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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the Bacterial Reverse Mutation test (Ames, OECD 471), lime oil (both cold-pressed and distilled) 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 items, Lime Oil Cold Pressed 1-Fold, Lime (Citrus aurantifolia), ext. and Lime Oil Distilled 1-Fold, Lime (Citrus aurantifolia), ext., were therefore considered to be non-mutagenic under the conditions of the tests.

 

In an in vitro chromosomal aberration test using a Chinese hamster fibroblast cell line (CHL) (OECD Guideline 473), 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.

 

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 lime 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. Lime oil has the lowest content of D-limonene in the group and contains several constituents that are not present in the other oils. A separate document is present in the dossier with respect to the presence of furocoumarins in citrus oils. The other constituents have been checked for genotoxic properties in the OECD-toolbox. No alerts for mutagenicity or carcinogenicity were found. It is concluded that the results from orange and lemon oil can be read across to lime oil.

 


Short description of key information:
Read across within citrus oils and major constituent limonene: not genotoxic

Endpoint Conclusion:No adverse effect observed (negative)
Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 March 2011 - 6 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):
All Salmonella strains (+S9 and -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 Salmonella strains (-S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50 μg/plate.
Salmonella strains TA100, TA1535 and TA98 (+S9): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
Salmonella strain TA1535 (+S9): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
E.coli strain WP2uvrA (+S9 and -S9): 5, 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 bacterial strains, initially from 150 μg/plate in the absence and presence of S9-mix. In Experiment 2 (preincubation methodology) the test item induced a toxic response to all of the bacterial strains, initially from 15 μg/plate (absence of S9-mix) and 150 μg/plate (presence of S9-mix). 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), lime oil (cold pressed) 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, Lime Oil Cold Pressed 1-Fold, Lime (Citrus aurantifolia), 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, Lime Oil Cold Pressed 1-Fold, Lime (Citrus aurantifolia), 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 bacterial strains, initially from 150 μg/plate in the absence and presence of S9-mix. In the preincubation assay the test item induced a 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 test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded 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, Lime Oil Cold Pressed 1-Fold, Lime (Citrus aurantifolia), ext., was considered to be non-mutagenic under the conditions of this test.

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 - 13 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):
All Salmonella strains (+S9 and -S9): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
E.coli strain WP2uvrA (+S9 and -S9): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.

Experiment 2 (pre-incubation method):
Salmonella strains TA100, TA98 and TA1535 (-S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
Salmonella strains TA100, TA98 (+S9) and Salmonella strain TA1537 (+S9 and -S9): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
E.coli strain WP2uvrA (+S9 and -S9): 15, 50, 150, 500, 1500, 2500, 5000 μg/plate.
Salmonella strain TA1535 (+S9): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.

Experiment 3 (plate incorporation method):
E.coli strain WP2uvrA (+S9 and -S9): 500, 1000, 1500, 2000 and 3000 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
Experiment 3: in agar (plate incorporation test) (performed in response to small but non-reproducible increases in revertant colony frequency noted for Escherichia coli strain WP2uvrA in the absence and presence of S9-mix.)

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 50 μg/plate and 1500 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 tester strains, initially from 50 and 500 μg/plate in the absence and presence of S9-mix, respectively. In Experiment 2 (pre-incubation methodology) the test item induced a similar toxic response with weakened bacterial background lawns initially noted from 15 μg/plate (absence of S9-mix) and 500 μg/plate (presence of S9-mix). 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. A third experiment (plate incorporation test) was performed in response to small but non-reproducible increases in revertant colony frequency noted for Escherichia coli strain WP2uvrA in the absence and presence of S9-mix.
Statistically significant increases in WP2uvrA revertant colony frequency were observed at sub-toxic dose levels (1500 μg/plate in the absence and presence of S9-mix) in the first Experiment only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, all of the experimental work demonstrated that the test item did exhibit toxicity regardless of exposure conditions. Therefore, the small increases in revertant colony frequency may be an artefact resulting from a modest level of toxicity to WP2uvrA. A possible mechanism may be that low level toxicity has caused a selective effect on the number of bacterial cells plated, resulting in an increase in non revertant tryptophan-dependent bacteria. Other statistically significant increases were observed in several tester strains; however, these responses were always accompanied by weakened bacterial background lawns and can, therefore, be disregarded.
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), lime oil (distilled) 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, Lime Oil Distilled 1-Fold, Lime (Citrus aurantifolia), 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, Lime Oil Distilled 1-Fold, Lime (Citrus aurantifolia), 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.

