Reaction mass of [(2S,3R,4S,5S,6R)-6-[[(2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxymethyl]-3,4,5-trihydroxyoxan-2-yl] (1S,2R,4aS,6aR,6aR,6bR,8R,8aR,9R,10R,11R,12aR,14bS)-8,10,11-trihydroxy-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 and [(2S,3R,4S,5S,6R)-6-[[(2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxymethyl]-3,4,5-trihydroxyoxan-2-yl] (4aS,6aR,6aS,6bR,8R,8aR,9R,10R,11R,12aR,14bS)-8,10,11-trihydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-12-11 to 2006-12-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: MADECASSOSIDE
Batch: 026
Amount: 10g (2 x 5 g)
State: solid
Solvent: Water
Purity: > or = 95%
Storage conditions: kepp away from the humidity

Target gene:

- Strain salmonella typhimurium TA98: Target mutation: His D 3025; Excision repair: uvrB; Plasmid: pKM101; Cell wall: rfa; Mutation type: Frameshift
- Strain salmonella typhimurium TA100: Target mutation: His G 46; Excision repair: uvrB; Plasmid: pKM101; Cell wall: rfa; Mutation type: Base-pair substitution
- Strain salmonella typhimurium TA102: Target mutation: His G 428; Excision repair: - ; Plasmid: pKM101; Cell wall: - ; Mutation type: Base-pair substitution
- Strain salmonella typhimurium TA1535: Target mutation: His G 46; Excision repair: uvrB; Plasmid: - ; Cell wall: rfa; Mutation type: Base-pair substitution
- Strain salmonella typhimurium TA1537: Target mutation: His D 3076; Excision repair: - ; Plasmid: pKM101; Cell wall: - ; Mutation type: Frameshift

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

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µ/plate

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

For TA98, TA100, TA102, TA1535 and TA1537 with S9 activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For TA1537 witout S9 activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

For TA102 without S9 activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For TA100 and TA1535 without S9 activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

For TA98 without S9 activation

Details on test system and experimental conditions:

*Cytotoxicity test: 
A potential cytotoxic effect of the test item that would interfere in the results was ruled out with the following test. Five concentrations and a negative control of the test item were tested in solmonella typhimurium TA100. The test item was mixed with top agar containing 2.5 mM Histidine / Biotin. The solution was then added to a plate containing minimal agar and incubated at about 37°C for about 48 hours.
Inhibition of growth by the test item suggects a cytotoxic activity. A cytotoxic effect at high concentrations only would require lower concentrations of the test item in the main test. Cytotoxic activity at lower concentrations could rule out the bacterial reverse mutation test for the evaluation of mutagenicity.

*Test performance (direct incorporation method):
Cultures of bacteria were incubated overnight with nutrient broth up to an absorbance (at 660nm) of approximately 1.2-1.4 OD. This OD indicates that bacteria are growing in the late exponential or early stationary phase of growth (approximately 10^9 bacteria/mL).
Plates were prepared with minimum agar medium. Medium was mixed and preheated to about 45°C, and then poured over the plate and cooled at room temperature.
Each bacterial strain was tested by triplicate, both, in the presence and absence or the metabolic system (S9). The bacterial syspension, the test item and PBS (-S9) or metabolic activation system mix (+S9) were mixed and temperature at about 37°C.

A solubility test was performed:
Solubility test Dilution
Liquid 100% 30% 10% 3% 1%
Solid (100µl/plate) 50mg/mL 16.67mg/mL 5mg/mL 1.67mg/mL 0.5mg/mL
Test item with cytotoxic activity: Other concentrations might be used

The solution was mixed with top agar and poured over the minimal agar medium plate. the top agar was allowed to solidify at room temperature before final incubation.
Plate were incubated at about 37°C for about 48 hours. The number of colonies per plate was then counted.
Two controls were included in the experiment:
- Negative control: Absolute negative control (spontaneous reversion rate)
- Positive control: Control mutagens were used for each strain and experimental conditions as shown in the following table

S. typhimurium TA98: - Without S9: 2-Nitrofluorene; 90 µg/plate
- With S9: 2-Aminoanthracene; 10 µg/plate
S. typhimurium TA100: Without S9: Sodium Azide 10 µg/plate
- With S9: 2-Aminoanthracene 10 µg/plate
S. typhimurium TA102: Without S9: Mitomycin C 0.75 µg/plate
- With S9: 2-Aminoanthracene 30 µg/plate
S. typhimurium TA1535: Without S9: Sodium Azide 30 µg/plate
- With S9: 2-Aminoanthracene 30 µg/plate
S. typhimurium TA1537: Without S9: 9-Aminoacridine 100 µg/plate
- With S9: 2-Aminoanthracene 30 µg/plate

Evaluation criteria:

Several criteria were used for determining a positive result: a dose reponse in the range tested and / or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Positive results from the bacterail reverse mutation test indicate that a test induces point mutations or reading frame shifts in the genomes of salmonella typhimurium.
Negative results from the test indicate that under the test conditions, the test item is not mutagenic and non-promutagenic in the tested species.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

The controls of the test were in concordance with the expected results:

- No cytotoxic effect was observed.

- Sterility test shawed no contamination during the study.

- All positive controls performed showed valid ratio (R) above 2.5.

- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data.

- No concentration of the test item showed a biological significant increase (R>=2.5) of the number of revertant either with or without S9 metabilic activation.

- No dose reponse was observed on none of the trested bacterial strains.

Conclusions:

The following conclusions can be inferred from the obtained results:
- No test item showed ratios (R) above 2.5 as compared to the negative control, either with or without S9 metabolic activation.
- No dose response was observed in non of the tested bacterial strains.
Based on the results obtained in this study, the test item Madecassoside was found to be NON MUTAGENIC and NON-PROMUTAGENIC under the test conditions.

Executive summary:

The present Bacterial Reverse Mutation Test (Ames test) was performed in order to evaluate the mutagenic potential of the test item. This study was conducted according to the European Directive 2004/10/CE and the Good Laboratory Practice (GLP) principles of Spain (Principios de Buenas Practicas de Laboratorio: RD 1369/2000). The test was performed in accordance with OECD Guideline 471 for the Testing of Chemicals (Bacterial Reverse Mutation Test. Adopted 21st July 1997) and the test Method B13/B14 of CommissionDirective 2000/32/EC.

Suspensions of 5 amino-acid requiring strains of salmonella typhimurium (TA98, TA100, TA102, TA1535, TA1537) auxotroph for an amino acid, were exposed to the test item in the presence and in the absence of an exogenous metabolic activation system.

After incubation, revertant colonies due to point mutation were counted and compared to the number of spontaneous colonies on solvent control plate (negative control). Similary, specific standard mutagens were tested and used as positive controls.

Based on the results obtained in this study, the test item Madecassoside was found to be NON MUTAGENIC and NON-PROMUTAGENIC under the test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)