Isobutyl isobutyrate

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:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl isobutyrate

EC Number:

202-612-5

EC Name:

Isobutyl isobutyrate

Cas Number:

97-85-8

Molecular formula:

C8H16O2

IUPAC Name:

2-methylpropyl 2-methylpropanoate

Specific details on test material used for the study:

Sponsor's identification: IBIB
Description: Colourless liquid
Batch number: GMNP0210302
Date received: 06 November 2003
Storage conditions room temperature in the dark

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The Salmonella typhimurium sfrains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst Escherichia coli strain WP2uvrX was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at -1960C. Prior to the master strains being used, characterisation checks were carried out to confirm the miino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 370C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

A preliminary assay was carried out to determine the toxicity of the test material -The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate.

Vehicle / solvent:

Dimethyl sulphoxide

Details on test system and experimental conditions:

Preliminary Toxicity Test

In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate. The assay was performed by dispensing measured aliquots (0.1 ml) of bacterial culture (TA100 or WP2uvrÆ) into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar and either 0.5 ml of S9-mix or phosphate buffer. 0.1ml of the test material formulation or vehicle was dosed directly onto the agar plates and immediately overlayed with the contents of the dosing tube. This procedure was repeated for each bacterial strain and for each concentration of test material both with and without S9-mix. Ten concentrations of the test material and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 370C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.


Mutation Test — Experiment 1 (Range-finding Test)
Five concentrations of the test material (50, 150, 500, 1500 and 5000 gg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar and either 0.5 ml of S9-mix or phosphate buffer. 0.1ml of the test material formulation or vehicle was dosed directly onto Vogel-Bonner agar plates and immediately overlayed with the contents of the dosing tube. This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.The positive controls were dosed using the standard plate incorporation method: 0.1 ml of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of positive control and either 0.5 ml of S9mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate).All of the plates were incubated at 370C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter

Mutation Test — Experiment 2 (Main Test)
The second experiment was performed using methodology as described for the range-finding test, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as the range-finding test (50 to 5000 ug/plate).

Evaluation criteria:

The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

Dimethyl suphoxide

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrX were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies generally within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains at 5000 ug/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. No test material precipitate was observed by eye on the plates at any of the doses tested in either the presence or absence of S9-mix. However, a slight oily precipitate was observed using a light dissection microscope at 5000 ug/plate.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.