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Genetic toxicity in vitro

Description of key information

Ames test (Wagner, 1999) : Negative in 5 strains (OECD 471)

HPRT (BASF, 2014) : Negative (OECD 476)

In vitro Micronucleus Test (RIFM, 2014): Negative (OECD 487)

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:
1999
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
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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 667 and 5000 µg/plate methyl ionone
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 1000 and 3333 µg/plate methyl ionone
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 1000 and 3333 µg/plate methyl ionone
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:
>= 1800 µg/plate methyl ionone
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 1800 µg/plate methyl ionone
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

Phase I: preliminary toxicity study: Concentrations at which cytotoxicity was oserved [µg/plate]

(converse results of two sources)

Results of FFHPVC Terpene Consortium:

   TA 98  TA 100  TA 1535  TA 1537  WP2 uvr A
 - S9    5000  3333  3333  
 + S9    667  1000  1000  

Results of RIFM monograph:

   TA 98  TA 100  TA 1535  TA 1537  WP2 uvr A
 - S9   >= 6667 >= 1000 >= 1000  
 + S9   >= 5000 >= 3333 >= 3333  

Phase II: mutagenicity study

(concordant results of two sources)

no evidence of mutagenicity.

Toxicity observed at concentrations of 1800 µg/plate with TA 100 and TA 1537.

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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. QA statement)
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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 September 2013 to 11 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2010
Deviations:
yes
Remarks:
in the S9-activated 4-hour exposure group, the % of cells with micronuclei was outside the historical solvent control range of 0 to 0.5% and in the preliminary toxicity assay HPBL cells were not treated for 4-h in the presence/absence of S9 activation.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Test Substance Synonyms: Methyl Ionone Alpha Extra; IPC 133720
Test Substance CAS Number reported: 7779-30-8
Test substance composition (according to CoA): Methyl ionone alpha, CAS 7779-30-8, 61.5% and Methyl ionone beta, CAS 127-43-5, 20.1%. As this fits the Boundary composition description, we consider the test item as being identical to the registered substance "Reaction mass of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one".
Test Substance Batch/Lot Number: HZ04827049
Test Substance Purity: 81.6% (Sum of main isomers per Certificate of Analysis)
Test Substance Molecular Weight: 206.29 (provided by Sponsor)
Test Substance Description: Clear colorless liquid
Storage Conditions: Room temperature , stored protected from light
Target gene:
In vitro Micronucleus in Mammalian cells
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 (Lot Nos. 3076 and 3095) was obtained from Molecular Toxicology Inc. (Boone, NC). Each bulk preparation of S9 was assayed by the supplier for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.
Immediately prior to use, the S9 was thawed and mixed with a cofactor pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 μL S9 per milliliter medium (RPMI 1640 serum-free medium supplemented with 100 units penicillin/mL and 100 μg streptomycin/mL and 2 mM L-glutamine).
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the doses tested ranged from 0.206 to 2060 μg/mL (10 mM). Substantial cytotoxicity [≥ 50 to 60% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was not observed at any dose level in the S9-activated 4-hour exposure group. Substantial cytotoxicity was observed at dose levels ≥ 61.8 μg/mL in the non-activated 4 and 24-hour exposure groups. Based on these findings, the doses chosen for the micronucleus assay ranged from 5 to 70 μg/mL for the non-activated 4 and 24-hour exposure groups, and from 20 to 650 μg/mL for the S9-activated 4-hour exposure group.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
A solubility test was conducted using water and DMSO. The vehicle was selected in order of preference that permitted preparation of the highest soluble or workable concentration, up to 50 mg/mL in aqueous solvents and up to 500 mg/mL in organic solvents.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
vinblastine
Details on test system and experimental conditions:
Species: Peripheral blood lymphocytes were obtained from a healthy non-smoking 39-year-old adult female on 24 September 2013 for the preliminary toxicity assay and from the same donor on 11 November 2013 for the repeat assay. For the initial assay, peripheral blood lymphocytes were obtained from a healthy non-smoking 29-year-old adult female on 08 October 2013. The donors had no recent history of radiotherapy, viral infection or the administration of drugs, and who had abstained from alcohol for 12 or more hours. This system has been demonstrated to be sensitive to the genotoxicity test for detection of micronuclei of a variety of chemicals.

