5-methylheptan-3-one

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

Study period:

24.10.2012-13.12.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study under GLP with full documentation according to international guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", adopted 21st July, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

June 1996 (Public draft)

Deviations:

no

Principles of method if other than guideline:

Additional guideline: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, June 2012

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5-methylheptan-3-one

EC Number:

208-793-7

EC Name:

5-methylheptan-3-one

Cas Number:

541-85-5

Molecular formula:

C8H16O

IUPAC Name:

5-methylheptan-3-one

Details on test material:

- Name of test material (as cited in study report): 5-methylheptan-3-one (Ethyl amyl ketone)
- Physical state: colourless, liquid
- Purity: Ethyl amyl ketone of 96.0 % min. mass purity (This data refers to the sum of isomeric C8 ketones)
- Lot/batch No.: Ref. Nr. 12023305
- Expiration date of the lot/batch: May 31, 2013
- Storage condition of test material: Store in a tightly closed container, cool and dry, in a well ventilated place. (In the testing laboratory the test item is stored at room temperature.)

Method

Target gene:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100: Main DNA target is GC
E. coli WP2 uvr A: Main DNA target is AT

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see table in "any other information on materials and methods"

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: see table in "any other information on materials and methods"

Metabolic activation:

with and without

Metabolic activation system:

Post mitochondrial supernatant (S9) prepared from livers of phenobarbital/beta-naphthoflavone-induced rats

Test concentrations with justification for top dose:

5000; 1581; 500; 158; 50; 15.8 µg/plate.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO); DMSO was found to be an appropriate vehicle for solubilising the test item up to 100 mg/mL.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamine

Remarks:

4 µg; Strain Salmonella TA98; non-activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2 µg; Strain Salmonella TA100 and TA1535; non-activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 µg; Strain Salmonella TA1537; non-activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

2 µl; Strain E.Coli WP2 uvrA; non-activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2 µg; all of Salmonella strains; activation

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

50 µg; E.Coli WP2 uvrA; activation

Details on test system and experimental conditions:

The study included a Preliminary Solubility Test, and a Preliminary Range Finding Test (Informatory Toxicity Test)

METHOD OF APPLICATION: Initial Mutation Test (Plate Incorporation Test) and Confirmatory Mutation Test (Pre-Incubation Test)

A) INITIAL MUTATION TEST (PLATE INCORPORATION TEST):
DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS:
The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.

B) CONFIRMATORY MUTATION TEST (PRE-INCUBATION TEST):
DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48h

NUMBER OF REPLICATIONS:
The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.

Statistics:

The colony numbers on the control, positive control and the test plates were determined (counted manually), the mean values and appropriate standard deviations and mutation rates were calculated.
Mutation rate = (Mean revertants at the test item (or control) treatments)/(Mean revertants of vehicle control)

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control;
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

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:

cytotoxicity

Remarks:

at 5000 µg/plate in S. typhimurium TA98, TA1535, TA1537 and TA100, at 1581 µg/plate in TA98

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

valid

Positive controls validity:

valid

Additional information on results:

In the Confirmatory Mutation Test the pre-incubation method was applied. This experiment was carried out using Salmonella typhimurium strains (TA98, TA100, TA1535, TA1537) and Escherichia coli WP2 uvrA strain, in presence and absence of metabolic activation (±S9 Mix) with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.
The examined concentrations were: 5000, 1581, 500, 158, 50 and 15.8 µg/plate.
The observed higher revertant colony numbers (when compared to the revertant colony numbers of the vehicle control) remained below the historical control data ranges and were far below the thresholds for being positive.

In the Confirmatory Mutation Test inhibitory effect of the test item was observed unequivocally in the Salmonella typhimurium strains that was indicated by decreased revertant colony counts and occasionally affected background lawn development.
Reduced or slightly reduced background lawn development and colony numbers below the historical control data ranges showed the inhibition at 5000 µg/plate in S. typhimurium TA98 and TA100, without and with addition of metabolic activation (±S9 Mix) and at 1581 µg/plate in TA98, with metabolic activation (+S9 Mix), in TA100, without metabolic activation (-S9 Mix).
The revertant colony numbers were below the revertant colony numbers of the vehicle control (but within the historical control data ranges) additionally reduced or slightly reduced background lawn development was obtained in S. typhimurium TA98 at 1581 µg/plate without metabolic activation (-S9 Mix), in TA1535 at 5000 (+S9 Mix); furthermore in TA1537 at 5000 µg/plate (±S9 Mix).
The obtained revertant colony numbers remained in the vehicle and in the historical control data range, but slightly reduced background lawn development showed the inhibition in S. typhimurium TA1535 at 5000 µg/plate, without metabolic activation (-S9 Mix).
The lower revertant colony numbers (in comparison with the revertant colony numbers of the vehicle control) were below the corresponding historical control data range, however the background lawn development was not affected in S. typhimurium TA100 at 500 and 50 µg/plate (+S9 Mix) and in E. coli WP2 uvrA (+S9 Mix).

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 no mutagenic activity

In conclusion, the test item Ethyl amyl ketone has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item Ethyl amyl ketone was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/ß-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

In the Range Finding Test as well as in the Initial Mutation Test the plate incorporation method was used.

Based on the results of the Solubility Test and the Range Finding Test the test item was dissolved in DMSO in a concentration of 100 mg/mL.

Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and used in the Initial and Confirmatory Mutation Tests: 5000; 1581; 500; 158; 50 and 15.8 µg/plate.

In the Initial and Confirmatory Mutation the test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.

The revertant colony numbers of vehicle control (Dimethyl sulfoxide, DMSO) plates with and without S9 Mix were mostly within the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Ethyl amyl ketone at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

The highest revertant colony number increase over the spontaneous rate of the vehicle control plates was observed in Salmonella typhimurium TA1537 at 158 µg/plate in the Initial Mutation Test (Plate Incorporation Test), with addition of metabolic activation (+S9 Mix). The mutation rate was: 1.43. This value was far below the genotoxicological threshold for being positive.

Inhibitory effect of the test item was obtained in the Confirmatory Mutation Test following the pre-incubation procedure. In general the pre-incubation procedures have equal or greater sensitivity than the plate incorporation assays and during the pre-incubation procedure the test compound, S9, and bacteria are incubated at higher concentrations than in the standard plate incorporation method.

The inhibitory effect of the test item was observed unequivocally in the examined Salmonella typhimurium strains and included the lower revertant colony numbers than the revertant colony numbers of the vehicle controls (that were often below the corresponding historical control data ranges) and reduced or slightly reduced background lawn development.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item Ethyl amyl ketone has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.