Isobutyl isobutyrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification: Isobutyl isobutyrate
Physical state/Appearance: Clear colourless liquid
Batch: TXTXOL
Purity: 98.998%
Expiry Date: 06 February 2018
Storage Conditions: Room temperature in the dark
Intended use/Application: Solvent

Species / strain / cell type:

lymphocytes:

Metabolic activation:

with and without

Metabolic activation system:

Rat S9

Test concentrations with justification for top dose:

Exposure Group Final concentration of test item Isobutyl isobutyrate (μg/mL)
4(20)-hour without S9 90, 180, 360, 380, 400, 420, 440
4(20)-hour with S9 (2%) 90, 180, 360, 480, 540, 560, 580, 600
24-hour without S9 180, 360, 440, 460, 480, 500, 540

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:

Preliminary Toxicity Test: male, aged 25 years
Main Experiment: female, aged 24 years
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Evaluation criteria:

Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985) (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including endoreduplicated cells) was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors

The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Key result

Species / strain:

lymphocytes: human lymphocytes

Remarks:

human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Isobutyl isobutyrate did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations

 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate. The dose levels selected for the Main Test were as follows:

 

Exposure Group	Final concentration of test item Isobutyl isobutyrate (μg/mL)
4(20)-hour without S9	90, 180, 360, 380, 400, 420, 440
4(20)-hour with S9 (2%)	90, 180, 360, 480, 540, 560, 580, 600
24-hour without S9	180, 360, 440, 460, 480, 500, 540

 

All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that induced optimum toxicity or marginally greater.

 

The test item, Isobutyl isobutyrate was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Identification: Isobutyl isobutyrate
Purity: 98.998%
Description: Clear colourless liquid
Receipt date: 06 February 2017
Lot number: TXTXOL
Expiry date: 06 February 2018
Storage Conditions: Room temperature in the dark

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Preliminary Toxicity Test

The dose range was set at 5.63 to 1442 μg/mL in all three exposure groups.

Main Mutagenicity Test

4-hour without S9: 30, 60, 120, 240, 260 μg/mL
4-hour with S9 (2%): 60, 120, 240, 320, 400, 480 μg/mL
24-hour without S9: 11.25, 22.5, 45, 90, 180, 240 μg/mL

Vehicle / solvent:

DMSO

Positive controls:

yes

Remarks:

Ethylmethanesulphonate at 400 μg/mL and 150 μg/mL, respectively, was used as the positive control. Cyclophosphamide at 1.5 μg/mL was used as the positive control in the presence of metabolic activation.

Details on test system and experimental conditions:

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at approximately 37 o C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Microsomal Enzyme Fraction
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test Procedure

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range was set at 5.63 to 1442 μg/mL in all three exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 105 cells/mL.


Mutagenicity Test

Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at 8 dose levels of the test item (30 to 320 μg/mL for the 4-hour –S9 exposure, 30 to 560 μg/mL in the 4-hour +S9 exposure and 5.63 to 300 μg/mL for the 24-hour –S9 exposure), vehicle and positive controls. 2 mL of S9-mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group) were added to each universal.

Evaluation criteria:

A mutation assay is considered acceptable if it meets the following criteria:

1. The majority of the plates, for both viability (%V) and TFT resistance, are analysable for each experiment.
2. The absolute viability (%V) at the time of mutant selection of the solvent controls is 65 to 120 %.
3. The total suspension growth of the solvent control following 4-hour exposure, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24-hour exposure the total suspension growth should be in the range of 32 to 180.
4. The in-house vehicle control mutant frequency is in the range of 50 – 170 x 10-6 cells. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution, or exposure to the metabolic activation system, may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10-6 mutant frequency per survivor are not acceptable and should be repeated.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

did not induce any increases in the mutant frequency

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At maximum recommended dose level of 5000 μg/mL

Vehicle controls validity:

valid

Remarks:

DMSO

Positive controls validity:

valid

Remarks:

Ethylmethanesulphonate and Cyclophosphamide

Conclusions:

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

Executive summary:

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 490 "In Vitro Mammalian Cell Gene Mutation Test. One main Mutagenicity Test was performed. In this main test, L5178Y TK mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 8 dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4-hour exposure groups both in the absence and 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 were in range of 30 -240 μg/mL.The maximum dose level used was limited by test item induced toxicity. No precipitate of the test item was observed throughout the main test. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. Optimum levels of toxicity were considered to have been achieved in all three exposure groups. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Three key OECD followed and GLP conducted in vitro genotoxicity studies were conducted for isobutyl isobutyrate. All studies, Ames assay, mouse lymphoma and chromosome aberration tests gave negative results. Therefore the test item is not requiring classification for genotoxicity under either UN GHS or EU CLP.