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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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
Study period:
11.2009 - 04.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Test Material Name: Diisobutyl Ketone
Chemical Name; 2,6-Dimethyl-4-heptanone
Synonyms: DIBK
Supplier, City, State (Lot, Reference Number): The Dow Chemical Company, Freeport, Texas (Lot# XA2355T643)

Method

Target gene:
Hypoxanthine-guanine-phosphoribosyltransferase (HPRT)
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO-K1-BH4

The cells are routinely maintained in Ham's F-12 nutrient mix supplemented with 5% (V/V) heat-inactivated (56°C, 30 minute), dialyzed fetal bovine serum, antibiotics, and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone, 0.25 µg/ml), and an additional 2 mM L-glutamine. The selection medium used for the detection of HGPRT- mutants will be Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 µM 6-thioguanine and 5% serum and the above-mentioned antibiotics.
Metabolic activation:
with and without
Test concentrations with justification for top dose:
preliminary toxicity assay: 0 (solvent control), 5.7, 11.3, 22.7, 45.3, 90.6, 181.3, 362.5, 725, and 1450 μg/ml

mutagenicity assay: 0 (solvent control), 200, 400, 600, 800, 1000, 1200, and 1450 μg/ml in the absence of S9 and 0 (solvent control), 100, 200, 400, 500, 600, 700, 800, and 1000 μg/ml in the presence of S9

Vehicle / solvent:
The test material was first dissolved in DMSO and further diluted (1:100) with the treatment medium to obtain the desired concentrations.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 20-methylcholanthrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 8 days
- Selection time (if incubation with a selection agent): 7 to 9 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency / colony formation
Evaluation criteria:
For an assay to be acceptable, the mutant frequency in positive controls should have been significantly higher than the solvent controls. An additional criteria was that the mutant frequency in the solvent controls should have been within reasonable limits of the laboratory historical control values and literature values. The test chemical was considered positive if it induced a statistically significant, dose related, reproducible increase in mutant frequency. The final interpretation of the data took into consideration such factors as the mutant frequency and cloning efficiencies in the solvent controls.
Statistics:
The frequency of mutants per 106 clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutant frequency variance. The actual plate counts are assumed to follow a Poisson distribution therefore the mean plate count was used as an estimate of variance. If the analysis of variance was significant at alpha = 0.05, a Dunnett's t-test was conducted, comparing each treated group and the positive control to the solvent control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed
if any of the pairwise comparisons of test material with the solvent control yielded significant differences.

Results and discussion

Test results
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:
not applicable
Positive controls validity:
valid
Additional information on results:
The pH and osmolality of treatment medium containing approximately 2589 μg/ml of the test material and medium containing 1% DMSO were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts). Alterations in the pH and osmolality of the culture medium have been shown to induce false positive responses in in vitro genotoxicity assays. There was no appreciable change in the pH at this concentration as compared to the culture medium with solvent alone and the slight drop in the osmolality was interpreted to be inconsequential to the conduct of the assay (culture medium with the test material, pH = 7.38, osmolality = 435 mOsm/kgH2O; culture medium with 1% DMSO, pH = 7.39, osmolality = 463 mOsm/kgH2O).
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

The results of the CHO/HGPRT forward gene mutation assay with diisobutyl ketone indicated that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.
Executive summary:

Diisobutyl ketone (2,6 dimethyl-4-heptanone) was evaluated in the in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S9) system. The concentrations ranged from 200 to 1450 μg/ml in the absence of S9 and from 100 to 1000 μg/ml in the presence of S9. The highest concentration for each activation system was based on the initial toxicity assay, where these concentrations resulted or exceeded relative cell survivals of 10-20%. The adequacy of the experimental conditions for detection of induced mutations was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays in the absence of S9 and 20-methylcholanthrene for assays in the presence of S9. Solvent control cultures were treated with the solvent used to dissolve the test material (i.e. dimethyl sulfoxide). The results of the CHO/HGPRT forward gene mutation assay with diisobutyl ketone indicated that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.