2-heptadecyl-1H-imidazole

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:

30 May 2017 - 24 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997

Deviations:

no

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:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit, Schwabach, Deutschland

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

2-heptadecyl-1H-imidazole

EC Number:

245-589-7

EC Name:

2-heptadecyl-1H-imidazole

Cas Number:

23328-87-2

Molecular formula:

C20H38N2

IUPAC Name:

2-heptadecyl-1H-imidazole

Method

Target gene:

his operon for (for S. typhimurium strains)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment for Toxicity: 0.316, 1.00, 3.16, 10.0, 31.6, 100, 250 and 500 µg/plate with and without metabolic activation for TA 98 and TA 100

Experiment 1: 3.16, 10.0, 31.6, 100, 250 and 500 µg/plate with and without metabolic activation
Experiment 2: 1.58, 5.00, 15.8, 50.0, 158 and 500 µg/plate with and without metabolic activation

The test substance concentrations used in the main experiment were chosen according to the results of the pre-experiment. Due to the limited solubility of the test item 500 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades.
As the results of the pre-experiment were in accordance with the criteria described above, these were reported as a part of the main experiment 1.

Vehicle / solvent:

- Vehicle/solvent used: tetrahydrofuran, VWR (Lot No. 17C051199)
- Justification for choice of solvent/vehicle: A solubility test with several solvents (distilled water, DMSO, ethanol, acetone and tetrahydrofuran) was performed. No solution and no homogenous suspension could be obtained with these solvents at the recommended maximum stock solution of 50 mg/mL. According to a Eurofins Study #174109 a stock solution of 5 mg/mL in tetrahydrofuran was used. The test item was freshly prepared as solution in tetrahydrofuran and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity. A correction factor of 1.07 was applied to consider the purity of the test item.

Controls

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar, plate incorporation test (pre-experiment and main experiment 1 and 2)

DURATION
- Exposure duration: at least 48 - 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants down to a mutation factor of approximately ≤ 0.5 or a clearing of the bacterial background lawn

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98 and TA 100 the number of reversions is at least twice as high
- if in tester strain TA 102 the number of reversions is at least 1.5-fold higher
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In two independent experiments several concentrations of the test substance were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. Due to the limited solubility of the test substance 500 µg/plate was selected as the maximum concentration. Since no homogenous suspension could be obtained with the tested solvents at the recommended maximum stock solution of 50 mg/mL, the suspension was diluted up to solubility of the test substance at a stock solution of 5 mg/mL. As at higher concentrations a part of the test substance retains at the bottom of the test tube and could not poured over the surface of a minimal agar plate, it was not possible to perform the test up to precipitating concentrations. No precipitation of the test substance was observed in any tester strain used in experiment 1 and 2 (with and without metabolic activation).

RANGE-FINDING/SCREENING STUDIES: Only strain TA 98 and TA 100 were tested in the pre-experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects of the test substance was observed in any strain.

Any other information on results incl. tables

Table 2: Results pre-experiment

Test Substance	Dose (µg/plate)	TA 98		TA 100
		Mutation Factor [toxicity]*		Mutation Factor [toxicity]*
		without S9	with S9	without S9	with S9
Solvent Control (Tetrahydrofuran)		1.0	1.0	1.0	1.0
4-NOPD	10.0	8.0	-	-	-
NaN3	10.0	-	-	11.1	-
2-AA	2.50	-	61.1	-	10.7
Test Substance	0.316	1.0	1.1	0.9	0.8
	1.00	0.9	1.1	1.1	0.7
	3.16	0.8	0.9	1.0	0.8
	10.0	0.9	1.1	1.1	1.0
	31.6	0.8	0.9	1.2	0.8
	100	0.8	1.2	1.1	0.8
	250	0.8	1.2	1.0	1.1
	500	1.1	1.0	1.0	1.2

