Orange, sweet, 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:

16-Aug-2010 to 26-Aug-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Performed under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Orange, sweet, ext.

EC Number:

232-433-8

EC Name:

Orange, sweet, ext.

Cas Number:

8028-48-6

Molecular formula:

This reference substance is a UVCB of the NCS type. It is a complex mixture of compounds and therefore molecular formula, molcular weight, and structural formula cannot be given.

IUPAC Name:

Essential oil of orange obtained from the peel of Citrus sinensis (Rutaceae) by expression and/or distillation, including cold pressed, distilled, terpenes and essence qualities

Constituent 2

Reference substance name:

Orange Oil Cold Pressed 1-Fold-Orange, ext. (Citrus sinensis, Rutaceae)

IUPAC Name:

Orange Oil Cold Pressed 1-Fold-Orange, ext. (Citrus sinensis, Rutaceae)

Constituent 3

Reference substance name:

8028-46-6

IUPAC Name:

8028-46-6

Details on test material:

- Name of test material (as cited in study report): Orange Oil Cold Pressed 1-Fold-Orange, ext. (Citrus sinensis, Rutaceae)- Physical state: Liquid- Lot/batch No.: Confidential- Stability under test conditions: Stable- Storage condition of test material: At room temperature 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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and B-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1:
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3300, and 5000 ug/plate
Main study: TA1535, TA1537 and TA98:
Without S9-mix: 1, 3, 10, 33, 99, and 198 ug/plate
With S9-mix: 10, 33, 99, 329, 988 and 3290 ug/plate
Experiment 2:
TA1535, TA1537 and TA98:
Without S9-mix: 1, 3, 10, 33, 100, and 150 ug/plate
With S9-mix: 10, 33, 100, 333, 1000, and 2000 ug/plate
WP2uvrA:
Without and with S9-mix: 100, 333, 1000, 3330, and 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was stable in ethanol and ethanol has been accepted and approved by authorities and international guidelines.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: sodium azide (5 ug/plate in saline for TA1535); 9-aminoacridine (60 ug/plate in water for TA1537); 2-nitrofluorene (10 ug/plate in DMSO for TA98); methylmethanesulfonate (650 ug/plate in DMSO for TA100); 4-nitroquinoline-N-oxide (10 ug/plate i

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Selection time (if incubation with a selection agent): at least 48 hours

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

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

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive if:
a) A two-fold (TA100) or more, or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

Mean and Standard Deviation

Results and discussion

Test results

Test results 1

Key result

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

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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 was observed up to and including the top dose of 5000 pg/plate.

RANGE-FINDING/SCREENING STUDIES: In test strain TA100, toxicity was observed at dose levels of 33 and 333 ug/plate and above in the absence and presence of S9-mix, resp. In test strain WP2uvrA, no toxicity was observed up to and including the top dose of 5000 pg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535, TA1537, and TA98: without S9: 100 ug/plate and above and with S9: 333 ug/plate and above.
TA100: without S9: 33 ug/plate and above, and with S9: 333 ug/plate and above
WP2uvrA: no toxicity was observed up to and including the top dose of 5000 ug/plate.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with metabolic activation and negative without metabolic activation

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. It is concluded that this test is valid and that Orange Oil Cold Pressed 1-Fold-Orange, ext. (Citrus sinensis, Rutaceae) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Orange Oil Cold Pressed 1-Fold-Orange, ext. (Citrus sinensis, Rutaceae) was tested in the Salmonella typhimurium reverse mutation assay with 4 histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in 2 independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and B-naphthoflavone). The study procedures were based on the most recent OECD guideline 471 and EC guidelines. Orange Oil Cold Pressed 1-Fold-Orange, ext. (Citrus sinensis, Rutaceae) was a clear yellow liquid. The test substance was dissolved in ethanol. In the dose range finding test the test substance was tested up to concentrations of 5000 ug/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test substance did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction in the bacterial background lawn, was observed in both test strains. Results of this dose range finding test were reported as part of the first experiment of the mutation assay. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay up to 198 and 3290 ug/plate in the absence and presence of 5% (v/v) S9-mix, resp. in test strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, the test substance was tested up to dose levels of 150 and 2000 ug/plate in the absence and presence of 10% (v/v) S9-mix, resp. in test strains TA1535, TA1537, TA98 and TA100. Test strain WP2uvrA was tested up to a concentration of 5000 ug/plate in the absence and presence of 10% (v/v) S9-mix. Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction in the bacterial background lawn, was observed in test strains TA1535, TA1537, TA98 and TA100. Orange Oil Cold Pressed 1-Fold-Orange, ext. (Citrus sinensis, Rutaceae) did not induce a significant dose-related increase in the number of revertant (His) colonies in each of the 4 test strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in test strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that Orange Oil Cold Pressed 1-Fold-Orange, ext. (Citrus sinensis, Rutaceae) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.