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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 October 2014 - 6 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD guideline 471 and under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Cymbopogon flexuosus
IUPAC Name:
Cymbopogon flexuosus
Details on test material:
- Name of test material (as cited in study report): Cymbopogon flexuosus
- Physical state: Liquid (colourless)
- Analytical purity: Confidential information
- Lot/batch No.: Confidential information
- Expiration date of the lot/batch: Confidential information
- Storage condition of test material: Room temperature in the dark

Method

Target gene:
Histidine locus (Salmonella typhimurium)
Tryptophan locus (Escherichia coli)
Species / strain
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):
- Type and identity of media: Vogel-Bonner Minimal Plates, Top Agar.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: E. coli WP2: uvrA DNA repair deficient
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 0.5-1500 µg/plate (based on results of experiment 1)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test item fully miscible in DMSO, but not in water (emulsion).
Controls
Untreated negative controls:
yes
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
other: 2-Aminoanthracene
Remarks:
Without S9: N-ethyl-N-nitro-N-nitrosoguanidine, 4-nitroquinoline-N-oxide, 9-aminoacridine. With S9: benzo(a)pyrene, 2-Aminoanthracene.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation)
Experiment 2: preincubation

DURATION
- Preincubation period: 20 minutes (Experiment 2 only)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: in triplicate

DETERMINATION OF MUTAGENICITY
- Method: Evaluate reduction in number of spontaneous revertants and negative effect on the growth of the bacterial background lawn (thinning).

DETERMINATION OF CYTOXICITY
- Method: Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
Evaluation criteria:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979)
2. A reproducible increase at one or more concentrations.
3. BIological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an ou-of-historical range response (Cariello and Piegorsch, 1996)).

ACCEPTANCE CRITERIA
- All bacterial strains must have demonstrated required characteristics as determined by their respective strains according to Ames et al. (1975)
- Tester strain cultures should exhibit characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- Tester strain culture density should be in the range of 0.9 to 9x10E9 bacteria/ml
- Positive control values must demonstrate intrinsic sensitivity of the test strains and integrity of the S9-mix
- A minimum of four non-toxic test item dose levels is allowed
- No evidence of excessive contamination
Statistics:
Not applicable

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
with plate incorporation methodology (experiment 1)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
with pre-incubation methodology (experiment 2)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON TEST RESULTS:
The results indicate that there were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in both experiments. A small, statistically significant increase in TA1537 revertant colony frequency was observed in the presence of S9 at 5 µg/plate in the second test (experiment 2), but this was considered to be of no biological relevance as there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at this concentration were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.5 times the concurrent vehicle control.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: Reduction in bacterial background lawn for all strains with and without S9 from 500 and 1500 µg/plate
- Experiment 2: Reduction in bacterial background lawn for all strains with and without S9 from 150 and 500 µg/plate
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate and all were found to be satisfactory. Amino acid supplemented top agar and S9-mix in both experiments were shown to be steril, as well as the test item formulation.

All positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Under the conditions of this study, Cymbopogon flexuosus was considered to be non-mutagenic. Therefore, the substance does not need to be classified as mutagenic according to the criteria outlined in Annex VI of DSD (67/548/EEC) and Annex I of CLP (1272/2008/EC).
Executive summary:

A bacterial reverse mutation assay (Ames test) was performed with Cymbopogon flexuosus according to OECD guideline 471 and under GLP conditions. S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 were selected as strains and the plate incorporation and preincubation method were used. The test substance was applied in concentrations ranging from 0.5 to 5000 µg/plate for the first (plate incorporation) experiment and 0.5 to 1500 µg/plate for the second (preincubation) experiment. Untreated, negative (vehicle) and positive controls were included.

The results indicate that there were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in both experiments. A small, statistically significant increase in TA1537 revertant colony frequency was observed in the presence of S9 at 5 µg/plate, but this was considered of no biological relevance as there was no evidence of a dose-response relationship or reproducibility.

Under the conditions of this study, Cymbopogon flexuosus was considered to be non-mutagenic. Therefore, the substance does not need to be classified as mutagenic according to the criteria outlined in Annex VI of DSD (67/548/EEC) and Annex I of CLP (1272/2008/EC).

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