3-methylbutyl isovalerate

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:

19-08-2015 to 23-09-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate (experiments 1 and 2); top dose chosen following the pre-experiment (experiment 1)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA with metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

Medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100 without metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

Medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

TA 1537, TA 98 without metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

Medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: spontaneous reversion rates

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Experiment 1

Without metabolic activation

	Dose/plate	TA 1535	TA 1537	TA98	TA100	WP2uvrA
DMSO		12±2	11±3	31±2	176±10	36±5
Untreated		9±1	10±6	31±8	181±6	51±4
Isoamyl isovalerate µg	3	11±4	11±3	30±5	175±11	38±5
	10	14±1	10±1	30±3	175±17	40±2
	33	14±2	9±3	34±3	181±5	43±5
	100	14±3	11±3	30±8	121±14	46±4
	333	10±3	11±2	23±5	80±11	40±3
	1000	11±1	11±2	28±4	66±8	37±1
	2500	10±2	13±3	27±2	65±6	42±1
	5000	9±2	9±4	22±3	71±4	45±7
NaN3	10 µg	1146±15			1978±228
4-NOPD	10 µg			362±4
4-NOPD	50 µg		98±8
MMS	2 µl					890±65

 

With metabolic activation

	Dose/plate	TA 1535	TA 1537	TA98	TA100	WP2uvrA
DMSO		13±1	9±1	41±12	125±16	49±8
Untreated		11±3	16±3	36±8	164±12	50±4
Isoamyl isovalerate µg	3	13±1	9±1	44±13	134±4	48±9
	10	12±2	9±2	32±8	111±7	53±5
	33	14±2	11±4	32±2	128±10	48±4
	100	12±3	14±2	36±3	139±21	61±4
	333	11±3	12±2	39±2	138±12	57±9
	1000	13±3	12±3	36±6	132±18	45±4
	2500	10±3	11±4	36±2	116±20	64±3
	5000	8±0	14±1	38±8	84±31	45±5
2_AA	2.5 µg	413±7	178±12	4167±483	3660±204
2_AA	10 µg					333±31

 

 Experiment 2

Without metabolic activation

	Dose/plate	TA 1535	TA 1537	TA98	TA100	WP2uvrA
DMSO		12±2	11±3	31±2	176±10	36±5
Untreated		9±1	10±6	31±8	181±6	51±4
Isoamyl isovalerate µg	3	11±4	11±3	30±5	175±11	38±6
	10	14±1	10±1	30±3	175±17	40±2
	33	14±2	9±3	24±3	181±5	43±5
	100	14±3	11±3	30±8	121±14	46±4
	333	10±3	11±2	23±5	80±11	40±3
	1000	11±1	11±2	28±4	66±8	37±1
	2500	10±2	13±3	27±2	65±6	42±1
	5000	9±2	9±4	22±3	71±4	45±17
NaN3	10 µg	1146±15			1978±228
4-NOPD	10 µg			362±4
4-NOPD	50 µg		98±8
MMS	2 µl					890±65

 

With metabolic activation

	Dose/plate	TA 1535	TA 1537	TA98	TA100	WP2uvrA
DMSO		11±4	10±4	35±10	130±13	42±8
Untreated		10±2	12±6	31±9	168±7	52±8
Isoamyl isovalerate µg	3	10±3	11±6	38±10	106±7	50±1
	10	9±2	11±3	26±10	138±8	40±6
	33	11±3	12±4	31±9	126±11	50±9
	100	10±4	10±3	34±4	109±16	43±5
	333	10±2	11±1	38±5	110±22	47±12
	1000	10±3	10±2	28±8	60±11	41±12
	2500	10±3	12±4	27±2	47±4	48±1
	5000	5±1*	11±2	30±10*	42±8*	42±6*
2_AA	2.5 µg	372±30	190±16	4072±483	2999±225
2_AA	10 µg					367±19

NaN3 2-AA 4-NOPD MMS

sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate

*Reduced background count

 

 

 

Applicant's summary and conclusion

Conclusions:

In a bacterial reverse mutation assay using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A with and without S9 metabolic activation the test substance was not mutagenic.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations in both experiments: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate. No precipitation of the test item occurred up to the highest investigated dose.

The plates incubated with the test item showed reduced background growth in nearly all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA1537, TA 98 and, TA 100. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.