*toxicity parameter: B = Background lawn reduced; N = No background lawn

Table 3: Results experiment 1

	EXPERIMENT 1 (Revertant colonies per plate ± SD)
	S9-Mix	Without
	Test Substance (µg/plate)	TA98	TA 100	TA 1535	TA 1537	TA 102
Distilled water		40 ± 2.3	89 ± 12.6	23 ± 1.2	17 ± 3.2	252 ± 14.0
Tetrahydrofuran		39 ± 8.1	75 ± 8.9	20 ± 2.1	20 ± 2.5	243 ± 8.0
Test Substance	3.16	30 ± 3.1	74 ± 12.7	21 ± 4.0	19 ± 2.0	212 ± 5.1
	10.0	37 ± 7.5	83 ± 14.8	22 ± 2.3	19 ± 1.5	205 ± 30.0
	31.6	32 ± 2.1	94 ± 8.5	21 ± 2.5	19 ± 2.6	195 ± 24.4
	100	30 ± 6.7	85 ± 3.1	19 ± 2.6	18 ± 3.6	201 ± 6.0
	250	32 ± 5.5	74 ± 11.0	18 ± 2.9	20 ± 3.5	255 ± 33.3
	500	42 ± 5.0	76 ± 10.8	21 ± 3.1	19 ± 4.2	292 ± 25.5
4-NOPD	10 (TA 98)/40 (TA 1537)	313 ± 9.2	-	-	119 ± 10.0	-
NaN3	10	-	834 ± 131.6	945 ± 44.8	-	-
MMS	1					2394 ± 318.7
	S9-Mix	With
	Test Substance (µg/plate)	TA98	TA 100	TA 1535	TA 1537	TA 102
Distilled water	-	39 ± 5.1	96 ± 9.1	19 ± 1.0	24 ± 4.6	241 ± 20.0
Tetrahydrofuran	-	37 ± 7.5	97 ±7.5	17 ± 2.6	25 ± 2.0	215 ± 7.2
Test Substance	31.6	33 ± 3.2	81 ± 17.1	14 ± 2.5	23 ± 2.0	451 ± 310.3
	100	39 ± 0.6	93 ± 26.1	18 ± 2.0	22 ± 1.5	265 ± 29.5
	316	32 ± 6.0	77 ± 22.9	17 ± 1.2	20 ± 1.5	226 ± 13.0
	1000	43 ± 6.0	82 ± 7.4	18 ± 3.0	23 ± 1.0	227 ± 12.1
	2500	43 ± 5.2	107 ± 8.3	20 ± 5.5	21 ± 3.2	209 ± 18.2
	5000	35 ± 9.0	121 ± 11.7	17 ± 3.5	20 ± 3.5	233 ± 35.7
2-AA	2.5/ 10 (only TA 102)	2242 ± 463.6	1039 ± 339.6	155 ± 6.7	208 ± 9.5	470 ± 229.4
	SD = Standard Deviation

 

 

Table 4: Results experiment 2

	EXPERIMENT 2 (Revertant colonies per plate ± SD)
	S9-Mix	Without
	Test Substance (µg/plate)	TA98	TA 100	TA 1535	TA 1537	TA 102
Distilled water		26 ± 4.5	126 ± 3.5	25 ± 1.0	13 ± 2.1	288 ± 28.0
Tetrahydrofuran		34 ± 4.0	99 ± 17.2	25 ± 3.5	15 ± 1.0	391 ± 4.2
Test Substance	1.58	31 ± 4.2	90 ± 8.4	37 ± 2.1	13 ± 2.6	314 ± 14.9
	5.00	29 ± 4.9	93 ± 10.1	34 ± 2.1	14 ± 2.6	347 ± 10.8
	15.8	25 ± 3.6	99 ± 9.1	27 ± 3.6	15 ± 2.5	358 ± 35.4
	50.0	29 ± 4.4	94 ± 8.5	31 ± 3.1	18 ± 2.3	403 ± 10.8
	158	25 ± 3.8	94 ± 6.2	31 ± 3.8	13 ± 1.0	390 ± 18.5
	500	29 ± 7.9	84 ± 5.5	30 ± 4.4	13 ± 1.0	406 ± 2.1
4-NOPD	10 (TA 98)/40 (TA 1537)	255 ± 67.3	-	-	212 ± 7.8	-
NaN3	10	-	901 ± 20.1	891 ± 46.5	-	-
MMS	1					2152 ± 31.5
	S9-Mix	With
	Test Substance (µg/plate)	TA98	TA 100	TA 1535	TA 1537	TA 102
Distilled water	-	27 ± 4.6	117 ± 19.4	29 ± 2.6	25 ± 5.0	406 ± 18.7
Tetrahydrofuran	-	25 ± 2.3	92 ± 8.7	26 ± 1.5	22 ± 2.1	380 ± 16.3
Test Substance	1.58	32 ± 4.0	105 ± 12.9	23 ± 2.5	25 ± 2.5	338 ± 23.0
	5.00	28 ± 8.7	108 ± 20.7	26 ± 2.5	25 ± 2.1	361 ± 2.6
	15.8	32 ± 8.1	100 ± 8.1	25 ± 1.0	24 ± 4.0	376 ± 9.3
	50.0	25 ± 4.4	115 ± 4.6	23 ± 4.5	23 ± 2.0	371 ± 12.5
	158	26 ± 4.0	91 ± 10.2	24 ± 7.2	22 ± 2.1	391 ± 6.1
	500	37 ± 2.6	106 ± 18.8	22 ± 7.0	23 ± 1.5	361 ± 12.5
2-AA	2.5/ 10 (only TA 102)	1407 ± 67.6	847 ± 151.9	247 ± 5.3	283 ± 10.0	1119 ± 59.9
	SD = Standard Deviation

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted test the test substance was not mutagenic in any of the five strains (TA 98, TA 100, TA 1535, TA 1537 and TA 102) tested with and without metabolic activation up to 500 µg/plate.