Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (Wagner, 1999) : Negative in 5 strains

HPRT (BASF, 2014) : Negative

Chromosomal Aberration in vitro (Gudi, 2000) : Ambiguous

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The preliminary toxicity study was not performed in duplicates and can not count as a full repeat experiment. Seeing the results obtained, it is not considered to have an effect on the outcome of the study.
Principles of method if other than guideline:
The test system was exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and updates by Maron and Ames (1983).
GLP compliance:
yes
Remarks:
Wagner III VO and Caruthers SM
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Lot No: 9709000929
Purity: 98%
Target gene:
his
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:
Aroclor-1254 induced rat liver S9 mix
Test concentrations with justification for top dose:
in the preliminary mutagenicity test: <= 5000 µg/plate methyl ionone
in the mutagenicity assay: 25 - 5000 µg/plate methyl ionone
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Key result
Species / strain:
other: S. typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the highest doses
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥1800μg per plate +/- S9
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥1800μg per plate
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no
Conclusions:
A bacterial reverse mutation assay was conducted on Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and Escherichia coli strain WP2uvrA in the presence and absence of S9 activation. Methyl ionone in dimethyl sulfoxide was tested at 25, 75, 200, 600, 1800, and 5000 ug/plate, and no positive response was observed. Methyl ionone was concluded to be negative in the bacterial reverse mutation assay.
Executive summary:

The test article, methyl ionone, was tested in the bacterial reverse mutation assay using S. typhimurium tester strains TA98, TA100, TA1535, TA1537 and E. coli tester strain WP2uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the preliminary toxicity assay, was used to establish the dose range for the mutagenicitiy assay. The second phase, the mutagenicity assay, was used to evaluate the mutagenic potential of the test article. Dimethyl sulfoxide was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in dimethyl sulfoxide at a maximum concentration of approx. 500 mg/mL. In the preliminary assay, the maximum dose tested was 5000 ug/plate; this dose was achieved using a concentration of 100 mg/mL and a 50 uL plating aliquot. No precipitate was observed. Toxicity was observed at greater than or equal to 6667 ug/plate and at 5000 ug/plate with tester strain TA100 in the absence and presence of S9 activation, respectively. Toxicity was observed at greater than or 1000 ug/plate and at greater than or equal to 3333 ug/plate in the absence of S9 activation with tester strains TA1535 and TA1537, resp. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 ug/plate. In the mutagenicity assay, no positive response was observed. Under the conditions of this study, test article, methyl ionone, was concluded to be negative in the Bacterial Reverse Mutation Assay.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
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)
Remarks:
BASF SE, 67056 Ludwigshafen, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F12 medium
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital- and β-naphthoflavone induced rats
Test concentrations with justification for top dose:
1st Experiment
without S9 mix (4-hour exposure period)
0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL
with S9 mix (4-hour exposure period)
0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL

2nd Experiment
without S9 mix (4-hour exposure period)
0; 1.25; 2.50; 5.00; 10.00; 20.00; 40.00; 80.00 μg/mL
with S9 mix (4-hour exposure period)
0; 1.25; 2.50; 5.00; 10.00; 20.00; 40.00; 80.00 μg/mL
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, dimethyl sulfoxide (DMSO) was used as vehicle, which has been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available.
- Final concentration of the vehicle in the culture medium: 1% (v/v)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
ethyl methanesulfonate (without metabolic activation), 7,12-dimethylbenz[a]anthracene (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium: Ham's F12 + 10% FCS

DURATION
- Preincubation period: 1 week, elimination of spontaneous HPRT-deficient mutants by pretreatment with "HAT" medium
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT (mutation assays): Hypoxanthine-free Ham's F12 medium supplemented with 6-thioguanine (10 μg/mL), 1% (v/v) glutamine (200 mM), and 10% (v/v) fetal calf serum (FCS)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Cytotoxicity
The cloning efficiency (CE, %) was calculated for each test group as follows:

CEabsolute = (total number of colonies in the test group) / (total number of seeded cells in the test group) x 100

CErelative = (CEabsolute of the test group) / (CEabsolute of the vehicle/negative control) x 100

The number of colonies in every flask was counted and recorded. Using the formula above the values of absolute cloning efficiencies were
calculated. Based on these values the relative cloning efficiencies of the test groups were calculated and given in percentage compared with the
respective CEabsolute value of the corresponding vehicle/negative control (vehicle/negative control = 100%).

