undecan-2-one; methyl nonyl ketone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

An increase in the number of revertants was not observed in the Salmonella typhimurium Reverse Mutation Assayand the Escherichia coli Reverse Mutation Assay neither with nor without S-9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions chosen here, it is concluded that Radia MNKE (MNK 2-undecanone) is not a mutagenic agent and does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

 

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

In vitro gene mutation study in mammalian cells:

Test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Mar 2017 - 13 Apr 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is with registered substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: EM97160115
- Expiration date of the lot/batch: 27 January 2018
- Purity test date: 07/07/2016
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Radia MNKE was dissolved in dimethyl sulfoxide.
- Final dilution of a stock liquid: not specified
FORM AS APPLIED IN THE TEST (if different from that of starting material):Radia MNKE was dissolved in dimethyl sulfoxide.

Target gene:

histidine locus
tryptophan locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Tester strains contained additional mutations: rfa: defective lipopolysaccharide cellcoat, gal :
mutation in the galactose metabolism, chl : mutation in nitrate reductase, bio : defective biotin synthesis, uvrB: deletion of the ultraviolet-repair B gene) .

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Dose range: Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of Radia MNKE used in the subsequent mutation assays was the level at which the test item inhibited bacterial growth or the level at which the test item exhibited limited solubility.
First mutagenicity test: 1.7, 5.4, 17, 52, 164 and 512 μg/plate.
Second mutagenicity test: 27 to 492 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: CR-191 2,5µg/plate in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding: optical density of bacteria 1.0 ± 0.1 at 700 nm (109 cells/ml)
DURATION
- Preincubation period: none, direct plate incorporation
- Exposure duration: 48 ± 4 h
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, reduction of the bacterial background lawn and/or the
presence of microcolonies

Rationale for test conditions:

According to the designated guidelines

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two(2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535,
TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up
experiment.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Dose-range finding test: precipitation of Radia MNKE on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate and at 5000 μg/plate at the end of the
incubation period.
First mutagenicity test: at the dose level of 512 μg/plate in the absence of S9-mix in the tester strains TA1535, TA1537, TA98 and TA100 and in the presence of 5% (v/v) S9-mix in the tester strains T
A1535, TA1537 and TA98, Radia MNKE did not precipitate on the plates.
Second mutagenicity test: Precipitation of Radia MNKE on the plates was observed at the start of the incubation period at concentrations of 2800 and 5000 μg/plate. At the end of the incubation period,
the test item precipitated on the plates at the highest dose level tested in the tester strains TA1535 and TA100 in the presence of S9-mix and in strains TA1537 and WP2uvrA in the absence and
presence of S9-mix. In addition, in tester strain TA98 the test item precipitated at dose levels of 154 μg/plate and upwards in the absence of S9-mix.
- Other confounding effects:In the second mutation experiment, no dose level with precipitate or toxicity was tested in the tester strain TA98 in the presence of S9-mix.
Evaluation: Although toxicity was observed at the dose levels of 164 and 512 μg/plate in the first mutation experiment, and precipitation was observed at 492 μg/plate in the tester strains TA1535,
TA1537 and TA100 in the second experiment, no toxicity or precipitate was observed at 492 μg/plate in the second mutation experiment. Since clear negative responses were observed in all tester strains tested, and in the first and second mutation experiments the test item was tested up to dose levels with toxicity or precipitate, the lack of a dose level with toxicity or precipitate had no influence on the study.

RANGE-FINDING/SCREENING STUDIES:
In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Radia MNKE precipitated on
the plates at the dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strain TA100 at dose levels of 17 and 164 μg/plate and upwards in the absence and presence of S9-mix, respectively. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Remarks on result:

other: Non-mutagenic potential

Conclusions:

An increase in the number of revertants was not observed in the Salmonella typhimurium Reverse Mutation Assayand the Escherichia coli Reverse Mutation Assay neither with nor without S-9 mix or
after the addition of a metabolizing system. Thus, under the experimental conditions chosen here, it is concluded that Radia MNKE (MNK 2-undecanone) is not a mutagenic agent and does not need to
be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

The objective of this study was to determine the potential of Radia MNKE and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 1.7 to 512 μg/plate in the absence of S9-mix in the tester strains TA1535, TA1537, TA98 and TA100 and in the presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 27 to 492 μg/plate in the tester strains TA1535, TA1537, TA98 and TA100 and at 492 to 5000 μg/plate in the tester strain WP2uvrA in the absence and presence of 10% (v/v) S9-mix. Radia MNKE did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp +) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. The negative and positive controls were valid, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that Radia MNKE is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary literature

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

5, 15, 25 μg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

Not specified

Evaluation criteria:

Not specified

Statistics:

Not specified

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 25 μg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: Non-mutagenic

Conclusions:

The test substance did not induce chromosomal aberrations in CHO cells in the presence and absence of metabolic activation system.

