Methyl 2-ethylhexanoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (1997).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 2-ethylhexanoate

EC Number:

212-429-2

EC Name:

Methyl 2-ethylhexanoate

Cas Number:

816-19-3

Molecular formula:

C9H18O2

IUPAC Name:

methyl 2-ethylhexanoate

Test material form:

other: liquid

Details on test material:

- Physical state: liquid
- Storage condition of test material: room temperature in the dark with desiccant

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Initial toxicity-mutation test:
B1 & B2: 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 μg per plate
Confirmatory mutagenicity test: 75, 200, 600, 1800 and 5000 μg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: Ethanol (EtOH) was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article formed a soluble and clear solution in ethanol (EtOH) at approximately 500 mg/mL, the maximum concentration tested.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72h at 37 +/- 2 degrees C

NUMBER OF REPLICATIONS:
- Initial toxicity-mutation assay: 2 plates per dose
- Confirmatory mutagenicity assay: 3 plates per does.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Evaluation of Results
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of 2 increasing concentrations of test article. Data-sets for strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3x times the mean vehicle control value. Data sets for strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2x times the mean vehicle control value.

Criteria for a Valid Test
The following criteria must be met for initial toxicity-mutation and the confirmatory mutagenicity assays to be valid. Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (ifa) and the deletion in the uvrB gene. Cultures of strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10- 50; TA100, 80- 240; TA1535, 5- 45; TA1537, 3- 21; WP2 uvrA, 10- 60. To ensure that appropriate numbers of bacteria are plated, strain culture titers must be greater than or equal to 0.3x109 cells/mL. The mean of each positive control must exhibit at least a 3x fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of 3 non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: 1) A >50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accomparued by an abrupt dose-dependent drop in the revertant count. 2) At least a moderate reduction in the background lawn.

Results and discussion

Test results

Test results 1

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:

not applicable

Positive controls validity:

valid

Test results 2

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Initial toxicity-mutation assay:
In the toxicity-mutation assay, the maximum dose tested was 5000 ug per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 uL plating aliquot. The dose levels tested were 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 ug per plate. Neither precipitate nor appreciable toxicity was observed. Based on the findings of the toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 ug per plate. In Experiment Bl (Initial Toxicity-Mutation Assay), no positive mutagenic responses were observed with tester strains TA98, TA100, TA1535 and WP2 uvrA in the presence of S9 activation and with tester strains TA1537 and WP2 uvrA in the absence of S9 activation. Due to a dosing error in which plates did not receive an aliquot of tester strain, tester strain TA98 in the absence of S9 activation was not evaluated but was retested in Experiment B2. Due to an unacceptable positive control value, tester strain TA1537 in the presence of S9 activation was not evaluated but was retested in Experiment B2. Due to toxicity on all the plates except for one vehicle control plate, tester strain TA100 in the absence of S9 activation was not evaluated but was retested in Experiment B2. Due to toxicity on one positive control plate and an overall unacceptable positive control value, tester strain T A1535 in the absence of S9 activation was not evaluated but was retested in Experiment B2. In Experiment B2 (Repeat Toxicity-Mutation Assay), no positive mutagenic response was observed with tester strain TA1537 in the presence of S9 activation and with tester strains TA98, TA100 and TA1535 in the absence of S9 activation.

Confirmatory mutagenicity assay:
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. B1 - initial toxcity-mutation assay

+/- S9	Test material concentration (ug/plate)	Number of revertants (colonies/plate)
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		Replicate	Mean	Replicate	Mean	Replicate	Mean	Replicate	Mean	Replicate	Mean
+S9	Vehicle	24	26	113	128	17	13	Not evaluated		14	14
		27		142		9				13
	2.5	20	23	133	134	19	19			21	19
		25		135		19				17
	7.5	30	28	140	138	16	18			12	14
		25		135		19				16
	25	26	26	134	132	12	14			10	12
		25		129		16				13
	75	21	23	128	126	22	16			17	13
		24		123		9				9
	200	20	20	141	123	16	14			12	11
		19		105		12				9
	600	19	22	108	112	12	13			10	11
		25		116		13				12
	1800	19	22	117	119	11	13			12	11
		24		121		14				10
	5000	19	22	96	116	13	13			18	13
		24		136		12				8
-S9	Vehicle	Not evaluated		Not evaluated		Not evaluated		8	6	12	11
								4		10
	2.5							6	5	16	12
								4		8
	7.5							5	7	11	13
								8		14
	25							6	4	16	17
								1		18
	75							9	8	11	10
								6		8
	200							7	7	15	13
								6		11
	600							1	6	8	12
								10		15
	1800							2	4	10	11
								5		11
	5000							3	3	12	13
								2		14
Positive control with S9	Name	2-aminoanthracene		2-aminoanthracene		2-aminoanthracene		Not evaluated		2-aminoanthracene
	Concentration (ug/plate)	1		1		1				10
	Replicates	150		615		76				57
		139		479		62				47
	Mean	145		547		69				52
Positive control without S9	Name	Not evaluated		Not evaluated		Not evaluated		9-aminoacridine		methanesulfonate
	Concentration (ug/plate)							75		1000
	Replicates							550		32
								63		42
	Mean							307		37