Mutant frequency
The number of colonies in every flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized to 10^6 cells seeded.
The uncorrected mutant frequency (MFuncorr.) per 10^6 cells was calculated for each test group as follows:

MFuncorr. = (total number of mutant colonies) / (number of seeded cells) x 10^6

The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency (MFcorr.):

MFcorr. = (MFuncorr.) / (CE2 absolute) x 100
Statistics:
MS EXCEL function RGP
The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0.
However, both, biological and statistical significance will be considered together.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
experiment 1: 50 µg/mL and above, experiment 2: 40 µg/mL and above
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the vehicle control groups and the positive substances ethyl methanesulfonate and 7,12-dimethylbenz[a]anthracene were within the histrorical control data range.

MUTANT FREQUENCY:
- No relevant increase in the number of mutant colonies was observed with or without S9 mix.
- The positive control substances ethyl methanesulfonate (without S9 mix; 300 μg/mL) and 7,12-dimethylbenz[a]anthracene (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies.

CYTOTOXICITY:
- Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were observed in both experiments in the absence and presence of S9 mix, at least at the highest applied concentrations.

CELL MORPHOLOGY:
- After 4 hours treatment the morphology and attachment of the cells was adversely influenced (grade > 2) in all experimental parts tested for gene mutations at least at the highest applied concentrations.

Table 1: Summary of results - experimental parts without S9 mix

 

Exp.

Exposure period [h]

Test groups [µg/mL]

S9 mix

Prec.*

Genotoxicity** MFcorr.
[per 106 cells]

Cytotoxicity***

CE1 [%]

CE2 [%]

1

4

Vehicle control1

-

n.d.

0.69

100.0

100.0

 

 

1.56

-

-

n.c.1

92.9

n.c.1

 

 

3.13

-

-

2.75

98.3

100.8

 

 

6.25

-

-

3.41

94.6

100.6

 

 

12.5

-

-

3.82

102.2

107.3

 

 

25.0

-

-

1.28

95.4

108.4

 

 

50.0

-

-

n.c.2

0.0

n.c.2

 

 

100.0

-

-

n.c.2

0.0

n.c.2

 

 

Positive control2

-

n.d.

89.30

81.3

95.3

 

 

 

 

 

 

 

 

2

4

Vehicle control1

-

n.d.

2.95

100.0

100.0

 

 

1.25

-

-

n.c.1

86.8

n.c.1

 

 

2.5

-

-

0.00

95.4

98.3

 

 

5.0

-

-

0.69

100.6

93.2

 

 

10.0

-

-

0.00

95.4

100.4

 

 

20.0

-

-

2.93

96.3

103.2

 

 

40.0

-

-

n.c.2

0.0

n.c.2

 

 

80.0

-

-

n.c.2

0.0

n.c.2

 

 

Positive control2

-

n.d.

121.64

79.2

80.8

 

 

Table 2: Summary of results - experimental parts wit S9 mix

 

Exp.

Exposure period [h]

Test groups [µg/mL]

S9 mix

Prec.*

Genotoxicity** MFcorr.
[per 106 cells]

Cytotoxicity***

CE1 [%]

CE2 [%]

1

4

Vehicle control1

+

n.d.

2.09

100.0

100.0

 

 

1.56

+

-

n.c.1

89.9

n.c.1

 

 

3.13

+

-

2.08

92.3

103.8

 

 

6.25

+

-

2.33

89.2

111.4

 

 

12.5

+

-

11.39

90.7

112.4

 

 

25.0

+

-

6.20

83.8

109.9

 

 

50.0

+

-

n.c.2

0.0

n.c.2

 

 

100.0

+

-

n.c.2

0.0

n.c.2

 

 

Positive control2

+

n.d.

