Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Methyl isoamyl ketone was not mutagenic/genotoxic under conditions used in these assays. Based on the key and supporting genotoxicity studies the chemicals does not raise the cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

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:
other: reliable without restrictions; study conducted according to established guidelines and performed according to GLPs.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His (-), Trp (-)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The tester strains were received directly from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley.
Additional strain / cell type characteristics:
other: The tester strains contain 2 additional mutations: rfa wall mutation and a deletion of the uvrB gene. Strains TA 98 and TA 100 also contain the R-factor plasmid, pKM101, which increases the sensitivity of these strains to mutagens.
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
The tester strain, WP2uvrA(pKM101) was received from the National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).
Additional strain / cell type characteristics:
other: The tester strain contains a uvrA DNA repair deficiency which enhances the strain's sensitivity to some mutagenic compounds. The strain also contains the R-factor plasmid, pKM101, which increases the sensitivity of these strains to mutagens.
Metabolic activation:
with and without
Metabolic activation system:
Liver microsomal enzymes (S9 homogenate) were purchased from Molecular Toxicology, Inc (Batch 0883).
Test concentrations with justification for top dose:
Dose Range-Finding Study:
6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330, and 5000 µg/plate

Mutagenicity Assay:
100, 333, 1000, 3330, 5000 µg/plate
Vehicle / solvent:
Dimethyl sulphoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoananthracene
Remarks:
2-aminoananthracene (Sigma Chemical Co., purity ≥97%) was used with tester strains TA 98, TA 100, TA 1535, TA 1537 at a concentration of 2.5 µg/plate and with tester strain WPuvrA(pKM101) at a concentration of 5.0 µg/plate with metabolic activity.
Positive control substance:
2-nitrofluorene
Remarks:
2-nitrofluorene (Aldrich Chemical Co., purity ≥98%) was used in tester strain TA 98 at a concentration of 1.0 µg/plate without metabolic activity.
Positive control substance:
sodium azide
Remarks:
Sodium azide (Sigma Chemical Co., purity ≥98%) used with tester strains TA 100 and TA 1535 at a concentration of 2.0 µg/plate without metabolic activity.
Positive control substance:
other: ICR-191
Remarks:
ICR-191 (Sigma Chemical Co., purity ≥98%) used with tester strain TA 1537 at a concentration of 2.0 µg/plate without metabolic activity.
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
4-nitroquinoline-N-oxide (Sigma Chemical Co., purity ≥99%) used with tester strain WPuvrA(pKM101) at a concentration of 2.0 µg/plate without metabolic activity.
Remarks:
Sterility Controls: The test substance at the greatest concentration tested was checked for sterility by plating a 50 μl aliquot on selective agar. Likewise, the S9 mix was also checked for sterility by plating 0.5 ml on selective agar.
Details on test system and experimental conditions:
Dose Range-Finding Study:
This study was performed using tester strains TA 100 and WP2uvrA(pKM101) with and without metabolic activation at concentrations up to 5 mg/plate.

Mutagenicity Assay:
The tester strains were exposed to the test substance via the plate incorporation methodology as described by Ames et al. (1975) and Maron and Ames (1983).

The plating procedure was the following: When S9 was not required, 100 µl of the tester strain and 50 µl of the vehicle or test substance dose was added to 2.5 ml of molten top agar. When S9 was required, 500 µl of S9 mix, 100 µl of tester strain, and 50 µl of vehicle were added to 2.0 ml of molten top agar. The mixture was vortexed and overlaid onto the surface of 25 ml of minimal bottom agar and after the mixture solidified the plates were inverted and incubated for 48 ± 8 hours at 37 ± 2°C. Positive control materials were plated using a 50 µl plating aliquot.

The condition of the bacterial background lawn was evaluated for evidence of cytotoxicity and test substance precipitate and was scored relative to the vehicle control plate. The number of revertant colonies per plate for the vehicle controls and all plates containing test substance were counted manually while the positive controls were counted by an automated colony counter with few exceptions.

