Decyl 2-ethylhexanoate

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:

21 July - 18 Aug 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

/ In the pre-incubation assay, 0.05 μL of test item or of the vehicle control was added to the top agar; no untreated and vehicle controls for positive controls (DMSO and Saline); less than 5 analysable concentrations in exp 2 for 4/5 strains

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

yes

Remarks:

/ In the pre-incubation assay, 0.05 μL of test item or of the vehicle control was added to the top agar; no untreated and vehicle controls for positive controls (DMSO and Saline); less than 5 analysable concentrations in exp 2 for 4/5 strains

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

yes

Remarks:

/ In the pre-incubation assay, 0.05 μL of test item or of the vehicle control was added to the top agar; no untreated and vehicle controls for positive controls (DMSO and Saline); less than 5 analysable concentrations in exp 2 for 4/5 strains

GLP compliance:

yes (incl. QA statement)

Remarks:

Health Care Inspectorate of the Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Decyl 2-ethylhexanoate

EC Number:

298-078-6

EC Name:

Decyl 2-ethylhexanoate

Cas Number:

93777-46-9

Molecular formula:

C18H36O2

IUPAC Name:

decyl 2-ethylhexanoate

Test material form:

liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Method

Target gene:

his operon, trp operon

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:

cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with Aroclor 1254 (500 mg/kg body weight)

Test concentrations with justification for top dose:

Based on a range-finding study (plate incorporation assay) (performed in tester strains TA 100 and E. coli WP2 uvr A; doses applied 1.7 - 5000 μg/mL), the following concentrations were used in the main experiments:

First experiment (all strains): 52, 164, 512, 1600 and 5000 μg/plate with and without metabolic activation (tested up to the limit concentration)
Second experiment (all strains): 52, 164, 512, 1600 and 5000 μg/plate with and without metabolic activation (tested up to the limit concentration)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in DMSO, ethanol was selected as the vehicle.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

other: ICR-191, 2-nitrofluorene (NF), 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation) (first experiment); preincubation (second experiment)
- Cell density at seeding (if applicable): 10^8 cells

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 ± 4 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of revertant colony number, bacterial background lawn and size of microcolonies

OTHER: Each S9 batch was characterised with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Evaluation criteria:

Acceptance criteria
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests
- the selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate
- not more than 5% of the plates are lost through contamination or some other unforeseen event

A reduction in the number of revertant colonies below the laboratory historical control data range, but no less than 20% compared to the number of revertants in the solvent control will be not considered to be caused by toxicity of the test item.

Evaluation criteria
When the test substance shows a biologically relevant increase in the number of revertant colonies of more than two times (TA100, and WP2uvrA) or three times (TA98, TA1535 and TA1537) compared to that of the solvent control at one or more concentrations with or without metabolic activation and/ or dose-related increase is detectable, the response is judged to be positive. Additionally, in case of an additional experiment, the positive response should be reproducible in at least one follow-up experiment.

The test item was considered to have shown no mutagenic activity in this study if it produces neither an biologically relevant increase in the number of revertants (as described above) nor a reproducible positive response at any of the dose groups, with or without metabolic activation.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

number of revertant colonies decreased extremely in exp. 2 at 5000 μg/plate (-S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

number of revertant colonies decreased extremely in exp. 2 at 1600 + 5000 μg/plate (-S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

number of revertant colonies decreased extremely in exp. 2 at 1600 + 5000 μg/plate (-S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

number of revertant colonies decreased slightly in exp.1 at 52 + 1600 μg/plate (-S9) (69%) and at 52 μg/plate (+S9) (73%) and extremely in exp.2 at 1600 + 5000 μg/plate (-S9) and slightly at 1600 + 5000 μg/plate (+S9) (68%)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitate was detected in both experiments on the plates of all examined strains with and without metabolic activation at least at the highest concentration tested (for more details, see table in section any other information on results).
- Other confounding effects: In exp. 2 without S9 mix the formation of microcolonies could be detected in the strains TA1535, 1537 and 100 at 1600 and 500 μg/plate and in TA98 at 5000 μg/plate.

RANGE-FINDING/SCREENING STUDIES: All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants. No toxicity appeared.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:

TA1535, -S9: 78 - 1381, mean ± SD: 785 ± 167
TA1535, +S9: 78 - 1058, mean ± SD: 228 ± 105

TA1537, -S9: 55 - 1565, mean ± SD: 653 ± 290
TA1537, +S9: 55 - 1112, mean ± SD: 387 ± 143

TA98, -S9: 410 - 2057, mean ± SD: 1155 ± 370
TA98, +S9: 263 - 1907, mean ± SD: 860 ± 323

TA100, -S9: 549 - 1848, mean ± SD: 892 ± 178
TA100, +S9: 620 - 2651, mean ± SD: 1404 ± 327

WP2uvrA, -S9: 127 - 1951, mean ± SD: 1263 ± 461
WP2uvrA, +S9: 85 - 1390, mean ± SD: 342 ± 165

- Negative (solvent/vehicle) historical control data:

TA1535, -S9: 4 - 36, mean ± SD: 14 ± 6
TA1535, +S9: 3 - 34, mean ± SD: 13 ± 5

TA1537, -S9: 3 - 25, mean ± SD: 7 ± 3
TA1537, +S9: 3 - 28, mean ± SD: 9 ± 4

TA98, -S9: 9 - 50, mean ± SD:17 ± 5
TA98, +S9: 9 - 57, mean ± SD: 25 ± 7

TA100, -S9: 63 - 153, mean ± SD: 100 ± 16
TA100, +S9: 60 - 156, mean ± SD: 103 ± 18

WP2uvrA, -S9: 12 - 68, mean ± SD: 26 ± 7
WP2uvrA, +S9: 12 - 70, mean ± SD: 32 ± 8

Any other information on results incl. tables

Table 1: Summary of test results (experiment 1, Plate Incorporation Method)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Frameshift type		Base-pair substitution type
		TA1537	TA98	TA100	TA1535	WP2 uvrA
–	Solvent control (ethanol)	13 ± 8	15 ± 2	117 ± 28	19 ± 6	22 ± 10
	52	4 ± 3	15 ± 2	92 ± 7	13 ± 8	26 ± 3
	164	8 ± 4	18 ± 4	88 ± 9	11 ± 1	21 ± 4
	512	7 ± 6	20 ± 5	86 ± 9 SP	12 ± 2	24 ± 5 SP
	1600	4 ± 1 SP	15 ± 3 SP	96 ± 9 SP	23 ± 5 SP	21 ± 4 SP
	5000	8 ± 1 SP	20 ± 3 SP	88 ± 7 SP	10 ±3 SP	15 ± 10 SP
	Positive controls (µg/plate)	ICR-191 (2.5)	NF (10)	MMS (650)	SAZ (5)	4-NQO (10)
	Mean No. of colonies/plate (average of 3 plates)	846 ± 70	1136 ± 258	1209 ± 71	886 ± 51	1430 ± 210
+	Solvent control (distilled water)	15 ± 7	34 ± 8	100 ± 8	13 ± 8	28 ± 18
	52	4 ± 4	26 ± 2	96 ± 4	11 ± 3	34 ± 7
	164	8 ± 3	30 ± 4	92 ± 15	9 ± 4	21 ± 6
	512	8 ± 7	32 ± 9	97 ± 11 SP	14 ± 5	26 ± 10 SP
	1600	9 ± 3 SP	31 ± 10 SP	91 ± 7 SP	13 ± 3 SP	28 ± 1 SP
	5000	11 ± 7 SP	36 ± 8 SP	89 ± 4 SP	16 ± 6 SP	26 ± 4 SP
	Positive controls (µg/plate)	2AA (2.5)	2AA (1)	2AA (1)	2AA (2.5)	2AA (15)
	Mean No. of colonies/plate (average of 3 plates)	389 ± 40	1140 ± 129	1044 ± 40	308 ± 39	460 ± 47

NF = 2-nitrofluorene

MMS = methyl-methanesulfonate

SAZ = sodium azide

4-NQO = 4-nitroquinoline N-oxide

2AA = 2-aminoanthracene

SP = slight precipitate

Table 2: Summary of test results (experiment 2, Pre-Incubation Method)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Frameshift type		Base-pair substitution type
		TA1537	TA98	TA100	TA1535	WP2 uvrA
–	Solvent control (ethanol)	6 ± 2	18 ± 0	88 ± 14	8 ± 1	20 ± 4
	52	12 ± 6	19 ± 3	78 ± 2	7 ± 2	21 ± 7
	164	5 ± 2	22 ± 10	90 ± 12	9 ± 2	24 ± 8
	512	5 ± 3	20 ± 8	75 ± 20	4 ± 1	18 ± 3
	1600	e MC	15 ± 6	e MC	e MC	18 ± 4
	5000	e SP MC	e SP MC	e SP MC	e SP MC	12 ± 3 s SP
	Positive controls (µg/plate)	NF (15)	NF (10)	MMS (650)	SAZ (5)	4-NQO (10)
	Mean No. of colonies/plate (average of 3 plates)	79 ± 13	1328 ± 270	724 ± 73	831 ± 9	192 ± 27
+	Solvent control (distilled water)	12 ± 4	31 ± 2	107 ± 9	12 ± 1	32 ± 6
	52	5 ± 2	28 ± 5	108 ± 4	7 ± 3	29 ± 6
	164	9 ± 5	30 ± 2	101 ± 10	8 ± 4	37 ± 8
	512	8 ± 1	29 ± 3	82 ± 9	13 ± 3	24 ± 9
	1600	5 ± 3	33 ± 9	88 ± 6	10 ± 4	28 ± 6
	5000	5 ± 2 SP	19 ± 0 SP	75 ± 9 SP	9 ± 2 SP	30 ± 1 SP
	Positive controls (µg/plate)	2AA (2.5)	2AA (1)	2AA (5)	2AA (2.5)	2AA (15)
	Mean No. of colonies/plate (average of 3 plates)	287 ± 36	628 ± 16	1328 ± 274	152 ± 22	574 ± 11

NF = 2-nitrofluorene

MMS = methyl-methanesulfonate

SAZ = sodium azide

4-NQO = 4-nitroquinoline N-oxide

2AA = 2-aminoanthracene

e = bacterial background lawn extremely reduced

s = bacterial background lawn slightly reduced

SP = slight precipitate

MC = microcolonies

Applicant's summary and conclusion

Conclusions:

Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells.