Tangor, Murcote, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative

Gene mutation in vitro (OECD TG 476): negative (read-across, Weight of Evidence)

Cytogenicity in vitro (OECD TG 473): negative (read-across, Weight of Evidence)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 March 2011 - 7 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-naphtone

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

Experiment 1:
- Salmonella strains TA100, TA1535, TA1537 (- and +S9), and TA98 (+S9): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
- Salmonella strain TA98 (-S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
- E. coli strain WP2uvrA (- and +S9): 15, 50, 150, 500, 1500, 5000 μg/plate.

Experiment 2:
Salmonella strains TA1535 (+S9), TA98 and E.coli strain WP2uvrA (-S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
Salmonella strains TA100 (- and + S9), TA98 and TA1537 (+S9): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
Salmonella strains TA1535 and TA1537 (-S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50 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. Two independent experiments were conducted.

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

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 was non-toxic 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 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 500 μg/plate -S9 and +S9, resp. No toxicity was noted for Escherichia 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

In a Bacterial Reverse Mutation test (Ames), tangerine oil did not induce 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, Tangerine Oil 1-Fold (Citrus reticulate Blanco), 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, Tangerine Oil 1-Fold (Citrus reticulate Blanco), 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 500 μg/plate –S9 and +S9, resp. No toxicity was noted for 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 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, Tangerine Oil 1-Fold (Citrus reticulate Blanco), ext, was therefore considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

Reason / purpose for cross-reference:

read-across: supporting information

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:
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.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

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. The justification for read across is provided in the attached background material of the chapter summary.

Reason / purpose for cross-reference:

read-across: supporting information

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:
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.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

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:
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.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

Reason / purpose for cross-reference:

read-across: supporting information

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:
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.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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.

Reason / purpose for cross-reference:

read-across: supporting information

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:
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.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

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:
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.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

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. 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 attached below

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'.

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:
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.

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

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. 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 attached below

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'.

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:
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.

Reference 10

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

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. 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 attached below

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'.

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:
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.

Reference 11

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

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. 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 attached below

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'.

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:
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.

Reference 12

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

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.

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. 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 attached below

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:

Interpretation of results:
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 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.

Reference 13

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

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. 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 attached below

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:

Interpretation of results:
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 conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For all citrus oils, the in vitro gene mutation study in bacteria (Ames test) was negative and the gene mutation study in mammalian cells with orange oil and lemon oil were also negative. The results of the gene mutation test in mammalian cells with orange oil and lemon oil test can be read across to tangerine 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. No cytogenicity was observed with 4 oils of this group. Based on the equal result in the Ames test (bridging study), this result can be read-across to Tangerine oil.

Justification for classification or non-classification

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