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EC number: 289-904-6 | CAS number: 90045-43-5 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Citrus paradisi M., Rutaceae.
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Link to relevant study records
- 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 - 1 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.
- 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
- 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
- Additional strain / cell type characteristics:
- not applicable
- 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):
Salmonella strain TA98 (-S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
Salmonella strains TA100, TA1535 and TA1537 (+S9 and -S9) and Salmonella strain TA98 (+S9): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
E.coli strain WP2uvrA (+S9 and -S9): 15, 50, 150, 500, 1500, 5000 μg/plate.
Experiment 2 (pre-incubation method):
All bacterial strains (-S9) and TA1535 (+S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
Salmonella strains TA100, TA98 and TA1537 (+S9): 1.5, 5, 15, 50, 150, 500, 1500 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 - 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)
- 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
- 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 150 μg/plate and at 5000 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 and 150 μg/plate in the absence and presence of S9-mix, resp. No toxicity was noted for strain E.coli strain WP2uvrA. In Experiment 2 (preincubation methodology) the test item induced a stronger 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'.
- Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
In the Bacterial Reverse Mutation test (Ames), grapefruit 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, with and without metabolic activation, or exposure method. The test item, Grapefruit Oil 1-Fold, Grapefruit (Citrus Paradisi M.), 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, Grapefruit Oil 1-Fold, Grapefruit (Citrus Paradisi M.), 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 and 150 μg/plate in the absence and presence of S9-mix, resp. No toxicity was noted for strain E.coli strain WP2uvrA.In the preincubation assay the test item induced a stronger 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, Grapefruit Oil 1-Fold, Grapefruit (Citrus Paradisi M.), ext, was considered to be non-mutagenic under the conditions of this test.
- 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.
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
- 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.; metabolic activation not included; 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: 0.063 mg/ml
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Remarks:
- untreated cells
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- 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
OTHER: Preliminary test: the maximum dose of the grapefruit 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
- 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'.
- Conclusions:
- Interpretation of results (migrated information):
negative without metabolic activation
In this in vitro chromosomal aberration test, without metabolic activation, the incidence of polyploid cells at 24 hr after treatment with grapefruit oil was 0.0 %. The incidence of cells with structural chromosome aberrations at 24 hr after treatment was 2.0 %. As grapefruit oil did not significantly induce chromosomal aberrations in CHL cells in vitro at three different concentrations (maximum dose: 0.063 mg/ml) in the absence of metabolic activation, and it is considered to be not clastogenic in this test. - Executive summary:
An in vitro chromosomal aberration test using a Chinese hamster fibroblast cell line (CHL) was carried out on grapefruit oil. The test was performed comparable to OECD Guideline 473. The cells were continuously exposed to grapefruit oil at three different doses for 24 and 48 hours without metabolic activation. The maximum dose of the grapefruit 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: 0.063 mg/ml. 100 Metaphases were examined. The incidence of polyploid cells at 24 hr after treatment was 0.0 %. The incidence of cells with structural chromosome aberrations at 24 hr after treatment was 2.0 %. Grapefruit 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:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- No data
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Acceptable basic data; comparable to OECD Guideline 473 with deviations; non GLP; no data on chemical identity. Furthermore, due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID.
- Justification for type of information:
- The justification for read across is provided in the attached background material of the chapter summary.
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
- 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.; metabolic activation not included; 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: 0.04 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
OTHER: Preliminary test: the maximum dose of the Lime 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
- 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'.
- Conclusions:
- 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 Lime oil was 0.0 %. The incidence of cells with structural chromosome aberrations at 24 hr after treatment was 4.0 %. Lime oil did not significantly induce chromosomal aberrations in CHL cells in vitro at three different concentrations (maximum dose: 0.04 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 Lime oil. The test was performed comparable to OECD Guideline 473. The cells were continuously exposed to Lime oil at three different doses for 24 and 48 hours without metabolic activation. The maximum dose of the Lime 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: 0.04 mg/ml. 100 Metaphases were examined. The incidence of polyploid cells at 24 hr after treatment was 0.0 %. The incidence of cells with structural chromosome aberrations at 24 hr after treatment was 4.0 %. Lime 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:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- 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'.
- Conclusions:
- A chromosomal aberration test in vitro without metabolic activation was performed on the read across source substance: Lime oil, the results are read across to the target substance Grapefruit oil.