Executive summary:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical Methyl nonyl ketone (CAS no. 112 -12 -9). The study was performed using CHO cells in the presence and absence of S9 metabolic activation system. The test chemical was soluble in DMSO and used at dose level of 5, 15 and 25 μg/ml. Cells were exposed for a period of 4 hours. Positive control chemicals were also included in the study, Ethylmethane sulphonate and N-nitroso-dimethylamine were used as the postive controls. Cytotoxicity started at a concentration of 25 μg/ml. The test substance did not induce chromosomal aberrations in CHO cells in the presence and absence of metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

data is from safety assessment reports

Qualifier:

according to guideline

Guideline:

other: mouse lymphoma cell forward mutation study

Principles of method if other than guideline:

Mouse lymphoma cell forward mutation study was performed to evaluate the mutagenic potential of the test chemical

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Test concentrations with justification for top dose:

0, 0.0032, 0.0042, 0.0056, 0.0075, 0.010, 0.013, 0.018, 0.024, 0.032, or 0.042 L/mL in the absence of S9, and 0, 0.013, 0.018, 0.024, 0.032, 0.042, 0.056, 0.075, 0.10, or 0.13 L/mL in the presence of S9

Vehicle / solvent:

not specified

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:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
- Number of independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment (sampling/recovery times):

Rationale for test conditions:

no data available

Evaluation criteria:

no data available

Statistics:

no data available

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Gene mutation tests in mammalian cells: There was no evidence of induced forward mutation at the TK locus at any of the doses tested with or without the S9 activation

Remarks on result:

other: not mutagenic

Conclusions:

There was no evidence of induced forward mutation at the TK locus at any of the doses tested with or without the S9 activation. Hence, the test chemical can be considered to be not mutagenic in nature.

Executive summary:

Mouse lymphoma cell forward mutation study was performed to evaluate the mutagenic potential of the test chemical Methyl nonyl ketone (CAS no. 112 -12 -9). Mouse lymphoma cells were used for the study. The dosing levels were 0, 0.0032, 0.0042, 0.0056, 0.0075, 0.010, 0.013, 0.018, 0.024, 0.032, or 0.042 L/mL in the absence of S9, and 0, 0.013, 0.018, 0.024, 0.032, 0.042, 0.056, 0.075, 0.10, or 0.13 L/mL in the presence of S9. The cultures were exposed to the test chemical for 4 hours. There was no evidence of induced forward mutation at the TK locus at any of the doses tested with or without the S9 activation. Hence, the test chemical can be considered to be not mutagenic in nature.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames assay:

1. The objective of this study was to determine the potential of Radia MNKE and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 1.7 to 512 μg/plate in the absence of S9-mix in the tester strains TA1535, TA1537, TA98 and TA100 and in the presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 27 to 492 μg/plate in the tester strains TA1535, TA1537, TA98 and TA100 and at 492 to 5000 μg/plate in the tester strain WP2uvrA in the absence and presence of 10% (v/v) S9-mix. Radia MNKE did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp +) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. The negative and positive controls were valid, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that Radia MNKE is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

2. in vitro Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical Methyl nonyl ketone (CAS no.:112 -12 -9). The study was performed using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 in the presence and absence of S9 metabolic activation system. The chemical was used at dose levels 3.3, 10, 33, 100, 333 and 1000 µg/plate. Plates were exposed to the test substance for a duration of 48 hours. The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

3. Gene mutation toxicity study was performed to determine the mutagenic nature of test read across substance octan-2 -one (CAS no.: 111 -13 -7). The study was performed using Salmonella typhimurium LT2 strains G46, TA1535, TA100, C3076, D3052, TA1538 and TA98 and E. coli strains WP2 and WP2 uvrA. In special cases, 4 additional strains are used for supplemental testing. These strains were TA92, TA94, CM881, and CM891. The test chemical was dissolved in DMSO. When appropriate, water or dimethoxyethane is used instead of dimethyl sulfoxide. Agar plates containing a concentration gradient of test com pound was prepared. Ten ml of minimal agar medium (not containing test compound) was poured into a square Petri dish (9 x 9 cm) which is tilted at a slight angle. The agar was then allowed to solidify into a wedge-shaped layer by standing at room temperature. Meanwhile, a 1000 -µg/ml mixture of test compound in agar was prepared by adding 10 ml of minimal agar to 0.1 ml of a 100 -mg/mI solution of test compound in dimethyl sulfoxide. When appropriate, water or dimethoxyethane was used instead of dimethyl sulfoxide. The cooled agar plates are then placed on a level surface, and an overlay of the 10 ml of agar containing the test compound was poured onto the plate to form a reversed wedge of agar on top of the first wedge. A concentration gradient of compound was produced by allowing the compound in the upper wedge to diffuse into the lower layer for 2 hr at room temperature. The concentration range in this plate is approximately 100 to 1000µg/ml. Three additional plates with concentration ranges of 10 to 100µg/ml, 1 to 10µg/ml and 0.1 to 1µg/ml are prepared. A streaking device consisting of 10 sterile 50µL pipets is next dipped into suspensions of the 10 test strains and allowed to fill by capillary action. The pipettes were then touched to the upper edge of the gradient and drawn across the plate. A wet trail of inoculum was observed. The plates were then incubated for 48 hr at 37°C. The study was performed in the presence and absence of rat liver enzyme metabolic activation system. Concurrent negative and positive controls were included in the study. Test substance failed to induce mutation in Salmonella typhimurium strains and E. coli strains in the gradient plate assay performed both in the presence and absence of metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

