Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Feb 2018 - 28 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
8 May 2017
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Histidine
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):
CELLS USED
- Source of cells: Trinova Biochem
- Suitability of cells: according to Dr. Bruce N. AMES
- Methods for maintenance in cell culture if applicable: Test strains were kept as lyophilisate pellets at 4°C. The lyophilisates of the test strains were used to inoculate the overnight cultures. Overnight cultures were grown in a water bath for 15 h at 37°C in Oxoid 2 nutrient broth.
- The final cell density was approximately 10^8 - 10^9 cells/mL.

MEDIA USED
- Type and identity of media: Oxoid 2 nutrient broth
- Properly maintained: Yes, regularly checked for genetic identity of the strains, their sensitivity to UV-radiation and crystal violet and their resistance to ampicillin and tetracycline.
Additional strain / cell type characteristics:
other: All: permeability to macromolecules TA98,100,1535,1537: no DNA excision repair TA98,100,102: R-factor plasmid TA102: plasmid, carrier of tetracycline resistance TA98,100,102: resistance to Ampicillin TA1535, TA1537: non-resistance to Ampicillin
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Preliminary cytotoxicity test: 10 to 100 000 µg/plate and 0.2 mL undiluted test item/plate
Signs of cytotoxicity were observed from 1000µg/plate (scarce background lawn), ad was therefore chosen as top dose in the main experiment.
Main study: 3.16, 10, 31.6, 100, 316 and 1000 µg/plate

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: well-known and compatible vehicle
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) or preincubation
- Cell density at seeding (if applicable): density was approximately 10^8 - 10^9 cells/mL.

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 h

DETERMINATION OF CYTOTOXICITY
- Method: scarce background lawn and/or reduction of the number of revertants by more than 50%
Rationale for test conditions:
According to OECD TG 471
Evaluation criteria:
The results of the negative and positive control cultures should be within the range of the historical data
The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20 - 60
TA100: 100 - 200
TA102: 240 - 320
TA1535: 10 - 35
TA1537: 3 - 20
Where concurrent negative or positive control data fall outside the range, they may be acceptable and considered for the inclusion into the historical control distribution as long as these data are not extreme outliers.

A test item is considered to show a positive response if
- the number of revertants is significantly increased compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- a concentration-related increase over the range tested in the number of the revertants per plate is observed.
- biological relevance of the results should be considered first.
Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of Salmonella typhimurium.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.
Statistics:
U-test according to MANN and WHITNEY and Spearman's rank correlation coefficient where applicable.

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
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
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Ten concentrations ranging from 10 µg Ginger CO2-se extract to 0.2 mL undiluted test item/per plate were tested. Pronounced to complete cytotoxicity (scarce background lawn and/or reduction of the number of revertants by more than 50%) was noted starting at a concentration of 1000 μg Ginger CO2-se extract/plate in both experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:

TA98
-S9: 158.8 (sd 26.2) min102 - max293
+S9: 157.6 (sd 26.9) min104 - max291
TA100
-S9: 954 (sd 102.1) min701 - max1365
+S9: 949.5 (sd 104.3) min703 - max1238
TA102
-S9: 1020.7 (sd 90.8) min756 - max1263
+S9: 1015.0 (sd 95.1) min759 - max1311
TA1535
-S9: 144.5 (sd 44.7) min62 - max406
+S9:144.5 (sd 47.4) min67 - max404
TA1537
-S9:75.0 (sd 26.9) min28 - max185
+S9:75.9 (sd 26.4) min31 - max184

- Negative (solvent/vehicle) historical control data:

TA98
-S9: 30.2 (sd 5.6) min20 - max49
+S9: 31.9 (sd 5.9) min20 - max49
TA100
-S9: 145.9 (sd 19.6) min95 - max200
+S9: 145.1 (sd 19.9) min100 - max209
TA102
-S9: 278.1 (sd 16.9) min245 - max323
+S9: 279.0 (sd 17.5) min203 - max324
TA1535
-S9: 19.7 (sd 4.4) min10 - max34
+S9:19.9 (sd 4.6) min10 - max36
TA1537
-S9: 6.7 (sd 1.8) min2 - max10
+S9:6.7 (sd 1.8) min2 - max10

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Applicant's summary and conclusion

Conclusions:
Based on the results of the in vitro gene mutation study in bacteria for Ginger CO2-SE extract, 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 CO2 -SE extract to induce gene mutations was examined in a OECD TG 471 study, under GLP. Ginger CO2 -SE extract was  examined in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, without and with metabolic activation. The first experiment was carried out as a plate incorporation test and the second as a preincubation test. Ginger CO2 -SE extract was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 10 to 100 000 µg Ginger CO2 -SE extract/plate in ethanol and 0.2 mL undiluted test item/plate were tested. Due to pronounced cytotoxicity at the concentration of 3160 µg Ginger CO2 -SE extract/plate, and cytotoxicity at the concentration of 1000 µg/plate, 1000 µg Ginger CO2 -SE extract per plate were chosen as top concentration for the main study.

Main study: Six concentrations ranging from 3.16 to 1000 µg Ginger CO2 -SE extract/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. Cytotoxicity (reduction of the number of revertants by more than 50% and/or scarce background lawn) was noted in all experiments without and with metabolic activation at the top concentration of 1000 µg Ginger CO2-se extract/plate in all test strains. No increase in revertant colony numbers as compared with control counts was observed for Ginger CO2 -SE extract under any condition.

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

Under the present test conditions, Ginger CO2 -SE extract tested up to a concentration of 1000 µg/plate that led to cytotoxicity, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.