Metabolic Activation System:
The homogenate was prepared from male Sprague-Dawley rats that had been injected (i.p.) with Aroclor 1254 (200 mg/ml in corn oil) at 500 mg/kg. The S9 mix was prepared immediately prior to use. Components of the S9 mix included: Water (0.70 ml), 1M NaH2PO4/Na2HPO4, pH 7.4 ( 0.10 ml), 0.25M Glucose-6-phosphate (0.02 ml), 0.10M NADP (0.04 ml), 0.825M KCL/0.2M MgCl2 (0.04 ml), and S9 homogenate (0.10 ml).
Evaluation criteria:
Dose Range-Finding Study:
Cytotoxicity, defined as a detectable decrease in the number of revertant colonies/plate and/or by a thinning or disappearance of the bacterial background lawn, was the main criterion evaluated in the dose range-finding study. The maximum dose for the mutagenicity assay was the dose at which no cytotoxicity was observed.

Mutagenicity Assay:
-Criteria for a Valid Assay:
Tester strain integrity was demonstrated by rfa wall mutation sensitivity towards crystal violet for Salmonella and pKM101 plasmid sensitivity towards ampicillin for both Salmonella and E coli. The characteristic number of spontaneous revertants was determined by the tester strain cultures exhibiting a characteristic number of spontaneous revertants per plate when plated along with the vehicle. The acceptable ranges are: TA 98: 8-60, TA 100: 60-240, TA 1535: 4-45, TA 1537: 2-25, and WP2uvrA(pKM101) 80-350.

-Positive Controls:
To demonstrate that the tester strains in the absence or presence of S9 mix were capable of identifying a mutagen, the mean value of a positive control needed to exhibit at least a 3-fold increase over the vehicle control.

-Cytotoxicity:
A minimum of 3 non-toxic doses were required to evaluate the assay data.

-Criteria for a Positive Response:
For a test substance to be considered positive, it had to produce at least a 2-fold (TA 98, TA 100, and WP2uvrA(pKM101) or 3-fold (TA 1535 and TA 1537) increase in the mean revertants per plate of at least one of the tester strains over the mean revertants per plate of the appropriate vehicle control. The increase had to be accompanied by a dose response to increasing concentrations of the test substance.

The results of the initial mutagenicity assay were confirmed in an independent experiment.
Statistics:
For all replicate platings, the mean revertants per plates and the standard deviation were calculated. Statistical analyses were not needed due to the absence of an increase in the number of revertant colonies at any dose level beyond the positive control.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
-Test Substance:
The test substance formed a solution in DMSO up to concentrations of 499 mg/ml. At 100 mg/ml, the most concentrated stock solution used in this study, the test substance formed a transparent colorless solution and remained dissolved in all succeeding solutions prepared for the study.

Dose Range-Finding Study:
No cytotoxicity was observed with either strain with or without metabolic activity as evidenced by a normal background lawn and no decrease in the number of revertants per plate. No test substance precipitate was observed on the plates at any of the doses tested.

Mutagenicity Assay:
All criteria for a valid assay were met, all data was acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of metabolic activity.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

It is concluded that, under the conditions of this test, methyl isoamyl ketone showed no evidence of mutagenic activity in this bacterial system with and without metabolic activation. Based on an absence of genotoxic/mutagenic effects in this study, methyl isoamyl ketone is not classified for “Germ Cell Mutagenicity” according to GHS.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535 and TA 1537 of S. typhimurium and strain WP2 uvr A pKM 101 of E. coli were exposed to methyl isoamyl ketone in DMSO at concentrations of 100, 333, 1000, 3330, 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method. The positive controls induced the appropriate responses in the corresponding strains and there was no evidence of induced mutant colonies over background. The results of the initial mutagenicity assay were confirmed in an independent second experiment.   

