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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene mutation in bacteria: (Bacterial Reverse Mutation Assay/Ames) (according to OECD 471): not mutagenic.
- In vitro Mammalian chromosome aberration test (equivalent or similar to OECD 473): not clastogenic without metabolic activation.
- In vitro Mammalian cell gene mutation test (according to OECD 476): negative.

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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
Target gene:
S. typhimurium: Histidine gene
E. coli: Tryptophan gene
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 / 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.
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
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
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.
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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Performed under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Not aplicable
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1 (3 hours treatment):
- without S9-mix: 1, 10, 20, 30, 35, 40, 41.5 and 43 ug/ml
- with S9-mix: 0.03, 0.1, 0.3, 1, 3, 10, 33, and 100 ug/ml
Experiment 2:
- without S9-mix: 3, 10, 15, 20, 25, 30, 35, and 40 ug/ml (24 h)
- with S9-mix: 0.03, 0.1, 0.3, 1, 3, 10, 33, and 100 ug/ml (3 h)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix: Methyl Methane Sulfonate; +S9 mix: Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 3 hours; Experiment II: -S9 mix: 24 hours; +S9 mix: 3 hours
- Expression time (cells in growth medium): No data yet
- Selection time (if incubation with a selection agent): No data yet

SELECTION AGENT (mutation assays): No data (as only preliminary results yet)

NUMBER OF REPLICATIONS: Single treated cultures/dose (8 doses); duplicate solvent controls.

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (CE); relative total growth (RTG)

OTHER EXAMINATIONS:
DETERMINATION OF SIZE DISTRIBUTION OF THE COLONIES (small and large TK-/- colony counts).
Evaluation criteria:
No data yet
Statistics:
No data yet
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see also 'Additional information on results'
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the culture medium at 100 ug/ml in both experiments.

RANGE-FINDING/SCREENING STUDIES: No data

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first experiment (concentrations up to 43 and 100 µg/ml in the absence and presence of 8% (v/v) S9-mix, resp., incubation time of 3 h) the test item was tested up to a cytotoxic level of 12% (RTG) in the absence of S9-mix. No toxicity was observed up to and including the dose level of 100 µg/ml in the presence of S9-mix. The test item precipitated in the culture medium at this dose level.
In the second experiment, the test item was tested up to concentrations of 40 and 100 µg/ml, but in the absence and presence of 12% (v/v) S9-mix, resp. The incubation times were 24 h and 3 h for incubations in the absence and presence of S9-mix, resp. The test item was tested up to a cytotoxic level of 41% (RTG) in the absence of S9-mix. No toxicity was observed up to and including the dose level of 100 µg/ml in the presence of S9-mix. The test substance precipitated in the culture medium at this dose level.
Conclusions:
Interpretation of results: negative with metabolic activation and negative without metabolic activation

n the absence and in the presence of S9-mix the test substance did not induce a significant increase in the mutation frequency in both experiments.
It is concluded that Orange Oil Cold Pressed 1-Fold-Orange, ext. (Citrus sinensis, Rutaceae) does not induce gene mutations in the cultured mammalian cells used, i.e. mouse lymphoma L5178Y test system, under the experimental conditions described in the report, as per guidance document, section 35, OECD 476.
Executive summary:

The study was performed in accordance with OECD Guideline 476 to investigate the potential of the substance Orange Oil Cold Pressed 1-Fold-Orange, ext. (Citrus sinensis, Rutaceae) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in 2 independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The test substance was a clear yellow liquid and was dissolved in ethanol. In the first experiment the substance was tested up to concentrations of 43 and 100 µg/ml in the absence and presence of 8% (v/v) S9-mix, resp. The incubation time was 3 h. The substance was tested up to a cytotoxic level of 12% (RTG) in the absence of S9-mix. No toxicity was observed up to and including the dose level of 100 µg/ml in the presence of S9-mix. The test substance precipitated in the culture medium at this dose level. In the second experiment the substance was tested up to concentrations of 40 and 100 µg/ml, but in the absence and presence of 12% (v/v) S9-mix, resp. The incubation times were 24 h and 3 h for incubations in the absence and presence of S9-mix, resp. The substance was tested up to a cytotoxic level of 41% (RTG) in the absence of S9-mix. No toxicity was observed up to and including the dose level of 100 µg/ml in the presence of S9-mix. The test substance precipitated in the culture medium at this dose level. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 11- and 23-fold for MMS in the absence of S9-mix, and by 20- and 17-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system functioned properly. In the absence and in the presence of S9-mix the test substance did not induce a significant increase in the mutation frequency in both experiments. It is concluded that Orange Oil Cold Pressed 1-Fold-Orange, ext. (Citrus sinensis, Rutaceae) does not induce gene mutations in the cultured mammalian cells used, i.e. the mouse lymphoma L5178Y test system, under the experimental conditions described in the report, as per guidance document, section 35, OECD 476.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Acceptable basic data; similar to OECD Guideline 473 with deviations; non GLP; no data on chemical identity.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Exposure time: 24 and 48 hrs.; treatment only without S9-mix; only 100 metaphases examined, no positive control; no duplicates included in test
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not relevant
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
Three doses; only maximum dose specified: 0.125 mg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
no
Positive control substance:
no
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 and 48 hrs.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hrs.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 0.2 μg/ml medium, last 2 hours of incubation
STAIN (for cytogenetic assays): Giemsa solution, 1.5 % , 12-15 min.

