic activation.

 

At the light of the results obtained during this study, we can conclude that the test item does not show any mutagenic nor pro-mutagenic activity, under the test conditions used.