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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test was conducted according to OECD Test Guideline No. 476, under GLP Standards, and QA. Klimisch 2 reliability has been assigned in accordance with (ECHA Practical Guide #6) due to the read-across purpose of this study.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Lemon, ext.
IUPAC Name:
Lemon, ext.
Constituent 2
Reference substance name:
Lemon Oil Cold Pressed 1-Fold-Lemon, ext. (Citrus limonum, Rutaceae)
IUPAC Name:
Lemon Oil Cold Pressed 1-Fold-Lemon, ext. (Citrus limonum, Rutaceae)
Details on test material:
- Name of test material (as cited in study report): Lemon Oil Cold Pressed 1-Fold-Lemon, ext. (Citrus limonum, Rutaceae)
- Physical state: Liquid
- Lot/batch No.: Confidential
- Stability under test conditions: stable
- Storage condition of test material: Confidential

Method

Target gene:
Thymidine kinase (TK) gene
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 Hepes buffered medium (basic medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
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:
Range finding:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 500 µg/ml in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.

Experiment 1 (3 hours exposure):
Without S9-mix: 0.3, 1, 3, 10, 30, 40, 50, 60, 70, 80, 90 and 100 μg/ml exposure medium.
With 8% (v/v) S9-mix: 0.3, 1, 3, 10, 33, 100, 150, 200, 250, 300, 350, 400, 450 and 500 μg/ml exposure medium.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 1, 3, 10, 30, 40, 50, 60 and 70 μg/ml exposure medium.
With S9-mix: 1, 10, 100, 200, 250, 300, 400 and 450 μg/ml exposure medium.

Experiment 2 (3 or 24 hours exposure):
Based on the results of the dose range finding test and experiment 1, the following dose levels
were selected for mutagenicity testing.
Without S9-mix: 1, 3, 10, 30, 40, 50, 55, 60, 65, 70, 75 and 80 µg/ml exposure medium (24 hour).
With 12% (v/v) S9-mix: 1, 3, 10, 30, 100, 200, 300, 400, 425, 450 and 500 μg/ml exposure medium (3 hour).
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 1, 3, 10, 30, 40, 50 and 55 µg/ml exposure medium.
With S9-mix: 1, 3, 10, 30, 100, 300, 400 and 500 μg/ml exposure medium.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controls
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 (+/- S9); Experiment II: -S9 mix: 24 hours; +S9 mix: 3 hours
- Expression time (cells in growth medium): For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the
treatment period. During this culture period at least 4 x 10E6 cells (if possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test substance the cells were plated for determination of the cloning efficiency (CEday2) and the
mutation frequency (MF).

SELECTION AGENT (mutation assays): TFT-selection

NUMBER OF REPLICATIONS: two independent experiments, 8 doses were selected to measure MF, duplicate solvent controls.

NUMBER OF CELLS EVALUATED:
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. 1 cell was added per well (2 x 96-well microtiter plates/concentration) in non selective medium.
For determination of the MF a total number of 9.6 x 10E5 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10E5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection).

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

OTHER EXAMINATIONS:
- DETERMINATION OF SIZE DISTRIBUTION OF THE COLONIES (small and large TK-/- colony counts).
Evaluation criteria:
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF (controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
No data

Results and discussion

Test results
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
Solubility: The test substance precipitated in the culture medium at 100 ug/ml and above in both experiments. The test substance was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 500 μg/ml.

RANGE-FINDING/SCREENING STUDIES:
At 3 hour treatment:
In the absence of S9-mix, the relative suspension growth was 52% at a test concentration of 33 µg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 100 μg/ml and above. In the presence of S9-mix, the relative suspension growth was 4% at the test substance concentration of 500 µg/ml compared to the relative suspension growth of the solvent control.
At 24 hour treatment:
In the absence of S9-mix, the relative suspension growth was 53% at the test substance concentration of 33 µg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 100 μg/ml and above.

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:
Experiment I:
In the absence of S9-mix, the dose levels of 0.3 to 40 μg/ml showed no cytotoxicity. Therefore, the dose level of 0.3 µg/ml was not regarded relevant for mutation frequency measurement. The dose
levels of 80 to 100 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the presence of S9-mix, the dose levels of 0.3 to 200 μg/ml showed no cytotoxicity. Therefore, the dose levels of 0.3, 3, 33 and 150 µg/ml were not regarded relevant for mutation frequency measurement. The dose levels of 300 to 400 μg/ml showed similar cytotoxicity. Therefore, the
dose level of 350 µg/ml was not regarded relevant for mutation frequency measurement. The dose level of 500 μg/ml was not used for mutation frequency measurement, since this dose level was too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 1, 3, 10, 30, 40, 50, 60 and 70 μg/ml exposure medium.
With S9-mix: 1, 10, 100, 200, 250, 300, 400 and 450 μg/ml exposure medium.

In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 91% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 90% compared to the total growth of the solvent controls.

Experiment II:
In the absence of S9-mix, the dose levels of 60 to 80 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix, the dose levels of 1 to 300 μg/ml showed no cytotoxicity. Therefore, the dose level of 200 µg/ml was not regarded relevant for mutation frequency measurement. The dose levels of 425 to 500 μg/ml showed similar cytotoxicity. Therefore, the dose levels of 425 and 450 µg/ml were not regarded relevant for mutation frequency measurement.

In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 91% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 38% compared to the total growth of the solvent controls.

CONTROLS
Mutation frequencies in cultures treated with positive control chemicals were increased by 11- and 16-fold for MMS in the absence of S9-mix, and by 12 - and 14 -fold for CP in the presence of S9-mix, in exp I and II respectively. 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.



Any other information on results incl. tables

In the absence of S9-mix, the test substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.
In the presence of S9-mix, the test substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.








Applicant's summary and conclusion

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

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 Lemon Oil Cold Pressed 1-Fold-Lemon, ext. (Citrus limonum, Rutaceae) does not induce gene mutations under the experimental conditions described in the report.
Executive summary:

The study was performed in accordance with OECD Guideline 476 to investigate the potential of the substance Lemon Oil Cold Pressed 1-Fold-Lemon, ext. (Citrus limonum, Rutaceae) to induce mutations at the mouse lymphoma thymidine kinase locus using the L5178Y mouse lymphoma cells. The assay was performed in two independent experiments in the absence and presence of S9-mix.

In the first experiment the substance was tested up to concentrations of 70 and 450 µg/ml in the absence and presence of 8% (v/v) S9-mix, resp. The incubation time was 3 h and the substance was tested up to a cytotoxic level of 91 and 90% (compared to solvent controls) in the absence and presence of S9-mix, resp. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

In the second experiment the substance was tested up to concentrations of 55 and 500 µg/ml in the absence and presence of 12% (v/v) S9-mix, resp. The incubation times were 24 h (-S9) and 3 h (+S9) and the substance was tested up to a cytotoxic level of 91 (-S9) and 38% (+S9), compared to solvent controls. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

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 16-fold for MMS in the absence of S9-mix, and by 12 - and 14 -fold for CP in the presence of S9-mix, in exp I and II respectively. 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 Lemon Oil Cold Pressed 1-Fold-Lemon, ext. (Citrus limonum, Rutaceae) does not induce gene mutations under the experimental conditions described in the report.