3-methylbutanone

Genetic toxicity: in vitro

Administrative data

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: The study was conducted according to established guidelines and performed according to GLPs.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

One minimal protocol deviation; this deviation had no effect on the outcome of the study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-2-butanone

IUPAC Name:

3-methyl-2-butanone

Constituent 2

Reference substance name:

3-methylbutanone

EC Number:

209-264-3

EC Name:

3-methylbutanone

Cas Number:

563-80-4

Molecular formula:

C5H10O

IUPAC Name:

3-methylbutan-2-one

Constituent 3

Reference substance name:

methylbutanone; methyl isopropyl ketone; MIPK

IUPAC Name:

methylbutanone; methyl isopropyl ketone; MIPK

Details on test material:

Test substance:
-Test substance (as cited in report): EC-2001-0250, MIPK
-Lot number: TXTXOL
-Date Received: September 17, 2001
-Physical Description: Transparent, colorless liquid
-Storage Conditions: Ambient temperature

Method

Target gene:

His (-), Trp (-)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The Salmonella typhimurium tester strains were received from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley.

Additional strain / cell type characteristics:

other: S. typhimurium (all strains): uvr B gene mutation, rfa wall mutation (TA 98 and TA 100): pKM 101 plasmid

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

The Escherichia coli tester strain was received from the National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).

Additional strain / cell type characteristics:

other: E. coli: uvr A gene mutation

Metabolic activation:

with and without

Metabolic activation system:

Liver microsomal enzymes (S9 homogenate) from Molecular Toxicology, Inc., lots #1302 (41.3 mg protein/mL) and 1281(40.4 mg protein/mL). Homogenate prepared from male Sprague-Dawley rats injected (i.p.) with Aroclor 1254 (200mg/mL in corn oil), 500mg/kg.

Test concentrations with justification for top dose:

Dose Rangefinding Assay (S. typhimurium TA 100 and E. coli):
6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate

Mutagenicity Assay:
All strains: 100, 333, 1000, 3330, and 5000 µg/plate

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Benzo[a]pyrene (Sigma Chemical Company, purity ≥97%) was used with tester strain TA 98 at a concentration of 2.5 µg/plate with metabolic activation.

Controls 2

Positive control substance:

other: 2-Aminoanthracene

Remarks:

2-Aminoanthracene (Sigma Chemical Company, purity ≥90%) was used with tester strains TA 100, TA 1535, TA 1537 at a concentration of 2.5 µg/plate and with tester strain WP2 uvr A at a concentration of 25.0 µg/plate, all with metabolic activation.

Controls 3

Positive control substance:

2-nitrofluorene

Remarks:

2-Nitrofluorene (Sigma Chemical Company, purity ≥98%) was used with tester strain TA 98 at a concentration of 1.0 µg/plate without metabolic activation.

Controls 4

Positive control substance:

sodium azide

Remarks:

Sodium azide (Sigma Chemical Company, purity ≥99%) was used with tester strains TA 100 and TA 1535 at a concentration of 2.0 µg/plate, both without metabolic activation.

Controls 5

Positive control substance:

other: ICR-191

Remarks:

ICR-191 (Sigma Chemical Company, purity ≥90%) was used with tester strain TA 1537 at a concentration of 2.0 µg/plate without metabolic activation.

Controls 6

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

4-Nitroquinoline-N-oxide (Sigma Chemical Company, purity ≥99%) was used with tester strain WP2 uvr A at a concentration of 1.0 µg/plate without metabolic activation.

Details on test system and experimental conditions:

An additional control was used in this assay.
Sterility Controls: The most concentrated test substance dilution was checked for sterility by plating a 50 µL aliquot on selective agar. The S9 mix was checked for sterility by plating 0.5 mL on selective agar.

S9 Mix:
The S9 mix was prepared immediately prior to use and contained the following components per mL of mix: H2O (0.70 mL), 1M NaH2PO4/Na2HPO4 (pH 7.4, 0.1 mL), 0.25M glucose-6-phosphate (0.02 mL), 0.10M NADP (0.04 mL), 0.825M KCL/0.2 MgCl2 (0.04 mL), S9 homogenate (0.10 mL).

-Dose Rangefinding study:
To determine cytotoxicity of the test material, the dose rangefinding study was performed with ten dose concentrations up to 5000 µg/plate (1 plate per dose) on tester strains TA 100 and WP2 uvr A in the presence and absence of metabolic activation.