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Minimal deviations from the signed protocol were without significant effect on the study outcome.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Physical Description: Transparent, colorless liquid
Storage Conditions: Ambient
Lot No.: 12-98
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The Chinese hamster ovary cells (CHO-WBL) were from a permanent cell line originally obtained from Dr. S. Wolff, University of California, San Francisco. The cells have been recloned to maintain karyotypic stability. This cell line has an average cycle time of 12-14 hr with a modal chromosome number of 21. Cells were grown in McCoy’s 5a culture medium (supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 units/ml penicillin and streptomycin), at approximately 37 ± 2°C, in an atmosphere of about 5 ± 1.5% CO2 in air.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix contained: NADP (1.5 mg/ml), isocitric acid (2.7 mg/ml), S9 homogenate (15.0 µl/ml). The S9 was from livers harvested 5 days post-dose from male Sprague-Dawley rats treated with 500 mg/kg Aroclor 1254 (Molecular Toxicology, Inc., Lot #855 and 897).
Test concentrations with justification for top dose:
-Initial Assay
With and without metabolic activity:
8.12, 11.6, 16.6, 23.7, 33.9, 48.4, 69.1, 98.7, 141, 202, 288, 412, 588, 840, and 1200 µg/ml

-Confirmatory Assay:
Without metabolic activity:
53.5, 107, 214, 285, 380, 506, 675, 900, and 1200 µg/ml
With metabolic activity:
380, 506, 675, 900, and 1200 µg/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Mitomycin C (Sigma Lot #116H2511), dissolved in water, was used in the non-activation series at concentrations of 0.75 and 1.5 µg/ml in the initial assay and 0.10 and 0.15 µg/ml in the confirmatory assay.
Positive control substance:
cyclophosphamide
Remarks:
Cyclophosphamide (Sigma Lot # 73H0846), dissolved in water, was used in the activation series at concentrations of 5.0 and 10.0 µg/ml in both the initial and confirmatory assay.
Details on test system and experimental conditions:
Aberrations Assay without Metabolic Activation:
Cultures were initiated by seeding ~1.2X10^6 cells per 75 cm2 flask into 10 ml culture media. One day after culture initiation, the cells were incubated at ~37°C with the test substance for 3 hours for the initial assay and 17.8 hours for the confirmatory assay.
-In the initial assay, the cells were refed with culture media and incubated until harvest time with 0.1 µg/ml Colcemid present during the last two hours.
-In the confirmatory assay, cells were refed with culture media with 0.1 µg/ml Colcemid and harvested 2 hours later.

Aberrations Assay with Metabolic Activation:
Cultures were initiated by seeding ~1.2X10^6 cells per 75 cm2 flask into 10 ml culture media. One day after culture initiation, the cells were incubated at ~37°C with the test substance and S9 mix for 3 hours for both the initial and confirmatory assay in culture media without Fetal Bovine Serum. The cells were washed twice with buffered saline and the cells were refed with culture medium. The cells were incubated until harvest time with 0.1 µg/ml Colcemid present during the last two hours.

Harvest:
Prior to harvest, visual observations of toxicity were made. These included an assessment of the percent confluence of the cell monolayer within the flasks and the presence of mitotic or dead cells floating in the medium. The cells were then trypsinized to collect mitotic and interphase cells and were treated with 0.075 M KCl hypotonic solution. The cultures were then fixed in a 3:1 methanol: glacial acetic acid fixative, allowed to air dry, and prepared for aberration analysis.

Slide Preparation and Staining:
Slides were prepared by dropping the harvested cultures on clean slides. The slides were stained with 5% Giemsa solution, allowed to air-dry and then permanently mounted.
Evaluation criteria:
Assay Acceptability:
An assay was acceptable for evaluation of test results only if all of the following criteria were satisfied:
-The negative controls and the vehicle control cultures must contain less than 5% cells with aberrations.
-The positive control result must be significantly higher (p ≤ 0.01) than the vehicle controls.
-The aberration results are negative and there is no significant reduction (approximately ≥ 50%) in confluence or mitotic index. The assay must include the highest applicable dose (10 mM or 5 mg/ml whichever is highest) or a dose exceeding the solubility limit in culture media.
-The assay must include at least three analyzable concentrations.
-If significant increases were observed but not in consecutive concentration, there should be a clear evidence of a dose-response.

Evaluation of a Positive Response:
The test substance was considered positive if a significant increase (p ≤ 0.01) in the number of cells with chromosomal aberrations is observed at one or more concentrations.

