Mandarin orange, ext.

Genetic toxicity: in vitro

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Mandarin oil, (Citrus Reticulata Blanco) ext

IUPAC Name:

Mandarin oil, (Citrus Reticulata Blanco) ext

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 / strain

Species / strain 1

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 2

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 results

Test results 1

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

Test results 2

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.