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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): The substance musk ketone did not induce mutagenicity in S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A in the presence or absence of phenobarbital/β-naphthoflavone-induced rat liver S9 metabolic activation (OECD 471, GLP).

In vitro cytogenicity (chromosome aberration) study in mammalian cells: the substance musk ketone was concluded to be negative for the induction of chromosome aberrations in the presence and absence of Aroclor 1254-induced rat liver S9 metabolic activation in CHO-K1 cells. (Equivalent or similar to OECD 473).

Gene mutation (mammalian cell gene mutation assay): there was no evidence of induced mutant colonies over background in L5178Y mouse lymphoma cells exposed to musk ketone  in the presence or absence of Aroclor-induced -induced rat liver S9 mammalian metabolic activation (Equivalent or similar to OECD 476/GLP).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 December 2016 - 07 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: International Flavors & Fragrances; 0008085963
- Expiration date of the lot/batch: 20 August 2018
- Purity: 99.8%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 °C on the day of each experiment. No correction for purity was required. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthaflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1 (plate incorporation; + and -S9): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate

Experiment 1 (pre-incubation; + and -S9): 15, 50, 150, 500, 1500, 5000 μg/plate
Vehicle / solvent:
The test item was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was selected as the vehicle.
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 (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation);
Experiment 2: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: Approximately 48 hours for both experiments

NUMBER OF REPLICATIONS: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the growth of the bacterial background lawn
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
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 (especially if accompanied by an out-of-historical range response.
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Species / strain:
other: S. typhimurium strains TA1535, TA1537, TA98 and TA100; E. coli WP2uvrA
Remarks:
Experiment 1
Metabolic activation:
with and 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:
valid
Species / strain:
other: S. typhimurium strains TA1535, TA1537, TA98 and TA100; E. coli WP2uvrA
Remarks:
Experiment 2
Metabolic activation:
with and 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:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Manual counts were performed at 5000 μg/plate because of test item precipitation (Experiment 1). Manual counts were performed at and above 1500 μg/plate because of test item precipitation. (Experiment 2).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: The historical ranges of the positive control reference items for 2015 and 2016 are presented in Appendix 1.
- Negative (solvent/vehicle) historical control data: Combined historical negative and solvent control ranges for 2015 and 2016 are presented in Appendix 1.
Conclusions:
In a reverse bacterial mutation assay (Ames test), musk ketone was negative with and without metabolic activatiion.
Executive summary:

In a reverse gene mutation assay in bacteria (QC11WT), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A were exposed to musk ketone (99.8%) in DMSO at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000μg/plate (plate incorporation; experiment 1) and 15, 50, 150, 500, 1500, 5000 μg/plate (20 minute pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (Phenobarbital/β-naphthoflavone-induced rat liver S9).

Musk ketone was tested up to the limit concentration (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Firmenich Incorporated (Princeton, N J, USA); 65659.9305.
- Purity: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at 2-6°C and protected from exposure to light.
Species / strain / cell type:
other: CHO-K1
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, Rockville, MD, USA
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
24 and 48 hrs; -S9: 4.3, 8.5, 17 and 34 µg/ml
4 hr; +S9: 1.25, 2.5, 5 and 10 µg/ml (24 and 48 hr harvest)
4 hr; +S9: 8, 10, 12 and 14 µg/ml (24 hr harvest)
Vehicle / solvent:
Acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
triethylenemelamine
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 3 x 10~5 cells/25cm2 flask (24-hr harvest) or 1 x 10~5 cells/25cm2 flask (48-hr harvest)

DURATION
- Exposure duration: In the non-activated study, the cells were continuously exposed to treatment in the incubator at 37 ±1°C prior to harvest at approximately 24 or 48 hr. In the S9 activated study, the cells were exposed to treatment in the incubator for 4 hr at 37 ±1°C.

- Expression time (cells in growth medium): After the exposure period, medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline, re-fed with complete medium and returned to the incubator until harvest at approximately 24 or 48 hr.

SPINDLE INHIBITOR (cytogenetic assays): Two hours prior to the scheduled cell harvest, Colcemid was added to duplicate flasks for each treatment condition (activated and non-activated studies) at a final concentration of 0.1 µg/ml and the flasks incubated for an additional 2 hr.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Metaphase cells with 20 + 2 centromeres were examined under oil immersion without prior knowledge of treatment groups.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Whenever possible, a minimum of 200 metaphase spreads (100/duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations. Chromatid and isochromatid gaps were recorded but not included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (The mitotic index was recorded as the percentage of cells in mitosis/500 cells counted); cloning efficiency.

