3-methylbutyl butyrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: “Kanpoan No. 287 -- Environment Protection Agency“ “Eisei No. 127 -- Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry“.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: gene mutation assay in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

3-methylbutyl isovalerate

EC Number:

211-536-1

EC Name:

3-methylbutyl isovalerate

Cas Number:

659-70-1

Molecular formula:

C10H20O2

IUPAC Name:

3-methylbutyl 3-methylbutanoate

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

other: Chinese hamster (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: Yes
- Cell cycle length, doubling time or proliferation index: doubling time 12 - 16 h in stock cultures

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL), 10 % FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. Cell culture incubation: humidified atmosphere with 1.5 % CO2 at 37 °C.
- Properly maintained: [yes]
- Checked for Mycoplasma contamination: [yes]
- Checked for karyotype stability: [yes)
- "Cleansed' against high spontaneous background: [yes]

Metabolic activation:

with and without

Metabolic activation system:

mammalian microsomal fraction S9 mix

Test concentrations with justification for top dose:

Doses applied in the gene mutation assay (concentrations marked with ¹ were chosen for the mutation rate analysis):

Experiment I (without S9, 4 h exposure): 1.7, 3.4 ¹, 6.8 ¹, 13.5 ¹, 27.0 ¹, 54.0 ¹, 108.0 µg/mL

Experiment II (with S9, 4 h exposure): 26.9 ¹, 53.8 ¹, 107.5 ¹, 215.0 ¹, 430.0* ¹, 860.0* µg/mL

* indicates Phase separation observed

The cultures at the lowest concentration without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines. The cultures at the highest concentration without metabolic activation were not continued based on exceedingly severe cytotoxicity. The cultures at the highest concentration with metabolic activation were not continued to avoid analysis of too many insoluble concentrations.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

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): 7 d
- Selection time (if incubation with a selection agent): 8 d

SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)

NUMBER OF REPLICATIONS: 5 to determine mutation (culture flasks with medium containing 6-TG per concentration)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Stained with 10 % methylene blue in 0.01 % KOH solution

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: The experimental part of the main experiment without metabolic activation was prematurely terminated as exceedingly severe cytotoxicity occurred already at low concentrations. This experimental part was repeated as experiment IA in a lower concentration range. Experiment IA was again terminated due to severe cytotoxicity and repeated as experiment IB with even lower concentrations. The data of experiment IB are reported as first experiment without metabolic activation.

Evaluation criteria:

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

Statistical Analysis: A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
experimental group p-value
experiment I, culture I without S9 mix, p-value: 0.492
experiment I, culture II without S9 mix, p-value: 0.025 S
experiment I, culture I with S9 mix, p-value: 0.453
experiment I, culture II with S9 mix, p-value: 0.855
S = significant trend

Results and discussion

Test results

Key result

Species / strain:

other: Chinese hamster (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No relevant and reproducible increase in mutant colony numbers/1E6 cells was observed in the main experiment up to the maximum concentration. The 95 % confidence interval was not exceeded.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the second culture without metabolic activation. This trend however, was judged as irrelevant as all of the absolute values of the mutation frequency remained within the 95 % confidence interval.
In the main experiment with and without S9 mix the range of the solvent controls was from 13.0 up to 18.1 mutants per 1E6 cells; the range of the groups treated with the test item was from 9.3 up to 26.9 mutants per 1E6 cells.
EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Applicant's summary and conclusion

Conclusions:

The test item did not induce gene mutations at the HPRT locus in V79 cells and therefore is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (1723 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiment was limited by the cytotoxicity and the solubility of the test item.

 

The experimental part of the main experiment without metabolic activation was prematurely terminated as exceedingly severe cytotoxicity occurred already at low concentrations. This experimental part was repeated as experiment IA in a lower concentration range. Experiment IA was again terminated due to severe cytotoxicity and repeated as experiment IB with even lower concentrations. The data of experiment IB are reported as the first experiment without metabolic activation.

 

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.