In this chromosomal aberration test in vitro without metabolic activation the incidence of polyploid cells at 24 hr after treatment with Lime oil was 0.0 %. The incidence of cells with structural chromosome aberrations at 24 hr after treatment was 4.0 %. Lime oil did not significantly induce chromosomal aberrations in CHL cells in vitro at three different concentrations (maximum dose: 0.04 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 Lime oil. The test was performed comparable to OECD Guideline 473. The cells were continuously exposed to Lime oil at three different doses for 24 and 48 hours without metabolic activation. The maximum dose of the Lime 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: 0.04 mg/ml. 100 Metaphases were examined. The incidence of polyploid cells at 24 hr after treatment was 0.0 %. The incidence of cells with structural chromosome aberrations at 24 hr after treatment was 4.0 %. Lime 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:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- No data
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Acceptable basic data; similar to OECD Guideline 473 with deviations; non GLP; no data on chemical identity. Furthermore, due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID.
- Justification for type of information:
- The justification for read across is provided in the attached background material of the chapter summary.
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
- 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
- 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'.
- Conclusions:
- 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:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- no data
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Acceptable basic data; comparable to OECD Guideline 473 with deviations; non GLP; no data on chemical identity. Furthermore, due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID.
- Justification for type of information:
- The justification for read across is provided in the attached background material of the chapter summary.
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
- 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 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
- 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'.
- Conclusions:
- 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.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Test was conducted according to OECD Test Guideline No. 476, under GLP Standards, and QA. Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID.
- Justification for type of information:
- The justification for read across is provided in the attached background material of the chapter summary.
- 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 (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
- 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:
- 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 in the cultured mammalian cells used, i.e. mouse lymphoma L5178Y test system, under the experimental conditions described in the report, as per guidance documents, section 35 OECD 476. - 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 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 Lemon Oil Cold Pressed 1-Fold-Lemon, ext. (Citrus limonum, 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 documents, section 35 OECD 476.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Test was conducted according to OECD Test Guideline No. 476, under GLP Standards, and QA. Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID.
- Justification for type of information:
- The justification for read across is provided in the attached background material of the chapter summary.
- 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
- 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:
- negative with metabolic activation
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:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Acceptable basic data; similar to OECD Guideline 473 with deviations; non GLP; no data on chemical identity. Furthermore, due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of the chapter summary.
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- 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'.
- Conclusions:
- A chromosomal aberration test in vitro without metabolic activation was performed on the read across source substance: Orange oil, the results are read across to the target substance Grapefruit oil.
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:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- Type of assay:
- in vitro mammalian chromosome aberration test
- 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'.
- Conclusions:
- A chromosomal aberration test in vitro without metabolic activation was performed on the read across source substance: Lemon oil, the results are read across to the target substance Grapefruit oil.
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.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- 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:
- An OECD Guideline 476 study was performed on the read across source substance: Lemon oil, the results are read across to the target substance Grapefruit oil.
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 in the cultured mammalian cells used, i.e. mouse lymphoma L5178Y test system, under the experimental conditions described in the report, as per guidance documents, section 35 OECD 476. - 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 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 Lemon Oil Cold Pressed 1-Fold-Lemon, ext. (Citrus limonum, 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 documents, section 35 OECD 476.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- 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:
- An OECD Guideline 476 study was performed on the read across source substance Orange oil, the results are read across to the target substance Grapefruit oil.
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. 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.
Referenceopen allclose all
The incidence of polyploid cells at 24 hours after treatment: 0.0 %
The incidence of cells with structural chromosome aberrations at 24 hours after treatment: 2.0 %
The incidence of polyploid cells at 24 hours after treatment: 0.0 %
The incidence of cells with structural chromosome aberrations at 24 hours after treatment: 4.0 %
The incidence of polyploid cells at 24 hours after treatment: 0.0 %
The incidence of cells with structural chromosome aberrations at 24 hours after treatment: 4.0 %
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 %
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 %
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 %
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 %
Additional information
In the Bacterial Reverse Mutation test (Ames, OECD 471), grapefruit 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, Grapefruit Oil 1-Fold, Grapefruit (Citrus Paradisi M.), ext,, was therefore considered to be non-mutagenic under the conditions of this test.
In an in vitro chromosomal aberration test using a Chinese hamster fibroblast cell line (CHL) (OECD Guideline 473), grapefruit 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.
Based upon an equal result in the tests of all oils from the Citrus category, the results of the available gene mutation tests in mammalian cells with a citrus oil can be read across to the other oils. For both orange oil and lemon oil, the gene mutation study in mammalian cells was negative. The results of these tests can be read across to grapefruit oil. This read across is considered valid because of:1) the similar origin of the oils in this group, 2) the dominant presence of D-limonene in all oils of this group, and 3) the same and similar other constituents.
Short description of key information:
Read across within citrus oils and major constituent limonene: not
genotoxic
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
Based on the available information, the substance grapefruit oil does not need to be classified for mutagenicity according to the criteria outlined in Annex I of 1272/2008/EC (CLP/EU-GHS) and Annex VI of 67/548/EEC.
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