242.56

85.6

81.2

 

 

 

 

 

 

 

 

2

4

Vehicle control1

+

n.d.

1.29

100.0

100.0

 

 

1.25

+

-

n.c.1

91.6

n.c.1

 

 

2.5

+

-

n.c.1

92.2

n.c.1

 

 

5.0

+

-

0.00

87.2

91.5

 

 

10.0

+

-

2.43

85.2

89.5

 

 

20.0

+

-

0.32

80.3

90.2

 

 

40.0

-

-

1.65

46.4

97.3

 

 

80.0

+

-

n.c.2

0.0

n.c.2

 

 

Positive control2

+

n.d.

169.99

99.1

76.2

 

 

*        Precipitation in culture medium at the end of exposure period

**      Mutant frequency MFcorr.: mutant colonies per 106cells corrected with the CE2 value

***    Cloning efficiency related to the respective vehicle control

n.c.1   Culture was not continued since a minimum of only four analysable concentrations are required

n.c.2   Culture was not continued due to strong cytotoxicity

1         DMSO 1% (v/v)

2         EMS 300 μg/mL

Conclusions:
Methyl ionone was found to be non mutagenic in mammalian cells in this HPRT.
Executive summary:

In the first of 2 hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus assays, duplicate cultures of Chinese hamster ovary cells (1x10(6) cells/ml) in flasks were incubated for 4 hours with 0.2 ml of 0 (vehicle),1.56, 3.13, 6.25, 12.50, 25.00, 50.00, or 100.00 μg test substance/ml in the presence of 4.0 ml S9 mix (liver homogenate from phenobarbital/-naphthoflavone-induced rats) in 16 ml Ham’s F-12 medium or in the absence of S9 mix in 20 ml Ham’s F-12 medium with 10% fetal calf serum. Prior to test substance treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment for 3-4 days with "HAT" medium (Ham's F12 medium supplemented with hypoxanthine, aminopterin, thymidine and 10% fetal calf serum), followed by incubation in Ham’s F-12 medium with 10% fetal calf serum for 3-4 days and then 1x10(6) logarithmically growing cells were incubated for 20-24 hours in 20 ml of Ham’s F-12 medium with 10% fetal calf serum in flasks. All incubations were performed at 37°C with a relative humidity of = 90% in a 5% (v/v) CO2 atmosphere. Concurrent positive controls (ethyl methanesulphonate for cultures without S9 and 7,12-dimethylbenz[a]anthracene for cultures with S9) were run. Following exposure, cells were rinsed several times with HBSS (Hanks balanced salt solution), 20 ml Ham’s F-12 medium with 10% fetal calf serum was added, and then flasks were left to stand for 3-4 days. For selection of mutants, 3x10(5) cells from each test group were seeded in 10 ml selection medium (hypoxanthine-free Ham's F12 medium supplemented with 6-thioguanine, 1% stable glutamine, and 10% fetal calf serum) in 75 cm2 flasks (6 flasks/concentration). After 6-7 days of incubation, medium was removed, remaining colonies were fixed with methanol, stained with Giemsa, and counted. Cytotoxicity was determined using cloning efficiency (survival and viability) determinations, which were obtained from incubation of 200 cells per concentration in 25 cm2 flasks using 5 ml Ham’s F-12 medium with 10% fetal calf serum followed by treatments described above. Absolute and relative cloning efficiency (%) were calculated. Mutant frequency per 10(6) cells was calculated and corrected for cloning efficiency. pH, osmolarity, solubility (precipitation), and cell morphology (microscopic examination) also were recorded. Statistical analyses were performed. The results were considered positive if there was an increase in the corrected mutation frequencies both above the concurrent negative control values and the lab’s historical negative control data range, any increase in mutant frequencies was reproducible, and there was a statistically significant increase in mutant frequencies with evidence of a dose-response relationship.