OTHER EXAMINATIONS:
- Determination of polyploidy: Polyploidy was evaluated as the percentage of polyploid cells/100 metaphase cells
counted.

Statistics:
P ≤0.025, one-sided Fisher's exact test.
Species / strain:
other: CHO-K1
Remarks:
24 hr
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: CHO-K1
Remarks:
48 hrs
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: CHO-K1
Remarks:
4 hr (24 hr harvest)
Metabolic activation:
with
Genotoxicity:
other: structural aberrations at 10 µg/ml only
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: CHO-K1
Remarks:
4 hrs (48 hr harvest)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: CHO-K1
Remarks:
4 hr (24 harvest repeat)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Specific cytotoxicity data was not provided. The authors indicated that cytotoxicity based on percent relative cloning efficiency was satisfactory to provide adequate metaphase cells for examination of chromosomal aberrations.
Conclusions:
Musk ketone is negative in the mammalian cytogenetic assay using CHO-K1 cells
Executive summary:

In an in vitro cytogenicity (chromosome aberration) study in mammalian cells (Api et al., 1996), CHO-K1 cells were exposed to musk ketone (>99%) in acetone. In the presence of metabolic activation (Aroclor 1254-induced rat liver S9), cells were exposed to 1.25, 2.5, 5 and 10 µg/ml for 4 hours (24 and 48 hr harvest) and 8, 10, 12 and 14 µg/ml  for 4 hours (24 hr harvest). In the absence of metabolic activation, cells were exposed to 4.3, 8.5, 17 and 34 µg/ml for 24 and 48 hours.

Cytotoxicity was satisfactory to provide adequate metaphase cells for examination of chromosomal aberrations. Positive controls induced the appropriate response. There was no evidence of a statistically significant increase in chromosomal aberrations without metabolic activation or with metabolic activation for 4 hours (48 hour harvest time point). At the dose level of 10.0 µg/ml, there was a statistically significant increase (12%) in structural aberrations, but not numerical aberrations, in the presence of metabolic activation at the 24-hr harvest time point.  In a confirmatory assay with dose levels of 8, 10, 12 and 14.0 µg/ml, in the presence of metabolic activation at the 24-hr harvest time point, there was no significant increase in structural or numerical aberrations. On the basis of the overall findings and the non-reproducibility at a single dose level, it was concluded that musk ketone is negative in the mammalian cytogenetic assay using CHO-K1 cells.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline [In vitro mammalian cytogenetics [Chromosome aberration]] OECD 473 for in vitro cytogenetic mutagenicity data.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 July 1993 to 13 December 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
OECD 476 (21 July 1997)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Firmenich Incorporated (Princeton, N J, USA); 65659.9305.
- Purity: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at 2-6°C and protected from exposure to light.
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Company, Research Triangle Park, NC, USA.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Maintained in Fischers’ Medium for Leukemic Cells of Mice with 0.1% Pluronics supplemented with 10% horse serum and 4mM L-glutamine. Gassed at 5 ±1% CO2 in air.
- Properly maintained: Yes
- Periodically 'cleansed' against high spontaneous background: Cleansed before use in the assay
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary test: 0.5 - 4000 µg/ml

Mutagenesis assays
-S9: 100 – 4000 µg/ml
+S9: 1.0 – 100 µg/ml

Mutagenesis assays: selected for cloning
-S9: 700 – 4000 µg/ml
+S9: 2.0 – 35 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone; lot no. 920807 (Fischer Chemical Company)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hrs + and – S9.
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): 10-12 days

SELECTION AGENT (mutation assays): Trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (preliminary test); relative total growth (main tests)



Evaluation criteria:
In the evaluation of the data, increases in mutant frequencies which occur only at highly toxic concentrations are not considered biologically relevant. The following criteria are considered:

Positive: A test article producing a concentration-related increase in mutant frequency with more than one dose level in the 10% or greater total growth range exhibiting a mutant frequency which is 2-fold greater than the background level will be considered positive.

Equivocal: A test article producing no concentration-related increase in mutant frequency but any one dose level in the 10% or greater total growth range exhibiting a mutant frequency which is 2-fold greater than the background level will be considered equivocal.

