5-methylheptan-3-one oxime

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: L5178T TK +/- Mouse Lymphoma Assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May 2014 to 14 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5-methylheptan-3-one oxime

EC Number:

245-010-8

EC Name:

5-methylheptan-3-one oxime

Cas Number:

22457-23-4

Molecular formula:

C8H17NO

IUPAC Name:

5-methylheptan-3-one oxime

impurity 1

Reference substance name:

(E)-3,4-dimethylhexan-2-one oxime

IUPAC Name:

(E)-3,4-dimethylhexan-2-one oxime

impurity 2

Reference substance name:

(Z)-3,4-dimethylhexan-2-one oxime

IUPAC Name:

(Z)-3,4-dimethylhexan-2-one oxime

Test material form:

liquid

Specific details on test material used for the study:

Batch: PE00083052
Purity: 96.3%

Method

Target gene:

This study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci.

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:

S9

Test concentrations with justification for top dose:

0, 5.59, 11.19, 22.38, 44.75, 89.5, 179, 358, 716, 1432 µg/mL

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Ethylmethanesulphonate without S9 activation Cyclophosphamide with S9 activation

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 5.59 to 1432 μg/mL in all three exposure groups. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (µg/mL)	%RSG(-S9) 4-Hour Exposure	%RSG(+S9) 4-Hour Exposure	%RSG(-S9) 24-Hour Exposure
0	100	100	100
5.59	94	93	97
11.19	86	101	108
22.38	84	94	96
44.75	80	101	93
89.5	78	9	73
179	54	72	25
358	0	0	0
716	0	0	0
1432	0	0	0

There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups. The onset of test item-induced toxicity was very sharp in all three of the exposure groups. A precipitate of the test item was observed at 1432 μg/mL in all three exposure groups at dosing. Based on the %RSG values observed, the maximum dose level in the subsequent mutagenicity experiment was limited by test item-induced toxicity.

 

Mutagenicity Test

Experiment 1

There was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of reductions in viability (%V) in the absence of metabolic activation, therefore indicating that residual toxicity had not occurred in this exposure group. In the presence of metabolic activation a reduction in viability can be observed, but only at a single dose level with excessive cytotoxicity (268.5 μg/mL). Based on the %RSG and RTG values observed, optimum levels of toxicity were achieved in the absence of metabolic activation only. Optimum levels of toxicity were not achieved in the presence of metabolic activation, due to the very steep toxicity curve of the test item.

Whilst optimum levels of toxicity were not achieved, a dose level in the presence of metabolic activation, that exceeded the upper limit of toxicity was plated for viability and 5-TFT resistance as sufficient cells were available at the time of plating (268.5 μg/mL). The excessive toxicity observed at 358 μg/mL in both the absence and presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 268.5 μg/mL in the presence of metabolic activation, exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances.

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6per viable cell, at any of the dose levels (including the dose levels that exceeded the upper limit of acceptable toxicity), in either the absence or presence of metabolic activation. With no evidence of any toxicologically significant increases in mutant frequency in either the absence or presence of metabolic activation in this Experiment, the test item was considered to have been adequately tested in the absence of metabolic activation. Precipitate of the test item was not observed at any of the dose levels.

As was seen previously, there was evidence of marked toxicity in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. In the presence of metabolic activation a reduction in viability can be observed, but only at a single dose level with excessive cytotoxicity (270 μg/mL). Based on the RTG and / or %RSG values observed, very near to optimum levels of toxicity were considered to have been achieved in both the absence and presence metabolic activation. In the presence of metabolic activation the highest test item dose level (270 μg/mL) had a RTG value of 0.08 and a %RSG of 11%, which was very close to optimum toxicity. However, marginally fell below the acceptable level of toxicity and was therefore excluded from statistical analysis. The excessive toxicity observed at and above 180 μg/mL in the absence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a modest effect on the toxicity of the test item.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6per viable cell, at any of the dose levels (including the dose levels that exceeded the upper limit of acceptable toxicity), in either the absence or presence of metabolic activation. It should be noted that all mutant frequency values were within the acceptable range for a vehicle control culture. With very near to optimum levels of toxicity achieved and no evidence of any toxicologically significant increases in mutant frequency in Experiments 1 and 2 inclusive, it was considered that the test item was adequately exposed. Precipitate of test item was not observed at any of the dose levels.

 

CONCLUSION

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Applicant's summary and conclusion

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests" adopted 21 July 1997, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods…….

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle, dimethyl sulfoxide (DMSO), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (2% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for viability and expression of mutant colonies were as follows:

 

Experiment 1

Group	Concentration of Stemone (μg/mL) plated for mutant frequency
4-hour without S9	22.38, 44.75, 89.5, 134.25, 179, 268.5
4-hour with S9 (2%)	22.38, 44.75, 89.5, 134.25, 179, 268.5

Experiment 2

Group	Concentration of Stemone (μg/mL) plated for mutant frequency
24-hour without S9	15, 30, 60, 90, 120, 150
4-hour with S9 (2%)	60, 90, 180, 210, 240, 270

 

Results……..

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of the test item was not observed at any of the dose levels in the Mutagenicity Test. The vehicle controls (DMSO) had mutant frequency values that wereconsidered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

 

Conclusion

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.