2-(2-heptadec-8-enyl-2-imidazolin-1-yl)ethanol

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:

24 Sep 2009 to 06 Nov 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-heptadec-8-enyl-2-imidazolin-1-yl)ethanol

EC Number:

202-414-9

EC Name:

2-(2-heptadec-8-enyl-2-imidazolin-1-yl)ethanol

Cas Number:

95-38-5

Molecular formula:

C18H34N2O - C24H46N2O

IUPAC Name:

2-(2-heptadec-8-en-1-yl-4,5-dihydro-1H-imidazol-1-yl)ethanol

Constituent 2

Reference substance name:

(Z)-2-(8-heptadecenyl)-4,5-dihydro-1H-imidazole-1-ethanol

EC Number:

244-501-4

EC Name:

(Z)-2-(8-heptadecenyl)-4,5-dihydro-1H-imidazole-1-ethanol

Cas Number:

21652-27-7

Molecular formula:

C22H42N2O

IUPAC Name:

2-(2-heptadec-8-en-1-yl-4,5-dihydro-1H-imidazol-1-yl)ethanol

Specific details on test material used for the study:

- Name of test material (as cited in study report): Amine O
- Analytical purity: 96.6%
- Lot/batch No.: 05774HP8
- Expiration date of the lot/batch: 04 May 2012
- Substance type: organic
- Physical state: Liquid, yellow to brown, clear
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Method

Target gene:

S. typhimurium: his
E. coli: trp

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

1st Experiment, SPT: 0; 22; 110; 550; 2750 and 5500 μg/plate, all strains

2nd Experiment, SPT: 0; 0.2; 1; 5; 25 and 50 μg/plate (TA 1535 ± S9 mix and E. coli - S9 mix); 0; 0.1; 0.5; 2.5; 12.5 and 25 μg/plate (TA 100, TA 1537, TA 98 ± S9 mix and E. coli + S9 mix). Due to strong bacteriotoxicity observed in the 1st experiment, the experimental part was repeated with lower dose groups.

3rd Experiment, PIT: 0; 0.2; 1; 5; 25 and 50 μg/plate (TA 1535 ± S9 mix and E. coli - S9 mix); 0; 0.1; 0.5; 2.5; 12.5 and 25 μg/plate (E. coli + S9 mix); 0; 0.05; 0.25; 1.25; 6.25 and 12.5 μg/plate (TA 100, TA 1537, TA 98 ± S9 mix)

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: Due to the limited solubility of the test item in water, DMSO was used as 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

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene (2-AA), 4-nitro-o-phenylenediamine (NOPD)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation; preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: at 37°C for 48 – 72 hours in the dark

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer

Evaluation criteria:

ACCEPTANCE CRITERIA
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain
- The sterility controls revealed no indication of bacterial contamination.
- The positive control items both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- The titer of viable bacteria was ≥ 108/mL.

ASSESSMENT CRITERIA
The test item 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 item is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

none

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: 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

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitation was found with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
In addition, the positive control items both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A strong bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 12.5 μg/plate onward.
In the preincubation assay clear bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 6.25 μg/plate onward.

Any other information on results incl. tables

1st Experiment (SPT)

	Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	31	32	101	102	15	14	8	9	47	48
22	9	12	67	76	14	14	3	2	51	15
110	0	0	0	0	0	0	0	0	0	0
550	0	0	0	0	0	0	0	0	0	0
2750	0	0	0	0	0	0	0	0	0	0
5500	0	0	0	0	0	0	0	0	0	0
positive control	502	836	724	973	775	184	351	176	863	213

2nd Experiment (SPT)

	Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control					18	17			41
0.2					18	17			44
1					16	17			37
5					19	17			39
25					15	15			41
50					3	6			8
positive control					1013	175			827

	Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	29	36	130	134			10	12		45
0.1	31	37	123	125			10	10		43
0.5	26	32	138	100			11	10		42
2.5	27	34	130	111			12	9		46
12.5	19	31	82	96			12	7		43
25	5	5	8	15			2	2		5
positive control	426	831	831	874			395	179		230

3rd Experiment (PIT)

	Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control					16	15			33
0.2					14	18			34
1					11	15			42
5					16	14			30
25					11	13			14
50					0	2			9
positive control					605	137			606

	Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	25	27	98	108			8	7
0.05	20	27	101	107			7	7
0.25	20	31	97	120			7	7
1.25	22	28	92	104			7	8
6.25	18	25	72	102			2	4
12.5	0	7	0	15			0	3
positive control	494	834	882	995			356	145

	Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control										36
0.1										31
0.5										31
2.5										33
12.5										24
25										16
positive control										234