A third experiment (plate incorporation test) was performed in response to small but non-reproducible increases in revertant colony frequency noted for Escherichia coli strain WP2uvrA in the absence and presence of S9-mix. 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 tester strains, initially from 50 and 500 μg/plate in the absence and presence of S9-mix, respectively. In Experiment 2 (pre-incubation methodology) the test item induced a similar toxic response with weakened bacterial background lawns initially noted from 15 μg/plate (-S9) and 500 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 biologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. Statistically significant increases in WP2uvrA revertant colony frequency were observed at sub-toxic dose levels (1500 μg/plate in the absence and presence of S9-mix) in the first Experiment only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, all of the experimental work demonstrated that the test item did exhibit toxicity regardless of exposure conditions. Therefore, the small increases in revertant colony frequency may be an artefact resulting from a modest level of toxicity to WP2uvrA. A possible mechanism may be that low level toxicity has caused a selective effect on the number of bacterial cells plated, resulting in an increase in non revertant tryptophan-dependent bacteria. Other statistically significant increases were observed in several tester strains; however, these responses were always accompanied by weakened bacterial background lawns and can, therefore, be disregarded. The test item, Lime Oil Distilled 1-Fold, Lime (Citrus aurantifolia), ext., was therefore considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
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. 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.
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:
Interpretation of results (migrated information):
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 gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
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. 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.
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:
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 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 cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
other: Publication
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.
Justification for data waiving:
other:
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'.

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 %

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 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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable basic data; similar to OECD Guideline 473 with deviations; non GLP; no data on chemical identity.
Justification for data waiving:
other:
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'.
The incidence of polyploid cells at 48 hours after treatment: 1.0 %

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

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

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

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

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
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'.

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 %

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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
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.; 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'.

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 %

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.

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

Additional information

Lime Oil Cold Pressed Genotoxicity: discussion on the presence of furocoumarins and consequences for risk assessment

Lime oil is one of the essential oils containing furocoumarins by nature, substances which can exert (photo)genotoxic effects. The furocoumarin content varies per quality and adds up to ~7.0% (typically 3%) in lime oil cold pressed. The initial approach for addressing the genotoxicity endpoint under REACH was with RA to the lemon oil that would be justified due to similarity in constituents. However, the furocoumarins’ level of lemon oil (up to 0.5%) is lower when compared to lime oil, and given the potential characteristics of these compounds, such a RA approach may be inappropriate for the endpoint of genetic toxicity.

 

In principle, phototoxicity and photogenotoxicity are not endpoints required under REACH. Nevertheless, in case any additional hazard is suspected outside the standard list of endpoints and in the CSR it is shown that exposure to lime oil in the presence of UV could occur, registrants shall address this hazard in order to ensure safety and safe use. The fact that some furocoumarins are classified for their carcinogenic properties in combination with its presence up to a total 7% requires the registrants of lime oil to perform a thorough investigation of the photo(geno)toxic effects of lime oil in order to ensure safe use.

IFRA Standards and EU restrictions

Currently, according to IFRA proposed standards (Literature\IFRA_Standard_Lime_Oil_Expressed.pdf), the limit for the lime oilper sein a finished leave-on product that will come in contact with the skin is0.7%. For rinse off products (including household cleaning products) and for non-skin contact products there is currently no restriction. Accordingly, a recommended limit for the furocoumarin 5-MOP (5-methoxypsoralen or bergapten), for all citrus oils, is 0.0015% (15 ppm) in skin contact leave one products, while there is no recommended restriction for rinse-off (including household cleaning products) and non-skin contact products. This value is equivalent to 0.0075% (75 ppm) in a fragrance compound used at 20% in the consumer product. These standards are set due to phototoxic effects of these materials. In particular for lime oil reference to some published document is made, according which lime oil exhibited phototoxic properties when tested on hairless mice, pigs and man (Urbach & Forbes, 1972[1]; as referenced in Opdyke, 1974[2]). The study is referenced as RIFM report, and it was asked through Geoffrey Hynes (genetic toxicologist from Givaudan); awaiting for response.

A position paper constructed by IFRA, on the presence of furocoumarins[3]in cosmetic finished products and in particular sun protection and sun bronzing products, is at the moment under evaluation on an EU level. The paper was evaluated by RHDHV, for potential use under REACH purposes. In summary, IFRA proposes a limit value of 5 ppm for non-rinse off and 50 ppm for rinse off cosmetic products, opposing to the proposed level by SCCP[4], of 1 ppm. It was concluded that this value cannot be reached as it is not relevant for the lime oil REACH registration, since none of the products are used for such purposes.

