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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro (OECD TG 471, Ames): Not mutagenic

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Ames
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 November 2005 - 7 Apr 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000/32/EC
GLP compliance:
yes (incl. certificate)
Remarks:
2 Dec 2002
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
These results were not indicative of severe toxicity to prevent the test material being tested up to the maximum recommended dose level of 5000 μg/plate.
Experiment 1 and 2: 50, 150, 500, 1500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water at 50 mg/ml but was miscible in dimethyl sulphoxide at the same concentration.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
mitomycin C
other: 2-Aminoanthracene (2AA), 1,8-Dihydroxyanthraquinone (DAN)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

BACTERIAL DENSITY: 1 to 9.9 x 10^9 bacteria per ml.

DURATION
- Exposure duration: 48 hours an 37C

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity: visible reduction in the growth of the bacterial background lawn.
Rationale for test conditions:
According to guideline
Evaluation criteria:
The reverse mutation assay may be considered valid ifthe following criteria are met:
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
- All tester strain cultures should be in the approximate range of 1 to 9.9 x 10^9 bacteria per ml.
- Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.
Statistics:
Statistical methods, as recommended by the UKEMS can be used as an aid to evaluation.
Kirkland D J (Ed) (1989) Statistical Evaluation of Mutagenicity Test Data. UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III, Cambridge University Press.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9-mix only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Observed in all strains and conditions at 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES: These results were not indicative of severe toxicity to prevent the test material being tested up to the maximum recommended dose level of 5000 μg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- For Positive historical control data and Negative (solvent/vehicle) historical control data refer to the attached table.

Conclusions:
Based on the results of the in vitro gene mutation study in bacteria, the ginger CO2-Total Extract does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The potential of Ginger CO2-Total Extract to induce gene mutations was tested in an in vitro gene mutation study in bacteria (OECD TG 471, GLP) plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at 50 - 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate under all conditions. Toxic effects, evident as a reduction in the background lawn, were observed in TA100 and TA1535 (with and without S9-mix) and TA1537 (without S9-mix only) at 5000 μg/plate. All controls were considered valid. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment, at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Based on these results Ginger CO2-Total Extract is considered to be non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro (OECD TG 471, Ames)

The potential of Ginger Extract CO2 -Total Extract to induce gene mutations was tested in an in vitro gene mutation study in bacteria (OECD TG 471, GLP) plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at 50 - 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate under all conditions.  Toxic effects, evident as a reduction in the background lawn, were observed in TA100 and TA1535 (with and without S9-mix) and TA1537 (without S9-mix only) at 5000 μg/plate. All controls were considered valid. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment, at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Based on these results Ginger CO2-Total Extract is considered to be non-mutagenic in this bacterial reverse mutation assay.

Justification for classification or non-classification

Based on the available data, the test substance Ginger oil CO2-Total Extract does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).