1. In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical Methyl nonyl ketone (CAS no. 112 -12 -9). The study was performed using CHO cells in the presence and absence of S9 metabolic activation system. The test chemical was soluble in DMSO and used at dose level of 5, 15 and 25μg/ml. Cells were exposed for a period of 4 hours. Positive control chemicals were also included in the study, Ethylmethane sulphonate and N-nitroso-dimethylamine were used as the postive controls. Cytotoxicity started at a concentration of 25μg/ml. The test substance did not induce chromosomal aberrations in CHO cells in the presence and absence of metabolic activation system.

2. In vitro cytogenicity/chromosomal aberration study was performed to evaluate the mutagenic potential of the read across substance Heptan-2 -one (CAS no.: 110 -43 -0, E.C. no.: 203 -767 -1). The study was performed according to OECD 473 Guidelines. Chinese hamster ovary cells were treated with the test chemical dissolved in DMSO at concentrations up to 1200 μg/mL in the presence and absence of metabolic activation.Aroclor 1254 -derived Sprague Dawley rats were used as metabolic activation system. The positive controls were mitomycin-C and cyclophosphamide, and the negative control was the solvent used i.e DMSO. Statistical analysis employed a Cochran-Armitage test for linear trends and Fisher's Exact Test to compare the percentage of cells with aberrations. No precipitate was observed at maximum concentration tested. No evidence of cytotoxicity was seen, so the highest cytotoxic concentration can be considered to be >1200 µg//ml .No statistically significant increases in the frequency of cells with structural chromosomal aberrations or polyploid cells were observed with any dose of the test item, either with or without S9 metabolic activation. Hence, the test chemical can be considered to be non mutagenic in nature.

In vitro gene mutation study in mammalian cells:

1. Mouse lymphoma cell forward mutation study was performed to evaluate the mutagenic potential of the test chemical Methyl nonyl ketone (CAS no. 112 -12 -9). Mouse lymphoma cells were used for the study. The dosing levels were 0, 0.0032, 0.0042, 0.0056, 0.0075, 0.010, 0.013, 0.018, 0.024, 0.032, or 0.042 L/mL in the absence of S9, and 0, 0.013, 0.018, 0.024, 0.032, 0.042, 0.056, 0.075, 0.10, or 0.13 L/mL in the presence of S9. The cultures were exposed to the test chemical for 4 hours. There was no evidence of induced forward mutation at the TK locus at any of the doses tested with or without the S9 activation. Hence, the test chemical can be considered to be not mutagenic in nature.

2. In vitro the genotoxic, clastogenic and mutagenic potential was evaluated for the test chemical 2-Nonanone (IUPAC name: nonan-2 -one). DNA damage was assessed by the alkaline single cell gel electrophoresis assay (comet assay) in Chinese hamster V79 cells.

V79 cells (2 × 10⁵) were seeded in duplicate duplicate into multiwell (six wells, 35mM diameter), cultivated overnight and then treated with test substance at 37◦C.for 4 h. The alkaline comet assay was performed. After lysis over night the cells were exposed to alkali for 60 min to permit DNA unwinding and expression of alkali labile sites.Electrophoresis was performed for 30 min at 25V and 300mA in an ice bath. All the steps were performed under dim light to prevent the occurrence of additional DNA damage. The extent of DNA migration was analysed using a fluorescence microscope and image analysis (Comet Analysis Software; Confocal Technologies, Liverpool, UK). For each cell, the tail moment (TM) was calculated and the median of the 54 randomly selected cells/experimental point (27 cells from each of two replicate slides) was determined.

2-Nonanone failed to induce gene toxicity in vitro in Chinese hamster V79 cells by Comet assay and hence is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the data available, the given test chemical does not exhibit gene mutation in vitro by Ames assay, In vitro mammalian chromosome aberration study and In vitro gene mutation study in mammalian cells. Hence, the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.