Lemon, ext.

Genetic toxicity: in vitro

Administrative data

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:

other: Acceptable basic data; comparable to OECD Guideline 473 with deviations; non GLP; no data on chemical identity.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1984

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Lemon, ext.

EC Number:

284-515-8

EC Name:

Lemon, ext.

Cas Number:

84929-31-7

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).

IUPAC Name:

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

Constituent 2

Reference substance name:

Lemon oil

IUPAC Name:

Lemon oil

Details on test material:

- Name of test material (as cited in study report): Lemon oil

Method

Target gene:

Not relevant

Species / strain

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

Controls

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 Lemon 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

Results and discussion

Test results

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'.

Any other information on results incl. tables

The incidence of polyploid cells at 24 hours after treatment: 1.0 %

The incidence of cells with structural chromosome aberrations at 24 hours after treatment: 2.0 %

Applicant's summary and conclusion

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 24 hr after treatment with Lemon oil was 1.0 %. The incidence of cells with structural chromosome aberrations at 24 hr after treatment was 2.0 %. Lemon 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 Lemon oil. The test was performed comparable to OECD Guideline 473. The cells were continuously exposed to Lemon oil at three different doses for 24 and 48 hours without metabolic activation. The maximum dose of the Lemon 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: 0.125 mg/ml. 100 Metaphases were examined. The incidence of polyploid cells at 24 hr after treatment was 1.0 %. The incidence of cells with structural chromosome aberrations at 24 hr after treatment was 2.0 %. Lemon 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.