4-methylpentan-2-one oxime

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-10-17 to 1989-11-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

1) only four of the five required strains were tested; 2) limited information on the test substance and methodological details were presented.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-methylpentan-2-one oxime

EC Number:

203-298-2

EC Name:

4-methylpentan-2-one oxime

Cas Number:

105-44-2

Molecular formula:

C6H13NO

IUPAC Name:

4-methylpentan-2-one oxime

Test material form:

liquid

Specific details on test material used for the study:

aSOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot # 37905-27-4
- Expiration date of the lot/batch:
- Date received: 1989-06-10

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate

Test concentrations with justification for top dose:

Dose range finding (TA100 with and without S9-mix): 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330, 6670 and 10000 µg/plate
Maion test (all strains with and without S9-mix): 0, 33.3, 66.7, 100, 333, 667, 1000, 3330, 6670 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test article formed a solution at 200 mg per ml, which was the most concentrated stock dilution of test article
prepared, and remained a solution at all dose levels tested in the mutagenicity assay, therefore DMSO was chosen as vehicle.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9: 2.5 µg/plate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9: 1 µg/plate for TA98

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9: 2 µg/plate for TA100 and TA1535

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

Without S9: 2 µg/plate for TA1537

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9: 1 µg/plate for TA1538

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
The test article was diluted and the S9 mix was prepared immediately before their use in any experimental procedure. When S9 mix was required, 0.5 ml of S9 mix was added to 13 x 100 mm glass culture tubes, pre-heated to 37 + 2°C. To these tubes were added 100 µl of appropriate tester strain and 50.0 µl of vehicle or test article dilution.
When S9 mix was not required, 0.5 ml of 0.1M phosphate buffer was substituted for the S9 mix. After vortexing, the mixture was allowed to incubate for
20 + 2 minutes at 37.2 2°C. Two ml of molten selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of 25 ml
of minimal bottom agar contained in a 15x100 mm petri dish. After the overlay had solidified, the plates were inverted and incubated for approximately 48
hours at 37 + 2°C.

DURATION
- Preincubation period: 20 min +- 2
- Exposure duration: 2 days

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: triplicate

COLONY COUNTING
Revertant colonies for a given tester strain and activation condition were counted either entirely by automated colony counter or entirely by hand. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

DETERMINATION OF CYTOTOXICITY
- Method: detectable as a decrease in the number of revertant colonies per plate and/or a thinning or disappearance of the bacterial background lawn.
The condition of the background bacterial lawn was evaluatedfor evidence of cytotoxicity due to the test article by using a dissecting microscope. The cytotoxicity was scored relative to the vehicle control plate and is noted along with the revertant counts for all plates at that dose level.

Evaluation criteria:

1. Tester Strains TA98 and TA100
For a test article to be considered positive, it must cause at least a 2-fold increase in the mean revertants per plate of at least one tester strain over the mean vehicle control value for that tester strain. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.

2. Tester Strains TA1535. TA1537 and TA1538
For a test article to be considered positive, it must cause at least a 3-fold increase in the mean revertants per plate of at least one tester strain over the mean vehicle control value for that tester strain. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.

Statistics:

no data

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

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:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
Dose levels to be tested in the mutagenicity assay were selected based on the results of the dose rangefinding study, Experiment 11068-A1, conducted on the test article using tester strain TA100 in both the presence and absence of S9 (one plate per dose). Ten dose levels of test article, from 10,000 to 10.0 µg were tested.
Cytotoxicity was observed at 3330 µg per plate and above both in the presence and absence of S9 as evidenced by the reduced number of revertants per plate
and the thinning of the bacterial background lawn.

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control substances induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 mix were validated.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The background lawn was moderately to extremely reduced at the highest dose tested in all tester strains

Any other information on results incl. tables

In Experiment 11068-B1, all data were acceptable and no positive increases in the number of histidine revertants per plate were observed with any of the tester strains either in the presence or absence of S9.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The test substance was evaluated for mutagenic potential in the Ames test using S. typhimurium strains TA98, TA100, TA1535 , TA1537 and TA1538 in the presence and absence of metabolic activation. The test substance was considered to be non-mutagenic under the conditions of this test.