1-(vinyloxy)dodecane

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:

2006-10-07 to 2006-11-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study conducted under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

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:

1-(vinyloxy)dodecane

EC Number:

212-141-7

EC Name:

1-(vinyloxy)dodecane

Cas Number:

765-14-0

Molecular formula:

C14H28O

IUPAC Name:

1-(ethenyloxy)dodecane

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: All Salmonella strains (rfa-minus) mutation, UV-sensitive "uvrB-minus"; For strain TA 98 and TA 100 R-factor plasmid pKM 101 (ampicillin resistance marker). E. coli WP2 uvrA with deficient excision repair.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9 mix

Test concentrations with justification for top dose:

20 µg - 5000 µg/plate (dosel levels for Standard Plate Test and Preincubation Test)

Vehicle / solvent:

- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, acetone was used as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with S9: 2-aminoanthracene (TA 1535, TA 100, TA 1537, TA 98, E.coli WP2 uvrA); without S9: N-methyl-N'-nitro-N-nitrosoguanidine (TA 1535, TA 100), 4-nitro-o-phenylendiamine (TA 98), 9-aminoacridine (TA 1537), 4-nitroquinoline-N-oxide (E. coli WP2 uvrA)

Details on test system and experimental conditions:

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

I) Standard plate test with Salmonella typhimurium

METHOD OF APPLICATION:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle
0.1 mL fresh bacterial suspension
0.5 mL S-9 mix (in tests with metabolic activation) or
0.5 mL phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 mL purified water
20 mL Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

DURATION:
- Exposure duration: After incubation at 37°C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

- SELECTION AGENT: his- medium

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


II) Standard plate test with Escherichia coli

METHOD OF APPLICATION:
Test tubes containing 2 mL portions of soft agar which consists of 100 mL agar (0.6% agar + 0.6% NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at 45°C and the remaining components are added in the following order:
0.1 mL test solution or vehicle
0.1 mL bacterial suspension
0.5 mL S-9 mix (in tests with metabolic activation) or
0.5 mL phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.

DURATION:
- Exposure duration: After incubation at 37°C for 48 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

- SELECTION AGENT: trp- medium

III) Preincubation Test

METHOD OF APPLICATION:
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.

DURATION:
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect was occasionally observed in the standard plate test depending on the strain and test conditions at 5000 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test substance was found from about 2500 ug/plate onward.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

I. Standard plate test

Table 1: Revertants (mean of three plates), without S9 mix

Dose µg/plate		TA1535	TA 100	TA1537	TA98	E. coli WP2 uvrA
Acetone		15	114	9	31	33
20 µg		16	116	10	28	31
100 µg		16	108	6	28	28
500 µg		14	96	8	30	37
2500 µg		12	103	7	26	29
5000 µg		8	90	4	24	29
Pos. contr.
MNNG (5.0 µg)		904	997
AAC (100 µg)				424
NOPD (10 µg)					595
4-NQO (5.0 µg)						591

 

Table 2: Revertans (mean of three plates), with S9 mix

Dose µg/plate	TA1535		TA 100	TA1537	TA98	E. coli WP2 uvrA
Acetone	18		125	9	36	38
20 µg	17		115	7	36	36
100 µg	16		112	11	32	31
500 µg	16		96	12	31	31
2500 µg	14		101	8	30	32
5000 µg	8		116	6	28	34
Pos. contr.
2-AA (2.5 µg)		129	878	145	758
2-AA (60 µg)						243

 

II. Preincubation test

 

Table 3: Revertants (mean of three plates), without S9 mix

Dose µg/plate		TA1535	TA 100	TA1537	TA98	E. coli WP2 uvrA
Acetone		16	113	9	28	33
20 µg		15	115	7	25	32
100 µg		16	113	9	25	33
500 µg		16	106	9	26	33
2500 µg		16	100	9	24	31
5000 µg		13	103	7	25	32
Pos. contr.
MNNG (5.0 µg)		642	848
AAC (100 µg)				403
NOPD (10 µg)					452
4-NQO (5.0 µg)						550

 

Table 4: Revertants (mean of three plates), with S9 -mix

Dose µg/plate	TA1535		TA 100	TA1537	TA98	E. coli WP2 uvrA
Acetone	18		116	10	34	37
20 µg	18		113	7	32	36
100 µg	14		112	10	29	31
500 µg	18		104	9	32	35
2500 µg	15		110	9	29	36
5000 µg	15		99	8	29	34
Pos. contr.
2-AA (2.5 µg)		130	773	128	620
2-AA (60 µg)						227