N-[3-(dimethylamino)propyl] amides C18-22 (even numbered)

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:

05 Apr - 13 May 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

/ Guideline study conducted according to the Japanese test guideline reported as "Standard of the Minister of Labor, based on the Industrial Safety and Health Law Article 57-2 Paragraph 1 (Notification No. 77 of JMOL on September 1, 1988) and Notification on partial revision of the standard (Notification No. 67 JMOL on June 2, 1997)", which in principle is similar to OECD TG 471.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Standard of the Minister of Labor, based on the Industrial Safety and Health Law Article 57-2 Paragraph 1 (Notification No. 77 of JMOL on September 1, 1988) and Notification on partial revision of the standard (Notification No. 67 JMOL on June 2, 1997)

Deviations:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]docosanamide

EC Number:

262-134-8

EC Name:

N-[3-(dimethylamino)propyl]docosanamide

Cas Number:

60270-33-9

Molecular formula:

C27H56N2O

IUPAC Name:

N-[3-(dimethylamino)propyl]docosanamide

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Solubility and stability of the test substance in the solvent/vehicle:stable in dimethylsulfoxid, acetone and tetrahydrofuran

FORM AS APPLIED IN THE TEST: liquid

Method

Target gene:

his operon, tryp 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 rats, treated with 5,6-benzoflavone

Test concentrations with justification for top dose:

Based on the results of the range-finding study (all strains; doses applied 5, 20, 78, 313, 1250, 5000 μg/mL), the following concentrations were used in the main experiment:
- 39, 78, 156, 313, 625, 1250 µg/plate (TA strains, without metabolic activation)
- 313, 625, 1250, 2500, 5000 µg/plate (WP2uvrA, with and without metabolic activation, TA strains with metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubility and stability properties.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

benzo(a)pyrene

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- Cell density at seeding:
in main test
TA100 and TA1535: 2.2 x 10^9
TA98: 3 x 10^9
TA1537: 1.7 x 10^9
WP2uvrA: 6.7 x 10^9

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: duplicate

Evaluation criteria:

The positive control should show a clear dose-dependend increase of at least two times compared to the negative control (spontaneous reversion rate). The negative and positive control as well as the test item should satisfy the acceptance 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 (value ± 2SD)
- no contamination in test material, S9 mix, Sodium phosphate buffer (analysis performed)
- the positive control led to an increase of at least two times compared to the negative control

Statistics:

No statistical analysis was performed. Mean values 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 compared to solvent control starting at 1250 μg/plate without S9 mix (24%)

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 compared to solvent control starting at 1250 μg/plate without S9 mix (46%)

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 compared to solvent control starting at 625 μg/plate without S9 mix (22 - 78%)

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 compared to solvent control starting at 625 μg/plate without S9 mix (14 - 71%)

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: Precipitate was detected in all examined strains with metabolic activation at all concentration tested and without metabolic activation starting at a concentration of 156 μg/plate (all TA strains) or 313 μg/plate (WP2uvrA).

RANGE-FINDING/SCREENING STUDIES: All bacterial strains showed negative responses over the entire dose range, i.e. no clear dose-dependent increase in the number of revertants of at least two times compared to the negative control. Precipitate was detected in all examined strains with and without metabolic activation starting at 313 μg/plate (for further details see "any other information on results incl tables").

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

TA98, -S9: 444 - 679, mean ± SD: 561 ± 58.8
TA98, +S9: 249 - 448, mean ± SD: 349 ± 49.8

TA100, -S9: 398 - 689, mean ± SD: 544 ± 72.8
TA100, +S9: 843 - 1457, mean ± SD: 1150 ± 153.5

TA1535, -S9: 352 - 720, mean ± SD: 536 ± 92.0
TA1535, +S9: 116 - 320, mean ± SD: 218 ± 51.0

TA1537, -S9: 254 - 895, mean ± SD: 574 ± 160.4
TA1537, +S9: 52 - 104, mean ± SD: 78 ± 13.1

WP2uvrA, -S9: 87 - 137, mean ± SD: 107 ± 14.8
WP2uvrA, +S9: 523 - 1101, mean ± SD: 812 ± 144.4

- Negative (solvent/vehicle) historical control data:

TA98, -S9: 15 - 31, mean ± SD: 23 ± 4.0
TA98, +S9: 18 - 40, mean ± SD: 29 ± 5.5

TA100, -S9: 90 - 133, mean ± SD: 111 ± 11
TA100, +S9: 96 - 147, mean ± SD: 122 ± 13

TA1535, -S9: 3 - 14, mean ± SD: 9 ± 2.7
TA1535, +S9: 4 - 14, mean ± SD: 9 ± 2.6

TA1537, -S9: 3 - 12, mean ± SD: 8 ± 2.3
TA1537, +S9: 2 - 15, mean ± SD: 9 ± 3.1

WP2uvrA, -S9: 14 - 33, mean ± SD: 23 ± 4.8
WP2uvrA, +S9: 15 - 35, mean ± SD: 25 ± 5.0

The ranges are calculated as mean ± 2xSD.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: toxic effects were examined with a stereoscopic microscope

Any other information on results incl. tables

Table 1: Summary of test results (Range-finding study, experiment 1, pre-incubation method)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 2 plates)
		Frameshift type		Base-pair substitution type
		TA1537	TA98	TA100	TA1535	WP2 uvrA
–	Solvent control (THF)	9	25	129	10	22
	5	8	22	132	8	23
	20	7	22	134	10	27
	78	10	30	130	9	24
	313	9 P	26 P	121 P	8 P	20 P
	1250	3 P T	7 P T	56 P T	6 P T	21 P
	5000	3 P T	11 P T	62 P T	5 P T	21 P
	Positive controls (µg/plate)	9AA (80)	AF-2 (0.1)	AF-2 (0.01)	NaN3 (0.5)	AF-2 (0.01)
	Mean No. of colonies/plate (average of 2 plates)	385	507	602	482	123
+	Solvent control (THF)	10	31	142	11	25
	5	9	34	141	9	26
	20	9	35	153	9	25
	78	7	32	145	9	20
	313	8 P	30 P	145 P	10 P	25 P
	1250	10 P	30 P	140 P	8 P	21 P
	5000	9 P	30 P	126 P	10 P	20 P
	Positive controls  (µg/plate)	B[α]P (5)	B[α]P (5)	B[α]P (5)	2AA (2)	2AA (10)
	Mean No. of colonies/plate (average of 2 plates)	85	384	1068	240	941

THF = tetrahydrofuran

AF-2 = 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

NaN₃= sodium azide

9AA = 9-aminoacridine

B[α]P = benzo[α]pyrene

2AA = 2-aminonathracene

P = precipitate

T = toxic effect observed

Table 2: Summary of test results (main study, experiment 2, pre-incubation method)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 2 plates)
		Frameshift type		Base-pair substitution type
		TA1537	TA98	TA100	TA1535	WP2 uvrA
–	Solvent control (THF)	7	29	132	9	26
	39	7	23	124	7	-
	78	8	29	117	8	-
	156	7 P	28 P	137 P	8 P	-
	313	7 P	29 P	116 P	8 P	22 P
	625	6 P T	27 P	102 P	7 P T	26 P
	1250	2 P T	22 P T	71 P T	2 P T	25 P
	2500	-	-	-	-	23 P
	5000	-	-	-	-	23 P
	Positive controls (µg/plate)	9AA (80)	AF-2 (0.1)	AF-2 (0.01)	NaN3 (0.5)	AF-2 (0.01)
	Mean No. of colonies/plate (average of 2 plates)	430	554	465	484	127
+	Solvent control (THF)	7	28	139	9	25
	313	8 P	32 P	144 P	10 P	25 P
	625	8 P	36 P	142 P	9 P	26 P
	1250	9 P	34 P	146 P	9 P	21 P
	2500	7 P	32 P	137 P	8 P	25 P
	5000	7 P	29 P	140 P	9 P	25 P
	Positive controls  (µg/plate)	B[α]P (5)	B[α]P (5)	B[α]P (5)	2AA (2)	2AA (10)
	Mean No. of colonies/plate (average of 2 plates)	91	334	1044	220	898

THF = tetrahydrofuran

AF-2 = 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

NaN₃= sodium azide

9AA = 9-aminoacridine

B[α]P = benzo[α]pyrene

2AA = 2-aminonathracene

P = precipitate

T = toxic effect observed