Preliminary Toxicity Test for Selection of Dose Levels:
HPBL were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. The precipitation in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality of the vehicle, the highest dose level, the lowest precipitating dose level, and the highest soluble dose level in treatment medium was measured. Dose levels for the micronucleus assay were based upon post-treatment toxicity (CBPI relative to the vehicle control).

Micronucleus Assays:
Five to ten dose levels were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. The precipitation in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose level evaluated for the micronucleus was based on 50 to 60% cytotoxicity (CBPI relative to the vehicle control). Two additional dose levels were included in the evaluation.

Treatment of Target Cells (Preliminary Toxicity Test and Definitive Assays):
The pH was measured at the highest test substance concentration prior to dosing. Treatment was carried out by refeeding the cultures with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure, to which was added 50 μL of test substance dosing solution or vehicle alone. In the definitive assay, positive control cultures were resuspended in either 5 mL of complete medium for the non-activated studies, or 5 mL of the S9 reaction mixture (4 mL serum free medium + 1 mL of S9 cofactor pool), to which was added 50 μL of positive control in solvent.
After the 4 hour treatment in the non-activated and the S9-activated studies, the cells were centrifuged, the treatment medium was aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium containing cytoB at 6.0 μg/mL and returned to the incubator under standard conditions. For the 24 hour treatment in the non-activated study, cytoB (6.0 μg/mL) was added at the beginning of the treatment.

Collection of Cells (Preliminary Toxicity Test and Definitive Assays):
Cells were collected after being exposed to cyto B for 24 hours (± 30 minutes), 1.5 to 2 normal cell cycles, to ensure identification and selective analysis of micronucleus frequency in cells that have completed one mitosis evidenced by binucleated cells (Fenech and Morley, 1986). The cyto B exposure time for the 4 hour treatment in the non-activated and the S9-activated studies was 20 hours (± 30 minutes).
Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and were stored overnight or longer at 2-8°C, or the slides were prepared immediately after harvest. To prepare slides, the cells were collected by centrifugation and if necessary, the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange and identified by the BioReliance study number, treatment condition, dose level, test phase, harvest date, activation system, and replicate tube design.

Cell Cycle Kinetics Scoring (Preliminary Toxicity Test and Definitive Assays):
For the preliminary toxicity test, at least 500 cells were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture), were evaluated to determine the CBPI at each dose level and the control. The CBPI was determined using the following formula:
CBPI = 1X Mononucleated cells + 2 x Binucleated cells + 3 x Multinucleated cells
Total number of cells scored
% Cytostasis (cytotoxicity) = 100 -100 {(CBPIt-1) /(CBPIc-1)}
t = test substance treatment culture
c = vehicle control culture

Micronucleus Scoring (Definitive Assays):
The slides from at least three test substance treatment groups were coded using random numbers by an individual not involved with the scoring process and scored for the presence of micronuclei based on cytotoxicity. A minimum of 2000 binucleated cells from each concentration (1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei.
Micronuclei in a binucleated cell (MN-BN) were recorded if they meet the following criteria:
• the micronuclei should have the same staining characteristics as the main nucleus
• the diameter of the micronuclei must be approximately 1/3 or less the diameter of a main nucleus
• the micronuclei must be non-refractile, located in the cytoplasm, no overlapping with nucleus and not connected by cytoplasmic bridges.
• the micronuclei are not linked or connected to the main nuclei;
• the micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary
Evaluation criteria:
VALIDITY CRITERIA
The frequency of cells with micronucleus induction in the vehicle control must be within the historical control range. The percentage of cells with micronucleus induction must be statistically increased (p ≤ 0.05, Fisher's exact test) in the positive control condition relative to the vehicle control. The Historical Control Data is included in Appendix III.