For the vehicle control, mutant frequency per 10(6) cells was 0.56 (uncorrected) and 0.69 (corrected) for cultures without S9 and 1.67 (uncorrected) and 2.09 (corrected) for cultures with S9. The positive control substances induced a clear increase in mutation frequencies. Osmolarity and pH values were not influenced by test substance treatment. In the absence and the presence of S9 mix, no precipitation in culture medium was observed up to the highest concentration. The acceptance criteria were fulfilled.

No mutagenic effects were observed in the culture treated with Methyl ionone. Cytotoxicity was observed at 50 and 100 μg test substance/ml.

Methyl ionone is considered to be non mutagenic in this HPRT.

In the second of 2 hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus assays, duplicate cultures of Chinese hamster ovary cells (1x10(6) cells/ml) in flasks were incubated for 4 hours with 0.2 ml of 0 (vehicle), 1.25, 2.50, 5.00, 10.00, 20.00, 40.00, or 80.00 μg test substance/ml in the presence of 4.0 ml S9 mix (liver homogenate from phenobarbital/-naphthoflavone-induced rats) in 16 ml Ham’s F-12 medium or in the absence of S9 mix in 20 ml Ham’s F-12 medium with 10% fetal calf serum. Prior to test substance treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment for 3-4 days with "HAT" medium (Ham's F12 medium supplemented with hypoxanthine, aminopterin, thymidine and 10% fetal calf serum), followed by incubation in Ham’s F-12 medium with 10% fetal calf serum for 3-4 days and then 1x10(6) logarithmically growing cells were incubated for 20-24 hours in 20 ml of Ham’s F-12 medium with 10% fetal calf serum in flasks. All incubations were performed at 37°C with a relative humidity of = 90% in a 5% (v/v) CO2 atmosphere. Concurrent positive controls (ethyl methanesulphonate for cultures without S9 and 7,12-dimethylbenz[a]anthracene for cultures with S9) were run. Following exposure, cells were rinsed several times with HBSS (Hanks balanced salt solution), 20 ml Ham’s F-

12 medium with 10% fetal calf serum was added, and then flasks were left to stand for 3-4 days. For selection of mutants, 3x10(5) cells from each test group were seeded in 10 ml selection medium (hypoxanthine-free Ham's F12 medium supplemented with 6-thioguanine, 1% stable glutamine, and 10% fetal calf serum) in 75 cm2 flasks (6 flasks/concentration). After 6-7 days of incubation, medium was removed, remaining colonies were fixed with methanol, stained with Giemsa, and counted. Cytotoxicity was determined using cloning efficiency (survival and viability) determinations, which were obtained from incubation of 200 cells per concentration in 25 cm2 flasks using 5 ml

Ham’s F-12 medium with 10% fetal calf serum followed by treatments described above. Absolute and relative cloning efficiency (%) were calculated. Mutant frequency per 10(6) cells was calculated and corrected for cloning efficiency. pH, osmolarity, solubility (precipitation), and cell morphology (microscopic examination) also were recorded. Statistical analyses were performed. The results were considered positive if there was an increase in the corrected mutation frequencies both above the concurrent negative control values and the lab’s historical negative control data range, any increase in mutant frequencies was reproducible, and there was a statistically significant

increase in mutant frequencies with evidence of a dose-response relationship.

For the vehicle control, mutant frequency per 10(6) cells was 2.78 (uncorrected) and 2.95 (corrected) for cultures without S9 and 1.11 (uncorrected) and 1.29 (corrected) for cultures with S9. The positive control substances induced a clear increase in mutation frequencies. Osmolarity and pH values were not influenced by test substance treatment. In the absence and the presence of S9 mix, no precipitation in culture medium was observed up to the highest concentration. The acceptance criteria were fulfilled.

No mutagenic effects were observed in the culture treated with Methyl ionone. Cytotoxicity was observed at 80 μg test substance/ml.

Methyl ionone is considered to be non mutagenic in this HPRT.

