Registration Dossier

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:
17 December 2014 - 13 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed according to OECD 471 and in compliance with GLP.

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cinnamon Bark Oil (CBO)
- Physical state: Yellow liquid
- Analytical purity: 100%
- Storage condition of test material: Room temperature in the dark

Method

Target gene:
Histidine and tryptophan locus
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 Agarplates, Top Agar
- Properly maintained: yes
- Periodically checked for viability, spontaneous reversion rate characteristics
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1: Plate Incorporation method: 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Experiment 2: Pre-incubation method: 1.5 to 1500 µg/plate (based on results experiment 1)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
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:
With S9-mix: 2-Aminoanthracene and Benzo(a)pyrene. Without S9-mix: N-ethyl-N-nitro-N-nitrosoguanidine, 9-Aminoacridine, 4-Nitroquinonile-N-oxide.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment 1: direct plate incorporation methodology (in agar).
Experiment 2: pre-incubation methodology.

DURATION
- Preincubation period: S9-mix 20 minutes (prior to exposure in experiment 2 only).
- Exposure duration: 48 hours (experiment 1 and 2).

NUMBER OF REPLICATIONS:
Preliminary test: no replication
Test for mutagenetic ity (exp 1 and 2) in triplicate.

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

DETERMINATION OF CYTOTOXICITY
- Method: Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plate.
Evaluation criteria:
EVALUATION CRITERIA
1. A dose-related increase in mutant frequency over the dose range tested
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
5. Fold increase greater than two times the concurrent solvent control for any tester strain.

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:
Individual plate counts, mean number of revertant comlonies per plate and the standard deviation are presented

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:
plate incorporation method (exp 1)
Cytotoxicity / choice of top concentrations:
no 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:
pre-incubation method (exp 2)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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.

RANGE-FINDING/SCREENING STUDIES:
Dose range for experiment 1 was predetermined at 1.5 to 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
Yes, results for positive, vehicle and untreated control reference items for 2012 and 2013 are presented.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: Reductions in growth of the bacterial background lawns of all tester strains from 1500 µg/plate (both with and without S9-mix). The test item was tested up to the maximum recommended level of 5000 µg/plate.
- Experiment 2: Reductions in growth of the bacterial background lawns in absence of S9-mix from 150 µg/plate (TA1537), 500 µg/plate (TA1535 and WP2uvrA) and 1500 µg/plate (TA100 and TA98). With S9-mix, reductions were noted from 500 µg/plate (TA100 and TA1537) and 1500 µg/plate (TA1535, WP2uvrA and TA98).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this test, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. Based on these results the test item Cinnamon bark oil can be considered non-mutagenic in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).
Executive summary:

The genotoxicity of the test substance Cinnamon bark oil was tested in bacterial strains TA1535, TA1537, TA98 and TA100 and E.coli WP2uvrA according to OECD guideline 471 (Ames test). Two experiments (using the plate incorporation methodology and pre-incubation methodology) were performed with concentrations of the test substance ranging from 1.5 to 5000 µg/plate, with and without metabolic activation. Negative, vehicle and positive controls were included as well. The frequency of revertant colonies was recorded.

The negative and positive controls were determined to be valid. All the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, confirming the activity of the S9-mix and the sensitivity of the bacterial strains. Cytoxicity was observed in both experiments by a reduction in growth of the bacterial background lawns, being more apparent in experiment 2 (using pre-incubation methodology). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose test item, either with or without metabolic activation or exposure method. 

Based on these results the test item Cinnamon bark oil can be considered non-mutagenic in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).