NUMBER OF REPLICATIONS: no data

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads

DETERMINATION OF CYTOTOXICITY
- Method: 50% cell-growth inhibition, estimated using a cell densitometer.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Preliminary test: the maximum dose of the orange oil sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer, representing the highest non-cytotoxic dose.
Evaluation criteria:
The results were considered to be negative if the incidence of chromosome aberrations was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels.
Statistics:
No data
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolarity: Previous studies indicated that the osmotic pressure of the medium generally rose with sample concentrations of more than 10 mM, so that the maximum dose might be limited to around this level, at which cytotoxic effects were not necessarily observed.

COMPARISON WITH HISTORICAL CONTROL DATA: no data
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The incidence of polyploid cells at 48 hours after treatment: 1.0 %

The incidence of cells with structural chromosome aberrations at 48 hours after treatment: 1.0 %

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation

In this chromosomal aberration test in vitro without metabolic activation the incidence of polyploid cells at 48 hr after treatment with Orange oil was 1.0 %. The incidence of cells with structural chromosome aberrations at 48 hr after treatment was 1.0 %. Orange oil did not significantly induce chromosomal aberrations in CHL cells in vitro at three different concentrations (maximum dose: 0.125 mg/ml) in the absence of metabolic activation, and was therefore considered not clastogenic in this test.
Executive summary:

A chromosomal aberration test in vitro using a Chinese hamster fibroblast cell line (CHL) was carried out on Orange oil. The test was performed comparable to OECD Guideline 473. The cells were continuously exposed to Orange oil at three different doses for 24 and 48 hours without metabolic activation. The maximum dose of the Orange oil sample was selected by a preliminary test, in which the highest non-cytotoxic dose was 0.125 mg/ml. 100 Metaphases were examined. The incidence of polyploid cells at 48 hr after treatment was 1.0 %. The incidence of cells with structural chromosome aberrations at 48 hr after treatment was 1.0 %. Orange oil did not significantly induce chromosomal aberrations in CHL cells in vitro at three different concentrations in the absence of metabolic activation and was therefore considered not clastogenic in this test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

For this endpoint 3 in vitro tests, all performed according/similar to OECD Test Guidelines, were selected as key studies:

- In a Bacterial Reverse Mutation Assay (OECD 471) Orange oil did not induce a significant dose-related increase in the number of revertant (His) colonies in each of the 4 Salmonella typhimurium test strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in Escherichia coli test strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded thatoil is not mutagenic in the S. typhimurium reverse mutation assay and in the E. coli reverse mutation assay.

- In a Chromosomal Aberration test in vitro (OECD 473) without metabolic activation Orange oil did not significantly induce chromosomal aberrations in CHL cells in vitro at three different concentrations (maximum dose: 0.125 mg/ml) in the absence of metabolic activation, and was therefore considered not clastogenic in this test.

- In the in vitro Mammalian Cell Gene Mutation Test (OECD 476) in the absence and in the presence of S9-mix theoil did not induce a significant increase in the mutation frequency in both experiments. It is concluded thatoil did not induce gene mutations in the cultured mammalian cells used, i.e. mouse lymphoma L5178Y test system, under the experimental conditions described in the report, as per guidance document, section 35, OECD 476.

 

Justification for classification or non-classification

In all 3 key genetic toxicity studies (3 in vitro tests)oil did not show any genotoxic potential. Therefore, it can be concluded that the substance is not mutagenic, and does not need to be classified for mutagenicity according to the criteria outlined in Annex I of 1272/2008/EC (CLP/EU-GHS) .