Evaluation of a Negative Response:
The test substance was considered negative if no significant increase was observed in the number of cells with chromosomal aberrations at any of the concentrations.
Statistics:
The experimental unit was the cell and therefore the percentage of cells with structural aberrations was the basis for evaluation. Statistical analysis employed a Cochran-Armitage test for linear trend and Fisher’s Exact Test to compare the percentage of cells with aberrations in treated cells to the results obtained for the vehicle controls.

Statistical analysis was also performed for cells exhibiting polyploidy and/or endoreduplication in order to indicate significant (p ≤ 0.01) increases in these events as indicators of possible induction of numerical aberrations. This study only evaluated structural aberrations and numerical aberrations.
Species / strain:
Chinese hamster Ovary (CHO)
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
Additional information on results:
Chromosomal Aberration Assay without Metabolic Activation
-Initial Assay and Confirmatory Assay
No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in cultures treated with 380, 506, 675, or 1200 µg/ml. The positive control elicited the proper response.

Chromosomal Aberration Assay with Metabolic Activation
-Initial Assay and Confirmatory Assay
No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in cultures treated with 412, 588, 840, and 1200 µg/ml. The successful activation by the metabolic system was demonstrated by the increased incidence of cells with chromosomal aberrations in the cultures induced with cyclophosphamide, the positive control agent



Summary of Chromosomal Aberrations Assay Treatment Schedule in Hours

 

Activation Condition

 

Test Substance

Wash

Colcemid®

Fixation

Initial Trial

 

 

 

 

 

 

 

- S9

0

3.0

18.0

20.1

 

+ S9

0

3.0

18.1

20.1

Confirmatory Trial

 

 

 

 

 

 

 

- S9

0

17.8

18.0

20.1

 

+ S9

0

3.0

18.0

20.0

Conclusions:
Methyl isoamyl ketone was considered negative for inducing chromosome aberrations in CHO cells with and without metabolic activation. Based on an absence of genotoxic/mutagenic effects in this study, methyl isoamyl ketone is not classified for “Germ Cell Mutagenicity” according to GHS.

Executive summary:

The objective of this in vitro assay was to evaluate the ability of EC 98-0268, MIAK to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic

activation.

The test substance was dissolved in dimethylsulfoxide (DMSO) at a concentration of 120 mg/ml for both the initial and confirmatory assays. The high dose achieved was 10.5 mM of the test

substance. All dosing was achieved using a dosing volume of 1.0% (1 ul/ml) and the solvent control cultures were treated with 10.0 ul/ml of DMSO. In the initial chromosomal aberrations assay, the treatment period was for 3.0 hours with and without metabolic activation and cultures were harvested 20.1 hours from the initiation of treatment. Concentrations of 8.12, 11.6, 16.6, 23.7, 33.9, 48.4, 69.1, 98.7, 141, 202, 288, 412, 588, 840, and 1200 ug/ml were tested with and without metabolic activation. Cultures treated with concentrations of 412, 588, 840, and 1200 ug/ml with and without metabolic activation

were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations was observed in the cultures analyzed.

In a confirmatory chromosomal aberrations assay, the treatment period was for 17.8 hours without metabolic activation and 3.0 hours with metabolic activation, and cultures were harvested 20.0 hours from the initiation of treatment. Concentrations of 53.5, 107, 214, 285, 380, 506, 675, 900, and 1200 ug/ml were tested without metabolic activation, and concentrations of 380, 506, 675, 900, and 1200 ug/ml were tested with metabolic activation. Cultures treated with concentrations of 380, 506, 675, and 1200 ug/ml without metabolic activation and 506, 675, 900, and 1200 ug/ml with metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations was observed in the cultures analyzed. EC 98-0268, MIAK was considered negative for inducing chromosome aberrations in CHO cells with and without metabolic activation.

