Amines, N-C16-18-alkyltrimethylenedi-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2008-05-14 - 2008-09-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

(Z)-N-9-octadecenylpropane-1,3-diamine

EC Number:

230-528-9

EC Name:

(Z)-N-9-octadecenylpropane-1,3-diamine

Cas Number:

7173-62-8

Molecular formula:

C21H44N2

IUPAC Name:

N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diamine

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): N-Oleyl-1,3-diaminopropane
- Physical state: liquid
- Analytical purity: N-Alkyl-1,3-diaminopropanes: 92.3%
N-Alkyl-amines: 7.7%
C-Chain-distribution (R = alkyl):
R = C16: 7%; C18: 91%; C20: 2%
- Lot/batch No.: S000902
- Production date of the test item: August 13, 2007
- Expiration date of the lot/batch: September 30, 2010
- Storage condition of test material: at room temperature
- Product (name): Duomeen OV
- Colour: Max 7 Gardner
- Melting Point: 9 - 20°C
- pH: basic

Method

Target gene:

histidine operon (his)

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

liver rat S9-mix (induced with ß-naphthoflavone and phenobarbital)

Test concentrations with justification for top dose:

Experiment 1:
0.00010, 0.000316, 0.00100, 0.00316, 0.0100, 0.0316 and 0.1 µL/plate
Experiment 2:
0.000158, 0.00050, 0.00158, 0.0050, 0.0158, 0.05 and 0.1 µL/plate (TA 98, TA 100, TA 1537, E. coli WP2 uvrA)
0.000050, 0.000158, 0.00050, 0.00158, 0.0050, 0.0158 and 0.05 µL/plate (TA 1535)

Vehicle / solvent:

Ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 10 µg/plate sodium azide (NaN3) (TA 100, TA 1535); 10 µg/plate (TA 98) and 40 µg/plate (TA 1537) 4-nitro-o-phenylene-diamine (4-NOPD); 1 µL/plate methylmethanesulfonate (MMS) (E. coli); 2.5 µg/plate for TA 98, 100, 1535, 1537 and 10 µg/plate for E. coli

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates in 2 experiments

DETERMINATION OF CYTOTOXICITY
- Method: background lawn or reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

ADDITIONAL INFORMATION
- Data recording:
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH. If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.

Evaluation criteria:

Criteria of validity:
A test is considered acceptable if for each strain:
1. the bacteria demonstrate their typical response to ampicillin (TA 98, TA 100)
2. the control plates with and without metabolic activation are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range):
TA 98: 18 - 54 (-S9) and 18 - 71 (+S9)
TA 100: 75 - 167 (-S9) and 81 - 168 (+S9)
TA 1535: 5 - 29 (-S9) and 6 - 31 (+S9)
TA 1537: 5 - 30 (-S9) and 6 - 36 (+S9)
E. coli WP2 uvrA: 35 - 92 (-S9) and 37 - 101 (+S9)
3. corresponding background growth on both negative control and test plates is observed
4. the positive controls show a distinct enhancement of revertant rates over the control plate

Evaluation of mutagenicity:
The mutation factor is calculated by dividing the mean value of the revertant counts through 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:
1. a clear and dose-related increase in the number of revertants occurs and/or
2. 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:
1. if in tester strains Ta 100 and E. coli WP2 uvrA the number of reversions is at least twice as high
2. if in tester strains TA 1535, TA 1537 and TA 98 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.

Statistics:

no data

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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 3

Species / strain:

S. typhimurium TA 98

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 4

Species / strain:

S. typhimurium 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 5

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 precipitation of the test item was observed in any of the 5 tester strains used in experiment 1 and 2 with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with tester strain TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as for the main experiment.
Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduetion in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
The test item was tested in the pre-experiment at the following concentrations:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate
Based on the pre-experiment results the following concentrations were used for the main experiments:
Experiment 1: 0.0001 - 0.1 µL/plate and Experiment 2: 0.000158 - 0.1 µL/plate (TA 98, TA 100, TA 1537, E. coli WP2 uvrA) and 0.00005 - 0.05 µL/plate (TA 1535)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects in experiment 1:
at 0.00316 µL/plate (-S9) and 0.0316 µL/plate (+S9) (TA 98 and TA 100);
at 0.010 µL/plate (-S9) and 0.1 µL/plate (+S9) (TA 1535);
at 0.01 µL/plate (-S9) and 0.0316 µL/plate (+S9) (TA 1537);
at 0.0316 µL/plate (-S9) and 0.1 µL/plate (+S9) (E. coli WP2 uvrA)

Cytotoxic effects in experiment 2:
at 0.0050 µL/plate (-S9) and 0.05 µL/plate (+S9) (TA 98, TA 100, TA 1535, TA 1537);
at 0.0158 µL/plate (-S9) and 0.1 µL/plate (+S9) (E. coli WP2 uvrA)

Remarks on result:

other: strain/cell type: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Ames Test Results - Experiment 1

With or without S9-Mix	Test substance concentration (dose/plate)	Mean number of revertant colonies per plate (triplicates)
		Base-pair substitution type			Frameshift type
		TA 100	TA 1535	E. coli WP2 uvrA	TA 98	TA 1537
-	A. dest.	95	20	53	28	6
-	Vehicle control (EtOH)	102	23	46	24	6
-	0.000100µL	96	17	42	29	9
-	0.000316 µL	67	16	45	23	10
-	0.00100 µL	94	26	46	26	11
-	0.00316 µL	67	24	46	24	7
-	0.0100 µL	41	13	53	15	4
-	0.0316 µL	0	0	0	0	0
-	0.1 µL	0	0	0	0	0
Positive controls - S9	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentrations (μg/plate)	10 µg	10 µg	1 µL	10 µg	40 µg
	Number of colonies/plate	1257	1145	407	395	99
		TA 100	TA 1535	E. coli WP2 uvrA	TA 98	TA 1537
+	A. dest.	99	19	56	33	10
+	Vehicle control (EtOH)	97	23	57	38	11
+	0.000100µL	108	29	56	40	9
+	0.000316 µL	103	23	46	36	7
+	0.00100 µL	112	25	63	44	8
+	0.00316 µL	114	26	62	43	11
+	0.0100 µL	129	25	69	41	13
+	0.0316 µL	62	13	66	21	6
+	0.1 µL	0	0	15	0	0
Positive controls + S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2.5 µg	2.5 µg	10 µg	2.5 µg	2.5 µg
	Number of colonies/plate	2209	243	204	3114	211

 

Table 2: Ames Test Results - Experiment 2

With or without S9-Mix	Test substance concentration (dose/plate)	Mean number of revertant colonies per plate (triplicates)
		Base-pair substitution type			Frameshift type
		TA 100	TA 1535	E. coli WP2 uvrA	TA 98	TA 1537
-	A. dest.	134	26	54	21	7
-	Vehicle control (EtOH)	134	31	51	30	7
-	0.000050µL	-	32	-	-	-
-	0.000158µL	126	24	56	29	9
-	0.0005 µL	124	28	53	23	7
-	0.00158 µL	117	19	50	22	12
-	0.005 µL	75	15	50	26	8
-	0.0158 µL	13	0	35	3	0
-	0.05 µL	0	0	0	0	0
-	0.1 µL	0	-	0	0	0
Positive controls - S9	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentrations (μg/plate)	10 µg	10 µg	1 µL	10 µg	40 µg
	Number of colonies/plate	1519	1537	447	528	112
		TA 100	TA 1535	E. coli WP2 uvrA	TA 98	TA 1537
+	A. dest.	105	21	56	39	13
+	Vehicle control (EtOH)	81	22	53	37	9
+	0.000050µL	-	28	-	-	-
+	0.000158µL	108	29	67	37	11
+	0.0005 µL	89	25	64	42	6
+	0.00158 µL	97	25	61	45	8
+	0.005 µL	103	17	51	40	8
+	0.0158 µL	112	22	50	41	17
+	0.05 µL	24	0	66	0	0
+	0.1 µL	0	-	2	0	0
Positive controls + S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2.5 µg	2.5 µg	10 µg	2.5 µg	2.5 µg
	Number of colonies/plate	1611	181	231	2440	328

NaN3= Sodium azide

MMS = Methyl methane sulfonate

4-NOPD = 4-Nitro-o-phenylene-diamine

2-AA = 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, N-Oleyl-1,3- diaminopropane did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, N-Oleyl-1,3-diaminopropane is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item N-Oleyl-1,3-diaminopropane was investigated for its potential to induce gene mutations according to the OECD guideline 471.

In two independent plate incorporation tests using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicates. The following concentrations of the test item were prepared and used in the experiments:

Experiment 1:

0.00010, 0.000316, 0.00100, 0.00316, 0.0100, 0.0316 and 0.1 µL/plate

Experiment 2:

0.000158, 0.00050, 0.00158, 0.0050, 0.0158, 0.05 and 0.1 µL/plate (TA 98, TA 100, TA 1537, E. coli WP2 uvrA) and

0.000050, 0.000158, 0.00050, 0.00158, 0.0050, 0.0158 and 0.05 µL/plate (TA 1535).

No precipitation of the test item was observed in any of the five tester strains used in experiment 1 and 2 with and without metabolic activation.

Toxic effects of the test item were noted in all tester strains evaluated in experiment 1 and 2.

In experiment 1 toxic effects of the test item were observed in tester strains TA 98 and TA 100 at doses of 0.00316 µL/plate and higher (without metabolic activation) and at doses of 0.0316 µL/plate and higher (with metabolic activation). In tester strain TA 1535 toxic effects of the test item were noted at doses of 0.01 µL/plate and higher (without metabolic activation) and at a dose of 0.1 µL/plate (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were seen at doses of 0.01 µL/plate and higher (without metabolic activation) and at doses of 0.0316 µL/plate and higher (with metabolic activation). In tester strain E. coli WP2 uvrA toxic effects of the test item were noted at doses of 0.0316 µL/plate and higher (without metabolic activation) and at a dose of 0.1 µL/plate (with metabolic activation).

In experiment 2 toxic effects of the test item were noted in tester strains TA 98, TA 100 and TA 1537 at doses of 0.005 µL/plate and higher (without metabolic activation) and at doses of 0.05 µL/plate and higher (with metabolic activation). In tester strain TA 1535 toxic effects of the test item were observed at doses of 0.005 µL/plate and higher (without metabolic activation) and at a dose of 0.05 µL/plate (with metabolic activation). In tester strain E. coli WP2 uvrA toxic effects of the test item were seen at doses of 0.0158 µL/plate and higher (without metabolic activation) and at a dose of 0.1 µL/plate (with metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with N-Oleyl-1,3-diaminopropane at any concentration level, neither in the presence nor absence of metabolic activation in experiment 1 and 2.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.