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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Report date:
Reference Type:
Mutagenicity studies on ketone solvents: methyl ethyl ketone, methyl isobutyl ketone and isophorone
O'donoghue JL, Haworth SR, Curren RD, Kirby PE., Lawlor T et al.
Bibliographic source:
Mutat. Res., 206, 149-161

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): Methyl Isobutyl Ketone
- Physical state: Liquid in amber bottle; color not determined
- Storage condition of test material: Room temperature under nitrogen


Target gene:
Thymidine kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver microsomes (S9)
Test concentrations with justification for top dose:
0.6, 1.4, 2.1, 2.9, or 3.7 µL/mL (-S9) and 1.4, 1.9, 2.5, 3.0, or 3.4 µL/mL (+S9).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: Ethylmethanesulfonate (-S9) and 7,12-Dimethylbenzanthracene (+S9)
Details on test system and experimental conditions:

- Exposure duration: 4 hours at 37 ºC
- Expression time (cells in growth medium): 2 days with a cell population adjustment at 24 and 48 hours

NUMBER OF REPLICATIONS: Duplicate (no repeat experiment performed).
Evaluation criteria:
The following criteria were used as guidelines in judging the significance of the activity of a test article in this system. In evaluating the results, it is considered that increases in mutant frequencies, which occur only at highly toxic concentrations, may be due to epigenetic events. Unfortunately, it is impossible to formulate criteria which would apply to all types of data which may be generated and therefore the scientist’s evaluation must be the final endpoint.

Positive:- if there is a positive dose response and one or more of the three highest doses exhibit a mutant frequency which is two-fold greater than the background level.
Equivocal:- if there is no dose response but any one or more doses exhibit a two-fold increase in mutant frequency over background.
Negative:- if there is no dose response and none of the test cultures exhibit mutant frequencies which are two-fold greater than background.

1. Some of the numbers generated by the test data, whether it is toxicity, mutant frequency, etc., are computed using non-rounded numbers. This may, in some instances, cause what appear to be errors in calculation if only the rounded numbers are used when checking the data.
2. All of the raw data generated by the assay and the original final report will be maintained in Microbiological Associates’ archives located in our Bethesda, Maryland facilities.
3. The stability of the test article under the actual experimental conditions used in this study was not determined by Microbiological Associates.
4. All test article stock solutions were freshly prepared immediately before their use in each procedure.
Not performed (number of revertant colonies were counted)

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
other: (-S9) = equivocal (defined in the study report as the absence of a dose-response relationship, but the presence of a two-fold increase in mutant frequency over background at any of the doses examined) and (+S9) = negative
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Any other information on results incl. tables

Chemical Manufacturers Association’s test article, methyl isobutyl ketone was tested in the L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay in the presence and absence of Aroclor induced rat liver S-9. Four mutagenesis assays were conducted. However, there were technical difficulties with three of the assays and the results were not provided in the study report. In the fourth assay, four non-activated cultures (-S9) that were cloned exhibited mutant frequencies which were more than twice the mean mutant frequency of the solvent controls. No clear dose-dependent response was noted in the cultures. None of the S9 activated cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls.

Applicant's summary and conclusion

Interpretation of results (migrated information):
ambiguous without metabolic activation
negative with metabolic activation
Executive summary:

In a non-GLP mammalian gene mutation assay (equivalent to OECD guideline 476), MIBK was tested at doses of 0, 0.6, 1.4, 2.1, 2.9, or 3.7 µL/mL in the absence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9) and at doses of 0, 1.4, 1.9, 2.5, 3.0, or 3.4 µL/mLin the presence of exogenous metabolic activation in mouse lymphoma L5178Y cells.  The experiment was conducted in duplicate; however, an independent repeat experiment was not performed. DMSO was used as the vehicle and ethylmethanesulfonate and 7,12-dimethylbenzanthracene were used as the positive control compounds in the absence and presence of metabolic activation, respectively. No cytotoxicity and no increase in the mutant frequency were observed at any MIBK concentration in the presence of metabolic activation. Cytotoxicity and equivocal genotoxicity (defined as a two-fold increase in the mutation frequency over solvent control levels at one or more dose levels but the absence of a dose-response) were noted at 3.7 µL/mL (the highest concentration tested) in the absence of metabolic activation. Incubation with positive control substances in the presence or absence of metabolic activation resulted in anticipated increases in the mutation frequencies.