Genetic toxicity in vivo

Description of key information

In vivo Micronucleus Test (Gudi, 2000) : Negative

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome and genome mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication/ study report which meets basic scientific principles
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
Procedure according to Heddle, 1973; Hayashi et al., 1994; Mavournin et al. 1990.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Details on species / strain selection:
ICR mice from Harlan Sprague Dawley, Inc.
Sex:
male/female
Details on test animals and environmental conditions:
Age 6 to 8 weeks old
Body weight range : Male: 25.0-29.6 g, Female: 25.2-29.7 g
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
Volume injected: 20 ml/kg bw
Duration of treatment / exposure:
Bone marrow was collected 24 and 48 hours after dose administration.
Frequency of treatment:
single application
Post exposure period:
24, 48 h
Remarks:
Doses / Concentrations:
462.5, 925, or 1850 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
not specified
Positive control(s):
- Cyclophosphamide
- Route of administration: IP injection
- Doses / concentrations: 2.5 mg/ml bw
Tissues and cell types examined:
erythrocytes for the bone marrow of the femurs
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
24 and 48 h

DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice, the femurs were exposed, cut just above the knee and the bone marrow was aspirated into a syringe containing fetal bovine serum. The cells were centrifuged; the supernatant was drawn off and then resuspended. The bone marrow suspension was spread onto a clean glass slide (two to four slides were prepared for each mouse). The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
2000 polychromatic erythrocytes were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
No effects, slight to moderate reduction (up to 35%) in the ratio of  
polychromatic erythrocytes to total erythrocytes were observed in some of  the 
test article-treated groups relative to the respective vehicle controls. 
A statistically significant increase in micronucleated  polychromatic 
erythrocytes (8 MNPCE/10000 PCE) was observed in male mice 24 hrs after 
treatment with 925 mg/kg. 
However, this response is not considered biologically relevant (each of the 
five animals had no more  than 3 MPCE, which are within the range of historical 
solvent control:  0-7 MN/2000 PCE/animal). 
No significant increase and no dose responsiveness increase was observed in any 
other test article treated group regardless of dose level, sex, or bone marrow 
collection time.
Conclusions:
The results of the assay indicate that under the conditions described in this report, the test articl, MEthyl ionone did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. Methyl ionone was concluded to be negative in the mouse micronucleus assay.
Executive summary:

A mouse micronucleus assay (Hayashi et al., Heddle, 1973; Mavournin et al., 1990) was conducted in male and female ICR mice (5/sex/dose). Methyl ionone in corn oil, the vehicle alone or the positive control (2.5 mg/ml cyclophosphamide in sterile distilled water) were administered by intraperitoneal injection at a constant volume of 20 ml/kg body weight. The test animals were dosed with 462.5, 925, or 1850 mg/kg body weight and bone marrow was collected 24 and 48 hours after dose administration. Mortality was observed in only 1/15 male mice receiving 1850 mg/kg. This animal was replaced at the time of bone marrow collection with a replacement animal that also

received 1850 mg/kg. Immediately following sacrifice, the femurs were exposed, cut just above the knee and the bone marrow was aspirated into a syringe containing fetal bovine serum. The cells were centrifuged; the supernatant was drawn off and then resuspended. The bone marrow suspension was spread onto a clean glass slide (two to four slides were prepared for each mouse). The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted. Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Slight to moderate reduction (up to 35%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. A statistically significant increase in micronucleated polychromatic erythrocytes (8 MNPCE/10000 PCE) in test article-treated group relative to the respective vehicle control group was observed in male mice 24 hrs after treatment with 925 mg/kg. However, this response is not considered biologically relevant, since each of the five animals had no more than 3 MPCE. These numbers of MNPCE are within the range of historical solvent control (0-7 MN/2000 PCE/animal). No significant increase and no dose responsiveness increase was observed in any other test article treated group regardless of dose level, sex, or bone marrow collection time. It was concluded that methyl ionone did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice.