Table 2. B2 - initial toxcity-mutation assay

+/- S9	Test material concentration (ug/plate)	Number of revertants (colonies/plate)
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		Replicate	Mean	Replicate	Mean	Replicate	Mean	Replicate	Mean	Replicate	Mean
+S9	Vehicle	Not evaluated		Not evaluated		Not evaluated		16	15	Not evaluated
								14
	2.5							9	9
								8
	7.5							9	11
								13
	25							13	12
								11
	75							6	10
								14
	200							10	12
								13
	600							9	6
								3
	1800							8	10
								11
	5000							15	13
								10
-S9	Vehicle	24	21	218	202	16	17	Not evaluated		Not evaluated
		17		185		17
	2.5	17	17	222	228	16	15
		16		234		13
	7.5	19	15	215	222	20	20
		11		228		19
	25	18	18	198	214	11	11
		18		229		10
	75	10	16	160	155	14	14
		22		150		14
	200	13	13	207	208	19	14
		13		209		8
	600	15	17	196	192	16	19
		18		188		22
	1800	13	13	194	187	14	14
		13		180		14
	5000	12	12	145	157	13	13
		11		169		12
Positive control with S9	Name	Not evaluated		Not evaluated		Not evaluated		2-aminoanthracene		Not evaluated
	Concentration (ug/plate)							1
	Replicates							88
								94
	Mean							91
Positive control without S9	Name	2-nitrofluorene		sodium azide		sodium azide		Not evaluated		Not evaluated
	Concentration (ug/plate)	1		1		1
	Replicates	239		662		254
		223		615		284
	Mean	231		639		269

Table 3. B3 - confirmatory mutagenicity assay

+/- S9	Test material concentration (ug/plate)	Number of revertants (colonies/plate)
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		Replicate	Mean	Replicate	Mean	Replicate	Mean	Replicate	Mean	Replicate	Mean
+S9	Vehicle	28	35	215	226	17	18	7	7	20	21
		40		227		22		7		19
		38		236		16		8		23
	75	32	32	213	202	17	18	7	9	18	19
		33		188		19		10		21
		32		205		18		10		18
	200	43	35	207	201	22	21	10	9	21	20
		33		186		23		9		23
		28		211		18		8		15
	600	35	34	193	203	15	17	10	11	19	17
		34		230		17		11		23
		33		187		20		11		10
	1800	23	29	206	194	16	14	10	9	18	19
		23		191		16		11		20
		40		185		11		7		18
	5000	22	27	197	199	14	16	6	5	20	17
		32		201		20		5		14
		27		200		13		5		18
-S9	Vehicle	30	35	230	220	16	18	9	10	15	19
		40		210		18		12		26
		35		220		20		9		17
	75	23	26	181	191	20	17	7	8	12	13
		18		199		17		11		13
		36		192		15		7		13
	200	33	34	196	210	17	23	11	11	10	14
		37		205		24		6		15
		32		230		27		15		17
	600	37	30	178	166	11	12	14	11	17	19
		30		155		15		13		22
		24		166		11		5		19
	1800	35	33	177	183	16	16	10	11	19	21
		28		157		14		13		23
		37		216		19		9		22
	5000	28	24	195	189	15	13	10	9	15	15
		24		191		16		10		11
		20		180		9		7		19
Positive control with S9	Name	2-aminoanthracene		2-aminoanthracene		2-aminoanthracene		2-aminoanthracene		2-aminoanthracene
	Concentration (ug/plate)	1		1		1		1		10
	Replicates	1200		1792		170		200		760
		476		2044		93		189		551
		319		1792		74		75		509
	Mean	665		1876		112		155		607
Positive control without S9	Name	2-nitrofluorene		sodium azide		sodium azide		9-aminoacridine		methyl methanesulfonate
	Concentration (ug/plate)	1		1		1		75		1000
	Replicates	184		749		248		157		131
		149		743		265		171		114
		207		717		240		796		122
	Mean	180		736		251		375		122

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, the test material did not induce gene mutations in the strains of S. typhimurium and E. coli used.

Executive summary:

The study was performed to the requirements of OECD Guideline 471 and ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (1997) under GLP, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA in both the presence and absence of S-9 mix. A preliminary test was performed to determine the toxicity of the test material. A range-finding study was performed to determine the doses used for the main test. In the main test, the plate incorporation method was used and was evaluated at a concentration of up to 5000 µg/plate. Positive controls appropriate for each strain, in the presence and absence of S9 -mix, were included. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The vehicle (ethanol) control plates gave counts of revertant colonies 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. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA in the presence and absence of S-9 mix.