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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria (OECD TG 471): negative

Chromosome aberration study in mammalian cells (OECD TG 473): negative

In vitro gene mutation study in mammalian cells (OECD TG 476): negative

Link to relevant study records

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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:
14 November 2012 - 7 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In compliance with GLP, according to OECD guideline 471 (Bacterial reverse mutation assay)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine or tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
- Type and identity of media: Vogel-Bonner Minimal Plates, Top Agar.
- Properly maintained: yes
- Periodically checked for viability, spontaneous reversion rate characteristics.
Additional strain / cell type characteristics:
other: E. coli WP2: uvrA DNA repair deficient
Metabolic activation:
with and without
Metabolic activation system:
S9
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: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
Without S9: N-ethyl-N-nitro-N-nitrosoguanidine, 4-nitroquinoline-N-oxide, 9-aminoacridine. With S9: benzo(a)pyrene, 2-Aminoanthracene.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment 1: plate incorporation methodology (in agar)
Experiment 2: pre-incubation methodology

DURATION
- Preincubation period: S9-mix 20 minutes (prior to exposure in experiment 2 only)
- Exposure duration: 48 hours (experiment 1 and 2)

NUMBER OF REPLICATIONS:
-Preliminary test: no replication
-Test for mutagenicity: in triplicate

DETERMINATION OF MUTAGENICITY
- Method: Evaluate reduction in number of spontaneous revertants and negative effect on the growth of the bacterial background lawn (thinning).

DETERMINATION OF CYTOXICITY
- Method: Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
Evaluation criteria:
EVALUATION CRITERIA
1. A dose-related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations
3. Biological relevance against in-house historical control ranges
4. Statistical analysis of data as determined by UKEMS
5. Fold increase greater than two times the concurrent solvent control for any tester strain.

ACCEPTANCE CRITERIA
- All bacterial strains must have demonstrated required characteristics as determined by their respective strains according to Ames et al. (1975)
- Tester strain cultures should exhibit characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- Tester strain culture density should be in the range of 0.9 to 9x10E9 bacteria/ml
- Positive control values must demonstrate intrinsic sensitivity of the test strains and integrity of the S9-mix
- A minimum of four non-toxic test item dose levels is allowed
- No evidence of excessive contamination
Statistics:
Not applicable
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
plate incorporation methodology (experiment 1)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
pre-incubation methodology (experiment 2)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES: The test item was toxic to TA100 from 500 µg/plate and to WP2uvrA from 1500 µg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: Reductions in growth of bacterial background lawns of all Salmonella strains from 500 µg/plate, E. coli from 1500 µg/plate (both with and without S9)
Experiment 2: Reductions in growth of bacterial background lawns of all Salmonella strains from 150 µg/plate without S9 and from 500 µg/plate with S9, E. coli from 500 µg/plate without S9 and at 1500 µg/plate with S9 (for E. coli only weakened background lawns).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
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. Therefore the test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The genotoxicity of the test substance Litsea cubeba essential oil was tested in bacteria according to OECD guideline 471 (Ames test) and under GLP conditions. Two experiments (plate incorporation methodology and pre-incubation methodology) were performed with concentrations of the test substance ranging from 1.5 - 5000 µg/plate, with and without metabolic activation. Negative, vehicle and positive controls were included as well. The frequency of revertant colonies was recorded.

The positive and negative control were valid: all positive control chemicals induced increase in frequency of revertant colonies and the increase observed for the negative control substance was considered acceptible. Cytotoxicity was observed in both experiments by a reduction in growth of the bacterial background lawns, being more apparent in the experiment using plate incorporation methodology. 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.

Based on the results of this Ames test, the test item Litsea cubeba essential oil was considered to be non-mutagenic under the conditions as specified.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March 2013 - 4 April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In compliance with GLP, according to OECD guideline 473 (genetic toxicology chromosome abberation test).
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: supplemented Eagle's Minimal Essential Medium (MEM)
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Exposure group 1: 5, 10, 20, 40, 60 and 80 µg/ml
Exposure group 2: 10, 20, 40, 80, 120 and 160 µg/ml
Exposure group 3: 5, 10, 20, 40, 60 and 80 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture media, DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in suspension

DURATION
- Preincubation period: 48 hours (for all three exposure groups)
- Exposure duration: 4 hours (for exposure group 1 and 2) and 24 hours (for group 3)
- Expression time (cells in growth medium): 20 hours (for groups 1 and 2)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (Colcemid 0.1 µg/ml)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures per dose level

NUMBER OF CELLS EVALUATED:
- Cromosome damage: first 100 consecutive well-spread methaphases from each culture were counted (or 50 cells if abberant frequency is large (30-50%)).
- Mitosis: 2000 lymphocyte cell nuclei per culture

DETERMINATION OF CHROMOSOME DAMAGE
- Method: metaphase counting. For cells with 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: if 69 chromosomes or more and incidence of polyploidy cells (%) is reported
Evaluation criteria:
- Test item: Dose-related decrease in mitotic index (cytotoxicity), significant increase in frequency of cells with aberrations and number of polyploid cells as compared to the (negative) control.
- Vehicle control: The frequeny of cells with chromosome abberations (excluding gaps) in the vehicle control cultures within the historical control data range.
- Positive control value: All positive control chemicals must induce positive responses (p<= 0.01).
Statistics:
Fisher's Exact Test.
Species / strain:
lymphocytes: human
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
- Effects of pH: No effect on pH in medium
- Effects of osmolality: Not increased by more than 50 mOsm
- Precipitation: No precipitate of the test item was observed at the end of the exposure period in any of the exposure groups.
- Other confounding effects: Heamolysis was observed at the end of the exposure period in the 4-hour exposure groups (1 and 2) at and above 40 and 80 µg/ml in the absence and presence of S9, respectively. No heamolysis was obeserved in the 24 hour exposure group (3).

RANGE-FINDING/SCREENING STUDIES: Maximum dose level selected for the main experiment was based on toxicity and was determined to be 80 µg/ml and 160 µg/ml for the 4(20)-hour exposure groups (1 and 2) and 80 µg/ml for the 24-hour exposure group.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Exposure group 1 (without S9): mitotic inhibition was 15%, 69% and 81% (without S9) at 40, 60 and 80 µg/ml, respectively.
Exposure group 2 (with S9): mitotic inhibition was 20% and 79% at the 80 and 120 µg/ml dose, respectively.
Exposure group 3 (24 hrs exposure): mitotic inhibition was 32% and 67% at 40 and 60 µg/ml, respectively.
Remarks on result:
other: other: not relevant
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the conditions of the study the test substance did not induce any toxicologically significant increase in frequency of cells with chromosome aberrations with or without metabolic activation. Therefore the test substance was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Genotoxicity of the test substance Litsea cubeba essential oil was determined in an in vitro chromosome aberration test according to OECD guideline 473 and under GLP conditions. Human lymphocytes were exposed to several concentrations of the test item ranging from 5 to 160 ug/ml in three groups, of which two had an exposure duration of 4 hours (with and without S9) and the other 24 hours. Positive and negative controls were included. Mitotic index was calculated and chromosome damage was evaluated by determining the frequency of cells with aberrations and the number of polyploid cells.

Qualitative assessment of the slides determined that there were metaphases suitable for scoring present in each of the exposure groups up to high exposure levels. Cytotoxicity was observed in all three exposure groups at higher doses (inhibition up to 79%). No significant changes in frequency of cells with aberrations and the number of polyploid cells was observed. Positive control was confirmed as a statistically significant increase in aberrations was noted in all items.

Under the conditions of the study the test substance did not induce any toxicologically significant increase in frequency of cells with chromosome aberrations with or without metabolic activation. Therefore the test substance was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 March 2013 - 8 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In compliance with GLP, according to OECD guideline 476 (In vitro mammalian cell gene muation tests).
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK+/-) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- TK +/- 3.7.2c
- Type and identity of media: supplemented RPMI 1640
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: not relevant
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1 (4 hrs exposure): 0.31 - 0.61 - 1.22 - 2.44 - 4.88 - 9.75 - 14.65 - 19.5 µg/ml without S9, 2.44 -4.88 - 9.75 - 19.5 - 29.25 - 39 - 58.5 - 78 µg/ml with S9
Experiment 2 (24 hrs exposure): 0.31 - 0.63 - 1.25 - 2.5 - 5 - 10 - 15 - 20 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, culture medium.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
cyclophosphamide (+S9) at 2 µg/ml, ethylmethanesulphonate (-S9) at 400 µg/ml (4hr) and 150 µg/ml (24hr)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium.

DURATION
- Exposure duration:
Experiment 1: 4 hours (with and without S9)
Experiment 2: 24 hours (without S9)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine (5-TFT)

NUMBER OF REPLICATIONS:
-Preliminary test: single
-Gene muation test: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG), relative suspension growth (RSG). Optimum toxicity is between 10-20% survival (80-90% toxicity).
Evaluation criteria:
- Test item: Statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value and a mutation frequency value that is greater than the corresponding vehicle control by the Global Evaluation Factor (GEF) of 126x10E-6 and demonstrates a positive linear trend.
- Vehicle control: Mutant frequency per survivor within range 50-170x10E-6 for the TK +/- locus in L5178Y cells
- Positive control: Induction of at least 3-5 times increase in mutant frequency as compared to the vehicle control
Statistics:
Statistical guideline recommended by UKEMS (Robinson et al 1989) were followed. Software used was Mutant 240C by York Electronic Research.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
after 4 hours exposure with and without S9
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
after24 hours exposure without S9
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate of the test item was not observed at any of the dose levels.

RANGE-FINDING/SCREENING STUDIES: Evidence of marked dose-related reductions in %RSG was found in all three exposure groups (4h -S9, 4h +S9 and 24h -S9) as compared to concurrent vehicle controls. A greasy/oily precipitate of the test item was observed at and above 625 µg/ml. Based on these values the maximum dose levels for the mutagenicity test were limited by test item-induced toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control mutant frequency value was within the acceptable range of 50-170 x 10E-6 viable cells. The positive control produced marked increases in mutant frequency/viable cell, indicating a well operating test system with and without S9.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: Evidence of marked dose-related toxicity following exposure to the test item with and without S9 was found, as indicated by the decreasing RTG and %RSG values. No effect on viability (%V) was found. It was concluded that the optimum levels of toxicity had been achieved in this test. Due to excessive toxicity at the highest dose level (78 µg/ml, with S9), this level was not plated for viability or 5-TFT resistance. Cytotoxicity of the positive control substance was acceptable.
Experiment 2: Evidence of marked dose-related toxicity following exposure to the test item was found, as indicated by the decreasing RTG and %RSG values. No effect on viability (%V) was found. It was concluded based on the RTG values that the optimum levels of toxicity had been achieved in this test. Due to excessive toxicity at and above 15 µg/ml, these levels were not plated for viability or 5-TFT resistance. Cytotoxicity of the positive control substance was acceptable.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the conditions of this test, the test article did not induce statistically significant (dose related) increase in the mutant frequency at the TK +/- locus in L5178Y cells at any dose level or experiment. Therefore, the test item Litsea cubeba is considered to be non-mutagenic.
Executive summary:

The genotoxicity of Litsea cubeba essential oil was tested in mouse L5178Y lymphoma cells according to OECD guideline 476 and under GLP conditions. Two experiments were performed, one with and without presence of S9 for 4 hours and one for 24 hours without presence of S9. The concentrations tested were 0.31 - 19.5 µg/ml (without S9, 4 hrs), 2.44 - 78 µg/ml (with S9, 4 hrs) and 0.31 - 20 µg/ml (without S9, 24 hrs). The mutation frequency at the TK+/- locus of the cells was determined to evaluate genotoxicity, relative suspension growth, cell viability and relative total growth were determined to check for cytotoxicity. Positive and vehicle controls were included.

The vehicle control mutant frequency value was within the acceptable range of 50-170 x 10E-6 viable cells. The positive control produced marked increases in mutant frequency/viable cell, indicating a well operating test system with and without S9. Evidence of marked dose-related (cyto)toxicity following exposure to the test item was found in both experiments (with or without S9), demonstrated by decreasing RTG and %RSG values. No effect on viability (%V) was found. The test article did not induce statistically significant (dose related) increase in the mutant frequency at the TK +/- locus in L5178Y cells at any dose level or experiment.

Under the conditions of this test, the test item Litsea cubeba essential oil is considered to be non-mutagenic.

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

Additional information

In vitro gene mutation study in bacteria (OECD TG 471)

The key study was an Ames test in bacteria according to OECD guideline 471. Two experiments (plate incorporation methodology and pre-incubation methodology) were performed with concentrations of the test substance ranging from 1.5 - 5000 µg/plate, with and without metabolic activation. 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. Therefore, Litsea cubeba was considered to be non-mutagenic under the conditions of this test.

Chromosome aberration study in mammalian cells (OECD TG 473)

The key study was an in vitro chromosome aberration test according to OECD guideline 473. Human lymphocytes were exposed to several concentrations of the test item ranging from 5 to 160 ug/ml in three groups, of which two had an exposure duration of 4 hours (with and without S9) and the other 24 hours. No significant changes in frequency of cells with aberrations and the number of polyploid cells was observed. Therefore, the test substance was considered to be non-clastogenic to human lymphocytes in vitro.

In vitro gene mutation study in mammalian cells (OECD TG 476)

The key study was an in vitro gene mutation test with mouse L5178Y lymphoma cells according to OECD guideline 476. Two experiments were performed, one with and without presence of S9 for 4 hours and one for 24 hours without presence of S9. The concentrations tested were 0.31 - 19.5 µg/ml (without S9, 4 hrs), 2.44 - 78 µg/ml (with S9, 4 hrs) and 0.31 - 20 µg/ml (without S9, 24 hrs). The test article did not induce statistically significant (dose related) increase in the mutant frequency at the TK +/- locus in L5178Y cells at any dose level or experiment. Therefore, the test item Litsea cubeba is considered to be non-mutagenic under the conditions of this test.

Justification for classification or non-classification

Litsea cubeba did not show any genotoxic potential in three in vitro genotoxicity tests. Therefore it can be concluded that the substance is not genotoxic and does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of 1272/2008/EC (CLP/EU-GHS).