-Mutagenicity Assay:
Since no cytotoxicity was observed in the rangefinding study, the highest dose used in the mutagenicity assay was the same dose as that tested in the rangefinding study. The results of the initial mutagenicity assay were confirmed in an independent experiment. All doses of the test material, the vehicle controls, and the positive controls were plated in triplicate. 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.

References:
Ames et al., 1975. Methods for detecting carcinogens and mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Research, 31:347-364.

Maron DM and Ames B, 1983. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research, 113:173-215.

-Plating Procedure:
When S9 mix was not required, 100 µL of tester strain and 50 µL of vehicle or test substance were added to 2.5 mL of molten selective top agar (45 ± 2 °C). When S9 mix was required, 500 µL of S9 mix, 100 µL of tester strain and 50 µL of vehicle or test substance were added to 2.0 mL of molten top agar. After the required components had been added, the mixture was vortexed, overlaid onto the surface of 25 mL bottom agar contained in a petri dish, and allowed to solidify. Plates were inverted and incubated for 52 ± 4 hours at 37 ± 2 °C. Positive control substances were plated using a 50 µL plating aliquot.

-Plate Scoring and Counting:
Plates that were not evaluated immediately following the incubation period were held at 5 ± 3 °C. The numbers of revertant colonies per plate were counted by an automated colony counter.

-Bacterial Background Lawn Evaluation:
The condition of the bacterial background lawn was evaluated both macroscopically and microscopically for indications of cytotoxicity and test substance precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate.

-Study Dates:
Initiation Date: September 18, 2001
Experimental Start Date: October 5, 2001
Experimental Termination Date: November 13, 2001

Evaluation criteria:

Dose Rangefinding study:
Cytotoxicity is detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn.

Mutagenicity Assay:
Assay Acceptance Criteria for a valid assay:
-All S. typhimurium tester strains must exhibit sensitivity to crystal violet to demonstrate the presence of the rfa wall mutation.
-Tester strains TA 98 and TA 100 must exhibit resistance to ampicillin to demonstrate the presence of the pKM 101 plasmid.
-All vehicle control cultures must exhibit their characteristic number of spontaneous revertants per plate. The acceptable values were: TA 98 (8-60), TA 100 (60-240), TA 1535 (4-45), TA 1537 (2-25), and WP2 uvr A (5-40).
-Tester strain culture density must reach 0.5 X 10^9 bacteria/mL to demonstrate that appropriate numbers of bacteria were plated.
-Positive controls with and without metabolic activation must exhibit a 3-fold increase over vehicle control.
-A minimum of 3 non-toxic doses are required to evaluate the assay data.

Assay Evaluation Criteria:
Tester Strains TA 98, TA 100 and WP2 uvr A:
For a test substance to be considered positive it must have produced at least a 2-fold increase in the mean revertants per plate relative to the respective vehicle control and must be accompanied by a dose response to increasing concentrations of the test substance.

Tester Strains TA 1535 and TA 1537:
For a test substance to be considered positive it must have produced at least a 3-fold increase in the mean revertants per plate relative to the respective vehicle control and must be accompanied by a dose response to increasing concentrations of the test substance.

Statistics:

For all replicate platings, the mean revertants per plate and the standard deviation were calculated.

Statistical analyses were not required due to the absence of an increase in the number of revertant colonies at any dose level. Results for the positive control materials were in line with historical data on those substances.

Results and discussion

Test results

Test results 1

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

not applicable

Positive controls validity:

valid

Additional information on results:

-Dose Rangefinding study:
No cytotoxicity was observed with either tester strain in the presence or absence of metabolic activation as evidenced by the lack of a dose-related decrease in the number of revertants per plate and a normal background lawn.

-Mutagenicity Assay:
In the initial and the confirmatory mutagenicity assay, all data were acceptable and no positive increases were observed in the mean number of revertants per plate with any of the tester strains with or without metabolic activation. All criteria for a valid assay were met.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

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 isopropyl ketone showed no evidence of mutagenic activity in this bacterial system with and without metabolic activation.

Based on the absence of genotoxic or mutagenic effects in this study with and without metabolic activation, this substance 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 of E. coli were exposed to methyl isopropyl ketone in DMSO at concentrations of 100, 333, 1000, 3330, or 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. There was no evidence of cytotoxicity in the rangefinding study or increases in induced mutant colonies over background in the mutagenicity assay, and the results of the initial assay were confirmed in an independent second experiment.