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

Additional information

The genetic toxicity of methylionone was analyzed in a bacterial reverse mutation assay according Ames et al. (Wagner, 1999). The bacteria strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were tested with and without metabolic activation by Aroclor-1254 induced rat liver S9 mix according to the plate incorporation method. Although some cytotoxicity in some strains at higher concentrations were noted, no mutagenic effect of methylionone was detected.

A second Ames test was performed with methyl ionone (Quest, 1980). Methyl ionone was tested in Salmonella typhimurium TA 1535, TA1537, TA1538, TA98, and TA100 with and without metabolic activation (S9) using 0 (solvent control), 0.01, 0.1, 1, or 10 μl/well of the test substance in dimethyl sulphoxide. The number of colonies on each plate was counted and the mean number of revertant colonies per treatment group was calculated. At the tested concentrations, no significant increase in the number of revertant colonies was reported with any of the strains, with or without S9. A slight increase was observed with strain TA1537 at 0.01 ul/plate, and due to this slight increase, a repeat test was conducted at concentrations of 0.005, 0.01, 0.05, and 0.1 ul/plate. No significant increase in the number of revertant colonies was reported at any of the tested concentrations.

It was concluded that no evidence of mutagenic potential of Methyl ionone was obtained in this bacterial test system at the dose levels used.

Methyl ionone 70 (CAS 1335-46-2) was tested in a HPRT test in CHO cells with and without metabolic activation (BASF SE, 2014). Cytotoxicity was found in the absence and presence of S9 mix, at least at the highest applied concentrations. The test substance did not cause any relevant increase in the mutant frequencies in two independent experiments.

In a study using a chromosome aberration assay the genetic toxicity in vitro was analyzed in Chinese hamster ovary cells (Gudi, 2000). The cells were incubated for 4 h or 20 h with 12.5, 25, and 50 ug/ml methyl ionone dissolved in DMSO with and without metabolic activation. 0.1 and 0.2 ug/ml mitomycin C was used as positive control in the non-activated assay and 10 and 20 ug/ml cyclophosphamide in the activated assay. After harvesting the cells, frequencies of cells with structural and numerical aberrations were calculated by analyzing a minimum of 200 metaphase spreads and scoring for chromatid-type and chromosome-type aberrations. The resulting findings were ambiguous, as an induction of structural chromosome aberrations was found in the absence of S9 metabolic activation, but not in the presence of S9 metabolic activation. Furthermore, it was negative in both the absence and presence of S9 metabolic activation for the induction of numerical chromosome aberrations.

The genetic toxicity in vivo was analyzed in mammalian erythrocyte micronucleus test (Gudi, 2000). Five male and five female IRC mice per dose were intraperitoneally injected with 20 ml/kg bw at concentrations of 462.5, 925, or 1850 mg/kg body weight methylionone in corn oil. 24 h and 48 h after dosing, animals were sacrificed and bone marrow cells were isolated from the femurs.

Only a light to moderate reduction (up to 35%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the methylionone-treated groups relative to the respective vehicle controls. A statistically significant increase in micronucleated  polychromatic erythrocytes (8 MNPCE/10000 PCE) was observed in male mice 24 hrs after treatment with 925 mg/kg. However, this response is not considered biologically relevant (each of the five animals had no more than 3 MPCE, which are within the range of historical solvent control:  0-7 MN/2000 PCE/animal). Also, no significant increase and no dose responsiveness was observed in any other test article treated group regardless of dose level, sex, or bone marrow collection time. Thus, methhylionone could be regarded as not mutagenic in vivo.


Short description of key information:
Genetic toxicity:
- in vitro in bacteria: negative (Ames test)
- in vitro in mammalian cells: negative (HPRT)
- in vitro in mammalian cells: ambigious (chromosome aberration, CHO cells)
- in vivo: negative (MNT)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Although ambiguous results were found in the chromosome aberration test in mammalian cells in vitro, those foundings were not confirmed in a cytogenicity test in vivo.

Moreover no genetic toxicity could be detected in bacteria as well as in mammalian cells in vitro (HPRT).

Thus, due to overall negative results for genetic toxicity, no classification is required.