Options for risk assessment for workers and consumers

Lime oil has been classified as a moderate skin sensitizer. No data is available which allows for setting a DNEL or DMEL for this health hazard. Therefore the risk is assessed using a qualitative approach. Sensitization is considered a moderate hazard in this approach. This results in the qualitative risk assessment for the sensitization hazard of Lime Oil listed inTable 1.

 

Table1Qualitative risk assessment for sensitization hazard of Lime Oil

 

Concentration of substance in mixture

Risk management measures

Risk conclusion

Worker

< 1%

-

Safe

> 1%

-       Minimise number of staff exposed;

-      Minimisation of manual phases;

-      Avoidance of contact with contaminated tools and objects;

-      Regular cleaning of equipment and work area;

-      Management/supervision in place to check that the RMMs in place are being used correctly and OCs followed;

-      Training for staff on good practice;

-      Good standard of personal hygiene

Safe

-      Segregation of the emitting process;

-      Effective contaminant extraction;

-      Good standard of general ventilation;

-      Substance/task appropriate gloves;

-      Skin coverage with appropriate barrier material based on potential for contact with the chemicals;

-      Substance/task appropriate respirator;

-      Optional face shield;

-      Eye protection.

Consumer product with potential skin contact

< 1%

-

Safe

> 1%

-

Use advised against

 

Currently no data is available that allows for setting a DNEL or DMEL for the potential phototoxicity and photogenotoxicity hazards of Lime Oil. This means that this hazard needs to be addressed in a qualitative manner. As no test data is available to allow for the classification of Lime Oil for these substances and Furocoumarins are present in a combined concentration of up to 7 percent and some Furocoumarins are deemed carcinogens the hazard class for the dermal risk is set at high. The volatility of Furocoumarins is very low. The effect reviewed here is related to the presence us sunlight (UV) and is no longer relevant once the substance is ingested. This combined with the fact that means that at room temperature the inhalation hazard is deemed negligible. Furthermore this results in the qualitative risk assessment for the phototoxicity and photogenotoxicity hazards of Lime Oil listed inTable 2.

 

Table2Qualitative risk assessment for Phototoxicity and Photogenotoxicity hazards of Lime Oil

 

Concentration of Lime Oil in mixture as used*

Risk management measures

Risk conclusion

Worker, indoor

Not restricted

None

Safe

Worker, outdoor**

< 0.07%

-

Safe

> 0.07%

-      Ensure all equipment well maintained;

-      Permit to work for maintenance work;

-      Regular cleaning of equipment and work area;

-      Management/supervision in place to check that the RMMs in place are being used correctly and OCs followed;

-      Training for staff on good practice;

-      Procedures and training for emergency decontamination and disposal;

-      Good standard of personal hygiene

Safe

-      Segregation of the emitting process;

-      Design closed system to allow for easy maintenance;

-      Substance/task appropriate gloves;

-      Skin coverage with appropriate barrier material based on potential for contact with the chemicals;

-      Optional face shield;

-      Eye protection.

Consumer indoor**

Not restricted

None

Safe

Consumer use outdoor without dermal contact

Not restricted

None

Safe

Consumer outdoor**

< 0.07%***

-

Safe

> 0.07%***

-

Use advised against

*This means that known dilution, as documented i.e. in the IFRA guidance for REACH Exposure scenarios for Fragrance Substances or developed SCEDs must be taken into account when determining the concentration. For most consumer product the dilution factor is >20, resulting in a safe use if the concentration is below 1% in the original product.

** Consumer, non-cosmetic uses with potential for dermal contact only. Examples are garden furniture cleaning products, window cleaning products.

 


[1]Urbach, F & Forbes, PD (1972). Report to RIFM, 23 March.

[2]Opdyke, DLJ (1974). Monographs on fragrance raw materials: lime oil Ex.Food and Cosmetic Toxicology, vol 12, pg 731

[3]International Fragrance Association (IFRA) position paper (2007). Furocoumarins in finished Cosmetic products.

[4]SCCP (2006) The Scientific Committee for Consumer Products’ Notes of Guidance for the Testing of Cosmetic Ingredients and Their Safety Evaluation, 6th Revision. Adopted by the SCCP during the 10th plenary of 19 December 2006

Justification for classification or non-classification

Based on the available information, the substance lime oil does not need to be classified for mutagenicity according to the criteria outlined in Annex I of 1272/2008/EC (CLP/EU-GHS).