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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Dec 2015 - 21 Dec 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
14 sept 2015
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine locus and tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Trinova Biochem GmbH
- Suitability of cells: according to OECD guideline

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Nutrient Broth 37°C
- Properly maintained: Yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I
All strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II
Strain TA 100 with and without S9 mix: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Other strains with and without S9 mix: 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine 4-NOPD, 2-aminoanthracene 2-AA
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation)
Experiment 2: preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
Rationale for test conditions:
According to OECD TG 471
Evaluation criteria:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
experiment I: 1000-5000 µg/plate with and without S9 mix, experiment II: 333 - 5000 µg/plate without S9 and 1000 - 5000 µg/plate with S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Experiment I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
experiment II without S9 mix 2500-5000µg/plate, with S9 mix 5000µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Experiment I and II
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 1537
Remarks:
Experiment I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
experiment II without S9 mix at 5000µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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: The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate in experiment I and from 1000 to 5000 μg/plate in experiment II. The undissolved particles had no influence on the data recording.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)

TA 1535 without S9 Mean SD Min Max
Solvent control 11 2.15 7 23
Untreated control 12 2.97 6 24
Positive control 1090 123.80 334 1372

TA 1535 with S9 Mean SD Min Max
Solvent control 12 2.14 7 21
Untreated control 12 2.71 7 26
Positive control 392 62.85 176 549

TA1537 without S9 Mean SD Min Max
Solvent control 10 1.83 6 18
Untreated control 10 2.29 6 20
Positive control 83 12.28 55 131

TA1537 with S9 Mean SD Min Max
Solvent control 13 3.27 7 27
Untreated control 14 3.72 7 25
Positive control 175 44.44 82 327

TA 98 without S9 Mean SD Min Max
Solvent control 24 3.75 16 36
Untreated control 26 4.72 15 43
Positive control 344 51.13 211 599

TA 98 with S9 Mean SD Min Max
Solvent control 33 5.55 18 51
Untreated control 36 5.83 17 56
Positive control 3822 857.83 319 5048

TA 100 without S9 Mean SD Min Max
Solvent control 155 24.19 84 194
Untreated control 174 21.92 90 206
Positive control 1956 279.93 658 2528

TA 100 with S9 Mean SD Min Max
Solvent control 145 31.81 81 204
Untreated control 170 23.62 93 212
Positive control 3606 676.07 722 4940

WP2uvrA without S9 Mean SD Min Max
Solvent control 41 5.72 27 63
Untreated control 42 6.01 31 63
Positive control 732 161.66 322 1066

WP2uvrA with S9 Mean SD Min Max
Solvent control 51 6.91 37 72
Untreated control 53 7.05 38 88
Positive control 362 72.26 212 858
Conclusions:
Based on the results of the in vitro gene mutation study in bacteria for Ginger Extract 46 % (HPLC, Sum Gingerols (6-,8-,10-) and Shogaols (6-,8-), the test substance 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 Extract 46 % (HPLC, Sum Gingerols (6-,8-,10-) and Shogaols (6-,8-) to induce gene mutations was tested in an in vitro gene mutation study in bacteria (OECD TG 471, GLP) to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. 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 3- 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate in experiment I and from 1000 to 5000 µg/plate in experiment II.  The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain TA 1537 without metabolic activation and in strains TA 98 and TA 100 with and without metabolic activation. 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 Hot Flavor CO2-TO extract is considered to be non-mutagenic in this bacterial reverse mutation assay.

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

Additional information

The potential of Ginger Extract 46 % (HPLC, Sum Gingerols (6-,8-,10-) and Shogaols (6-,8-) to induce gene mutations was tested in an in vitro gene mutation study in bacteria (OECD TG 471, GLP) to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. 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 3- 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate in experiment I and from 1000 to 5000 µg/plate in experiment II.  The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain TA 1537 without metabolic activation and in strains TA 98 and TA 100 with and without metabolic activation. 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 Hot Flavor CO2-TO extract is considered to be non-mutagenic in this bacterial reverse mutation assay.

Justification for classification or non-classification

Based on the available information Ginger Hot Flavor CO2-TO 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).

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