Ethyl 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:

14.07.2015 - 15.10.2015

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:

Ethyl isovalerate

EC Number:

203-602-3

EC Name:

Ethyl isovalerate

Cas Number:

108-64-5

Molecular formula:

C7H14O2

IUPAC Name:

ethyl 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 (experiment 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

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

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 DMSO

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:

S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100 bacteria

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

Test results 2

Key result

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

Any other information on results incl. tables

Experiment 1

Without metabolic activation

	Dose/plate	TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
DMSO		9±2	11±1	24±5	140±3	39±5
Untreated		11 ± 2	8 ± 1	27 ± 2	160 ± 10	44 ± 5
Test item	3 µg	8 ± 2	11 ± 2	24 ± 6	135 ± 5	40 ± 3
	10 µg	11 ± 3	12 ± 2	24 ± 3	158 ± 11	39 ± 2
	33 µg	10 ± 5	11 ± 3	19 ± 5	139 ± 13	43 ± 8
	100 µg	8 ± 1	10 ± 2	18 ± 4	153 ± 9	38 ± 8
	333 µg	9 ± 3	9 ± 3	21 ± 2	126 ± 5	42 ± 7
	1000 µg	9 ± 1	11 ± 4	27 ± 6	127 ± 6	40 ± 3
	2500 µg	7 ± 2	5 ± 2	20 ± 2	70 ± 4	38 ± 11
	5000 µg	7 ± 1	5 ± 2	18 ± 4	46 ± 7*	28 ± 4
NaN3	10 µg	919 ± 66			2106 ± 122
4-NOPD	10 µg			413 ± 9
4-NOPD	50 µg		90 ±5
MMS	2.0 µL					783 ± 68

 

With metabolic activation

	Dose/plate	TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
DMSO		10 ± 2	11 ± 4	34 ± 8	117 ± 10	46 ± 8
Untreated		14 ± 4	9 ± 4	36 ± 2	144 ± 10	48 ± 10
Test item	3 µg	11 ± 3	12 ± 4	34 ± 5	115 ± 11	46 ± 7
	10 µg	9 ± 1	12 ± 5	35 ± 3	107 ± 10	51 ± 7
	33 µg	9 ± 1	12 ± 4	28 ± 6	110 ± 15	46 ± 6
	100 µg	9 ± 1	13 ± 4	31 ± 4	116 ± 15	45 ± 4
	333 µg	11 ± 2	12 ± 3	29 ± 3	101 ± 4	43 ± 2
	1000 µg	8 ± 2	11 ± 2	34 ± 1	113 ± 6	43 ± 4
	2500 µg	10 ± 3	9 ± 3	33 ± 2	86 ± 9	37 ± 7
	5000 µg	9 ± 1	9 ± 2	26 ± 2	63 ± 11	44 ± 1
2-AA	2.5 µg	399 ± 8	225 ± 34	4168 ± 319	3201 ± 160
2-AA	10 µg					370 ± 37

 

Experiment 2

Without metabolic activation

	Dose/plate	TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
DMSO		11 ± 2	8 ± 3	22 ± 4	176 ± 13	34 ± 4
Untreated		11 ± 3	11 ± 2	32 ± 14	189 ± 23	40 ± 6
Test item	3 µg	12 ± 2	7 ± 1	23 ± 4	159 ± 12	35 ± 4
	10 µg	10 ± 4	7 ± 1	22 ± 4	175 ± 23	36 ± 2
	33 µg	10 ± 4	6 ± 1	26 ± 4	171 ± 20	33 ± 4
	100 µg	10 ± 4	10 ± 1	24 ± 8	164 ± 6	34 ± 7
	333 µg	9 ± 4	8 ± 1	22 ± 3	131 ± 14	37 ± 8
	1000 µg	8 ± 2	7 ± 2	21 ± 5	91 ± 13	28 ± 4
	2500 µg	6 ± 1*	7 ± 2	27 ± 1	72 ± 11	27 ± 1*
	5000 µg	4 ± 2*	7 ± 2*	5 ± 2*	0 ± 0	22 ± 2*
NaN3	10 µg	1173 ± 24			1895 ± 196
4-NOPD	10 µg			428 ± 28
4-NOPD	50 µg		103 ± 19
MMS	2.0 µL					580 ± 90

 

With metabolic activation

	Dose/plate	TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
DMSO		9 ± 1	10 ± 3	28 ± 2	146 ± 14	43 ± 7
Untreated		12 ± 3	12 ± 2	40 ± 9	212 ± 8	49 ± 8
Test item	3 µg	9 ± 3	12 ± 2	31 ± 3	143 ± 6	44 ± 6
	10 µg	10 ± 4	9 ± 5	33 ± 9	151 ± 40	47 ± 14
	33 µg	13 ± 1	9 ± 4	33 ± 2	148 ± 23	50 ± 11
	100 µg	9 ± 2	10 ± 4	29 ± 3	139 ± 8	52 ± 7
	333 µg	10 ± 1	13 ± 4	33 ± 5	136 ± 14	50 ± 8
	1000 µg	10 ± 4	8 ± 3	33 ± 4	109 ± 4	47 ± 12
	2500 µg	13 ± 3	8 ± 3	24 ± 4	57 ± 7	32 ± 5
	5000 µg	13 ± 1*	7 ± 3*	26 ± 7	40 ± 4	21 ± 2*
2-AA	2.5 µg	372 ± 72	174 ± 11	3958 ± 855	4259 ± 350
2-AA	10 µg					298 ± 8

 * Reduced background count

Applicant's summary and conclusion

Conclusions:

The test substance is negative in Bacterial Reverse Mutation Assay for S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A with and without S9 metabolic activation, hence is classified as 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 all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains used except strains TA 1537 and WP2 uvrA. 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.