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Toxicological information

Genetic toxicity: in vitro

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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:
14 November 2012 - 7 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: In compliance with GLP, according to OECD guideline 471 (Bacterial reverse mutation assay)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Eucalyptus citriodora
IUPAC Name:
Eucalyptus citriodora
Details on test material:
- Name of test material (as cited in study report): Eucalyptus citriodora
- Physical state: Liquid
- 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 or 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 Plates, Top Agar.
- Properly maintained: yes
- Periodically checked for viability, spontaneous reversion rate characteristics.
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:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1:
- Salmonella strains and E.coli strain (without S9): 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate
- E.coli strain (with S9) 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
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: 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 mutagenicity: 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
2. A reproducible increase at one or more concentrations
3. Biological relevance against in-house historical control ranges
4. Statistical analysis of data as detemined by UKEMS
5. Fold increase greater then 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:
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:
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: The test item was toxic to TA100 and WP2uvrA initially from 500 µg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: Reductions in growth of bacterial background lawns of all Salmonella strains and E.coli (without S9) from 500 µg/plate, E. coli from 1500 µg/plate (with S9)
Experiment 2: Reductions in growth of bacterial background lawns of all Salmonella strains from 150 µg/plate without S9 and from 500 µg/plate with S9, E. coli from 500 µg/plate with and without S9 (for E. coli only weakened background lawns).
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

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 or exposure method. Therefore the test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The genotoxicity of the test substance Eucalyptus citriodora was tested in bacteria according to OECD guideline 471 (Ames test) and under GLP conditions. Two experiments (plate incorporation methodology and pre-incubation methodolog) were performed with concentrations of the test substance ranging from 1.5 - 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 positive and negative control were valid: all positive control chemicals induced increase in frequency of revertant colonies and the increase observed for the negative control substance was considered acceptible. Cytotoxicity was observed in both experiments by a reduction in growth of the bacterial background lawns, being more apparent in the experiment using plate incorporation methodology. 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 or exposure method.

Based on the results of this Ames test, the test item Eucalyptus citriodora was considered to be non-mutagenic under the conditions as specified.

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