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: study conducted according to GLPs and also according to OECD Guideline 476 and the Commission Regulation (EC) 440/2008 B:17 guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase Locus (TK +/-)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Large stocks of the cleansed L5178Y cell line were stored in liquid nitrogen at the testing facility. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate made by Harlan; protein concentration 32.3 mg/mL (Lot 020709). Homogenate prepared from male Sprague-Dawley rats induced by applications of 80 mg/kg bw/day Phenobarbital (i.p.) and β-Naphthoflavone (p.o.) each on 3 consecutive days.
Test concentrations with justification for top dose:
Experiment I
With and without metabolic activation: 75.0, 150, 300, 600, 1200 µg/mL

Experiment II
With metabolic activation: 75.0, 150, 300, 600, 1200 µg/mL
Without metabolic activation: 150, 300, 600, 900, 1200 µg/mL
Vehicle / solvent:
DMSO (Merck, purity 99.9%, lot K40013731 920)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Methyl Methane Sulfonate (MMS) (Sigma, purity>99%, lot 76296KJ) dissolved in DMSO was used without metabolic activation at concentrations of 19.5 µg/mL (0.18 mM, experiment I) and 13.0 µg/mL (0.12 mM, experiment II).
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Cyclophosphamide (CPA) (Aldrich, purity ≥ 98%, lot 097 K 1311) dissolved in 0.9% saline was used with metabolic activation at concentrations of 3.0 µg/mL (10.7 µM, experiments I and II) and 4.5 µg/mL (16.1 µM, experiments I and II).
Details on test system and experimental conditions:
S9 Mix:
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to give a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 4 mM NADP, in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

Pre-Test:
A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. 1x10^7 cells were exposed to each concentration of methyl isoamyl ketone for 4 and 24 hours without and 4 hours with metabolic activation. The cell density was determined immediately after treatment and at each day of the growth period and adjusted to 3x10^5 cells/mL. Cells were resuspended and allowed to grow for two days. The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period according to the method of Clive and Spector (1975). Concentrations of up to 1200 µg/mL (10 mM) were used in the pretest.

Experimental Performance:
In the mutation experiment, 1X10^7 cells/flask suspended in 10 mL RPMI medium with 3% horse serum (15% horse serum in the second experiment) were exposed to various concentrations of methyl isoamyl ketone in the presence or absence of metabolic activation. After 4 hours (24 hours in the second experiment) the test item was removed by centrifugation (425X g, 10 min), washed twice with "saline G,” resuspended in 30 mL complete culture medium and incubated for an expression and growth period of 48 hours. The cell density was determined each day and adjusted to 3X10^5 cells/mL.

One sample of the cells was taken at the end of treatment (4 and 24 hours, respectively), and diluted and seeded into microtiter plates to determine the viability of the cells after treatment (cloning efficiency 1). Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4X10^3 cells in selective medium with Trifluorothymidine (TFT) (Serva). The viability (cloning efficiency 2) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37 ° ± 1.5 °C in 4.5% CO2/95.5% water saturated air for 10-15 days, and were then evaluated.

Size Distribution of the Colonies:
The numbers of colonies were counted manually and the colony size distribution was determined in the controls and at all concentrations of the test item. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the large colonies).

Study dates:
Experimental Starting Date: August 31, 2009
Experimental Completion Date: October 12, 2009
Evaluation criteria:
Acceptability of the assay:
A mutation assay is considered acceptable if it meets the following criteria:
- All plates, from either the cloning efficiency or the TFT resistance-testing portion of the experiment are analysable.
-The absolute cloning efficiency at the time of mutant selection (CE) is 65-120 %.
-The total suspension growth of the solvent control is 8-32 or following the 24 hour treatment, is 32-180.
-The range of the solvent control mutant frequency is in the range of 50- 170 x 10^-6.
-The positive controls should yield an absolute increase in total MF. At least 40% of the induced mutation frequency (IMF) should be reflected in the small colony MF and the positive controls should induce at least 150 small colonies.
-The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures.
-The highest concentration of the test item should be 10 mM or 5000 µg/mL, unless limited by parameters of the guidelines.

Evaluation of Results
A test item is classified as mutagenic if:
-the induced mutation frequency exceeds a threshold of 126 colonies per 10^6 cells above the solvent control
-the mutation frequency is dose-dependent
-The mutagenic response is reproducible if it occured in both parallel cultures.