Negative: A test article producing no concentration-related increase in mutant frequency and no 2-fold or greater increase above the background in dose levels in the 10% or greater total growth range will be considered negative.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
30 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: The osmolality of the solvent control was 410 mOsm/kg and the osmolality of the top dose, 4000 µg/ml, was 363 mOsm/kg.


RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test based on cell population growth relative to the solvent controls. Cells were exposed to solvent alone and 9 concentrations of the test article ranging from 0.5 – 4000 µg/ml for 4 hrs in the absence and presence of metabolic activation. A high dose of 5000 µg/ml could not be achieved in acetone due to a lack of solubility. Cell population density was determined 24 and 48 hrs after exposure.

Conclusions:
Under the conditions of this MLA in L5178Y cells, musk ketone was negative in the presence and absence of Aroclor-induced rat liver S9 metabolic activation.
Executive summary:

In a mammalian cell gene mutation assay (thymidine kinase MLA; 23793), mouse lymphoma L5178Y cells cultured in vitro were exposed to musk ketone (>99%) in acetone at concentrations of  700 – 4000 µg/ml in the absence and 2.0 – 35 µg/ml in the presence of mammalian metabolic activation (Aroclor-induced rat liver S9) for 4 hours.  

Musk ketone was tested up to cytotoxic concentrations in the presence of metabolic activation and up to precipitating concentrations in the absence of metabolic activation. The positive controls induced the appropriate response.  There was no evidence of induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for OECD 476 for in vitro mutagenicity (mammalian cell gene mutation) data.  

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test)

There is one in vitro gene mutation study (reverse gene mutation assay/Ames test in bacteria) available.

In a reverse gene mutation assay in bacteria (OECD 471/GLP), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A were exposed to musk ketone (99.8%) in DMSO at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000μg/plate (plate incorporation; experiment 1) and 15, 50, 150, 500, 1500, 5000 μg/plate (20 minute pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (Phenobarbital/β-naphthoflavone-induced rat liver S9). Musk ketone was tested up to the limit concentration (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

In vitro cytogenicity (chromosome aberration) study in mammalian cells:

There is one in vitro cytogenicity (chromosome aberration) study in mammalian cells available.

In an in vitro cytogenicity (chromosome aberration) study in mammalian cells (Equivalent or similar to OECD 473), CHO-K1 cells were exposed to musk ketone (>99%) in acetone. In the presence of metabolic activation (Aroclor 1254-induced rat liver S9), cells were exposed to 1.25, 2.5, 5 and 10 µg/ml for 4 hours (24 and 48 hr harvest) and 8, 10, 12 and 14 µg/ml  for 4 hours (24 hr harvest). In the absence of metabolic activation, cells were exposed to 4.3, 8.5, 17 and 34 µg/ml for 24 and 48 hours. Cytotoxicity was satisfactory to provide adequate metaphase cells for examination of chromosomal aberrations. Positive controls induced the appropriate response. There was no evidence of a statistically significant increase in chromosomal aberrations without metabolic activation or with metabolic activation for 4 hours (48 hour harvest time point). At the dose level of 10.0 µg/ml, there was a statistically significant increase (12%) in structural aberrations, but not numerical aberrations, in the presence of metabolic activation at the 24-hr harvest time point.  In a confirmatory assay with dose levels of 8, 10, 12 and 14.0 µg/ml, in the presence of metabolic activation at the 24-hr harvest time point, there was no significant increase in structural or numerical aberrations. On the basis of the overall findings and the non-reproducibility at a single dose level, it was concluded that musk ketone is negative in the mammalian cytogenetic assay using CHO-K1 cells.

Gene mutation (mammalian cell gene mutation assay):

There is one gene mutation (mammalian cell gene mutation assay) available.

In a mammalian cell gene mutation assay (Equivalent or similar to OECD 476/GLP), mouse lymphoma L5178Y cells cultured in vitro were exposed to musk ketone (>99%) in acetone at concentrations of  700 – 4000 µg/ml in the absence and 2.0 – 35 µg/ml in the presence of mammalian metabolic activation (Aroclor-induced rat liver S9) for 4 hours.  Musk ketone was tested up to cytotoxic concentrations in the presence of metabolic activation and up to precipitating concentrations in the absence of metabolic activation. The positive controls induced the appropriate response.  There was no evidence of induced mutant colonies over background.

All three studies are negative and the results are suitable for use in the human health hazard assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance musk ketone (CAS No. 81-14-1) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.