EVALUATION CRITERIA
Toxicity induced by treatment was based upon CBPI and was reported for the cytotoxicity and micronucleus portions of the study. The percent frequency of micronucleated binucleated (MN-BN) cells was determined out of at least 2000 total binucleated cells per dose levels and reported for each treatment group.
Statistical analysis of the percentage of micronucleated cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent micronucleated cells of each treatment group with that of the vehicle control. Due to negative results, the Cochran-Armitage test was not required to measure dose-responsiveness.
The test substance was considered positive if it induced a statistically significant and dose-dependent increase in the frequency of MN-BN cells (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.
Statistics:
Statistical analysis of the percentage of micronucleated cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent micronucleated cells of each treatment group with that of the vehicle control. Due to negative results, the Cochran-Armitage test was not required to measure dose-responsiveness.
Key result
Species / strain:
lymphocytes: Human Peripheral Blood Lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Under the conditions of the assay described in this report, the test item was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.
Executive summary:

The test substance that is analytically identical to the boundary composition of this dossier, was tested in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity was performed to establish the dose range for testing in the micronucleus test. The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance. In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation.

Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

In the preliminary toxicity assay, the doses tested ranged from 0.206 to 2060 μg/mL (10 mM). Substantial cytotoxicity [≥ 50 to 60% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was not observed at any dose level in the S9-activated 4-hour exposure group. Substantial cytotoxicity was observed at dose levels ≥ 61.8 μg/mL in the non-activated 4 and 24-hour exposure groups. Based on these findings, the doses chosen for the micronucleus assay ranged from 5 to 70 μg/mL for the non-activated 4 and 24-hour exposure groups, and from 20 to 650 μg/mL for the S9-activated 4-hour exposure group.

In the initial micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 60 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 200 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 40 μg/mL in the non-activated 24-hour exposure group. In the S9-activated 4-hour exposure group due to inconsistent cytotoxicity, the micronucleus assay was repeated at doses ranging from 5 to 650 μg/mL. In the repeat assay, substantial cytotoxicity was observed at dose levels ≥ 100 μg/mL in the S9-activated 4-hour exposure group.

The highest dose analyzed under each treatment condition produced 50 to 60% reduction in CBPI, which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test).

The results for the positive and negative controls indicate that all criteria for a valid assay were met.

Based on the findings of this study, the test substance was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Methyl Ionone is not mutagenic, not aneugenic and not clastogenic according to the data available.

Additional information

Mutagenicity in Bacteria (Ames tests):

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.

BASF, 2014 - HPRT (OECD 487, GLP)

Methyl ionone 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.

RIFM, 2014 - Micronucleus in vitro (OECD 487, GLP):

The test substance that is analytically identical to the boundary composition of this dossier, was tested in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity was performed to establish the dose range for testing in the micronucleus test. The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance. In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation.

Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

In the preliminary toxicity assay, the doses tested ranged from 0.206 to 2060 μg/mL (10 mM). Substantial cytotoxicity [≥ 50 to 60% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was not observed at any dose level in the S9-activated 4-hour exposure group. Substantial cytotoxicity was observed at dose levels ≥ 61.8 μg/mL in the non-activated 4 and 24-hour exposure groups. Based on these findings, the doses chosen for the micronucleus assay ranged from 5 to 70 μg/mL for the non-activated 4 and 24-hour exposure groups, and from 20 to 650 μg/mL for the S9-activated 4-hour exposure group.

In the initial micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 60 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 200 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 40 μg/mL in the non-activated 24-hour exposure group. In the S9-activated 4-hour exposure group due to inconsistent cytotoxicity, the micronucleus assay was repeated at doses ranging from 5 to 650 μg/mL. In the repeat assay, substantial cytotoxicity was observed at dose levels ≥ 100 μg/mL in the S9-activated 4-hour exposure group.

The highest dose analyzed under each treatment condition produced 50 to 60% reduction in CBPI, which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test).

The results for the positive and negative controls indicate that all criteria for a valid assay were met.

Based on the findings of this study, the test substance was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.

Justification for classification or non-classification

Based on the results of the genetic tests, the substance does not need to be classified for genotoxicity according to EU CLP (EC 1272/2008, and its amendments).