Historical variability of the mutation rates in solvent controls and the mutation rates of all solvent controls were taken into consideration.
Whenever a test item is considered mutagenic, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies, clastogenic effects are indicated.
Statistics:
A linear regression was performed (SYSTAT11, SYSTAT Software, Inc., Richmond, CA, USA) to assess whether a dose dependent increase of mutant frequencies occurred. The number of mutant colonies obtained for the groups treated with the test item are then compared to the solvent control groups; trends are judged as significant whenever the p-value is below 0.05.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The assay was performed in two independent experiments, using two parallel cultures. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation.

No precipitation of the test substance was noted at any dose concentration. No toxicity was noted at any dose concentration nor was there a reproducible or statistically dose dependent increase of the mutation frequency compared to the solvent control. The positive controls induced the appropriate responses.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the experimental conditions reported methyl isoamyl ketone did not induce mutations in the lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Based upon the following study, methyl isoamyl ketone is not classified under GHS.
Executive summary:

In a mammalian cell gene mutation assay, cultures of mouse lymphoma L5178Y cells were exposed to methyl isoamyl ketone in DMSO at concentrations up to 1200 µg/mL in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses as indicated by a distinct increase in induced total mutant colonies. With the test substance, however, the threshold above the mutation frequency of the corresponding solvent control was not exceeded at any of the test points with or without metabolic activation. It was concluded that, under the conditions of the study, methyl isoamyl ketone showed no evidence of inducing mutations in the lymphoma thymidine kinase locus assay using the cell line L5178Y in both the absence and presence of metabolic activation. 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The mutagenic/genotoxic potential of methyl isoamyl ketone has been characterized in a series of well conducted bacterial and mammalian in vitro mutagenicity tests. In a bacterial reverse mutation assay conducted according to EU Method B.13/14 and equivalent or similar to OECD Guideline 471, there was no increase in mutation frequency in any strain of Salmonella typhimurium or Escherichia coli at concentrations up to 5000 μg/plate in the presence or absence of metabolic activation and no evidence of cytotoxicity or precipitate at the highest dose tested. In a supporting bacterial reverse mutation assay conducted by a method similar to OECD Guideline 471, there was no increase in mutation frequency in any strain of Salmonella typhimurium at concentrations up to 50 μl/plate in the presence and absence of metabolic activation. The two highest doses tested, i.e., 25 and 50 μl/plate were cytotoxic to strains TA 1535, TA 1537 and TA 1538. These concentration levels were 5-10X higher than the recommended maximum test concentrations for soluble non-cytotoxic substances in current OECD guidelines. 

In an in vitro chromosome aberration assay conducted according to OECD Guideline 473, there was no evidence of an increase in the number of CHO cells with chromosomal aberrations or polyploidy in the presence or absence of metabolic activation, even at dose levels that showed clear cytotoxicity. In a supporting study which was conducted at several dose levels that were significantly higher than those that caused cytotoxicity in the key study, chromosomal aberrations were observed in the presence and absence of metabolic activation but only at extremely cytotoxic doses. 

In a mammalian cell gene mutation assay, cultures of mouse lymphoma L5178Y cells were exposed to methyl isoamyl ketone in DMSO at concentrations up to 1200 µg/mL in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses as indicated by a distinct increase in induced total mutant colonies. With the test substance, however, the threshold above the mutation frequency of the corresponding solvent control was not exceeded at any of the test points with or without metabolic activation. It was concluded that, under the conditions of the study, methyl isoamyl ketone showed no evidence of inducing mutations in the lymphoma thymidine kinase locus assay using the cell line L5178Y in both the absence and presence of metabolic activation. In a non-guideline in vitro cell transformation assay conducted according to acceptable scientific standards, there was no increase over controls in the number of foci of transformed cells, even at dose levels that caused cytotoxicity. For all studies, vehicle and positive controls induced the appropriate responses. 

Methyl isoamyl ketone was not mutagenic/genotoxic under conditions used in these assays.

Justification for classification or non-classification

Although no in vitro or in vivo germ cell mutagenicity/genotoxicity studies were available for review, the total weight of evidence available indicates that methyl isoamyl ketone is not expected to induce heritable mutations in the germ cells of humans and is not classified for “Mutagenicity/genotoxicity” according to GHS.