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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:
7 March 2011 - 6 May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed according to OECD guideline 471 and under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
not applicable
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): Mandarin oil, (Citrus Reticulata Blanco) ext
- Physical state: Yellow liquid
- Analytical purity: Not applicable – natural complex substance
- Lot/batch No.: Confidential information
- Expiration date of the lot/batch: Confidential information
- Storage condition of test material: Approx. 4°C in the dark under nitrogen

Method

Target gene:
S. typhimurium: Histidine gene
E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction from rats induced with phenobarbitone/ß-naphtoflavone
Test concentrations with justification for top dose:
Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

Experiment 1 (plate incorporation method):
All Salmonella strains (+S9 and -S9): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
E.coli strain WP2uvrA (+S9 and -S9): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.

Experiment 2 (pre-incubation method):
Salmonella strain TA100 (+S9): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
All bacterial strains (-S9), Salmonella strains TA1535, TA98 and TA1537 (+S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
E.coli strain WP2uvrA (+S9): 15, 50, 150, 500, 1500, 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: sponsor informed that test item was not soluble in distilled water.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) for TA100, TA1535, and WP2uvrA, 9-aminoacridine (9AA) for TA1537, 4-Nitroquinoline-1-oxide (4NQO) for TA98; +S9: 2-Aminoanthracene (2AA) for TA100, TA1535, TA1537, and WP2uvrA, Benzo(a)pyrene (BP) for TA98.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation test)
Experiment 2: preincubation test

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

SELECTION AGENT (mutation assays): agar containing Histidine or Tryptophan

NUMBER OF REPLICATIONS: Doses of the test substance were tested in triplicate in each strain.

NUMBER OF CELLS EVALUATED: All tester strain cultures should be in the range of 0.9 to 9*10E9 bacteria/ml

DETERMINATION OF CYTOTOXICITY
- Method: Preliminary toxicity test, measuring number of revertant colonies and effects on the growth of the bacterial background lawn.
Evaluation criteria:
A test item will be considered non-mutagenic (negative) in the test system if the criteria below are not met:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979)
- A reproducible increase at one or more concentrations
- Biological relevance against in-house historical control ranges
- Statistical analysis of data as determined by UKEMS (Mahon et al, 1989)
- Fold increase greater that two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
Statistics:
Not applicable (see evaluation criteria)

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
Species / strain:
E. coli WP2 uvr A
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation observed.

RANGE-FINDING/SCREENING STUDIES: The test item was initially toxic to TA100 from 50 μg/plate and from 1500 μg/plate to WP2uvrA.

COMPARISON WITH HISTORICAL CONTROL DATA: The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory).

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first experiment (plate incorporation methodology), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains, initially from 50 μg/plate in the absence and presence of S9-mix. No toxicity was noted for E.coli strain WP2uvrA. In Experiment 2 (pre-incubation methodology) the test item induced a toxic response to all of the bacterial strains, initially from 15 μg/plate (-S9) and 150 μg/plate (+S9). The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type, exposures with or without S9-mix and experimental methodology. The test item was tested up to the maximum recommended dose level of 5000 μg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.
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 with metabolic activation
negative without metabolic activation

In the Bacterial Reverse Mutation test (Ames), mandarin oil did not show biologically significant increases in the frequency of revertant colonies for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The test item, Mandarin oil, (Citrus Reticulata Blanco) ext, was therefore considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item, Mandarin oil, (Citrus Reticulata Blanco) ext,using both the plate incorporation and pre-incubation methods at up to 7 dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The tests were performed according to OECD guideline 471.

In the plate incorporation assay the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains, initially from 50 μg/plate in the absence and presence of S9-mix. No toxicity was noted for E.coli strain WP2uvrA. In the pre-incubation assay the test item induced a toxic response to all of the bacterial strains, initially from 15 μg/plate (-S9) and 150 μg/plate (+S9). The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type, exposures with or without S9-mix and experimental methodology. The test item was tested up to the maximum recommended dose level of 5000 μg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method. The test item, Mandarin oil, (Citrus Reticulata Blanco) ext, was therefore considered to be nonmutagenic under the conditions of this test.