N,N'-bis(3-aminopropyl)piperazine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference Type:

publication

Title:

Mutagenicity Test Data of Existing Chemical Substances

Author:

Japan Chemical Industry Ecology-Toxicology & Information Center (JETOC)

Year:

1996

Bibliographic source:

Japan Chemical Industry Ecology-Toxicology & Information Center (JETOC)

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis(3-aminopropyl)piperazine

EC Number:

230-589-1

EC Name:

N,N'-bis(3-aminopropyl)piperazine

Cas Number:

7209-38-3

Molecular formula:

C10H24N4

IUPAC Name:

3-[4-(3-aminopropyl)piperazin-1-yl]propan-1-amine

Test material form:

liquid

Specific details on test material used for the study:

purity ≥ 98 %

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium strains: TA 98, TA 100, TA 102, TA 104 TA 1535, TA 1537, TA 1538 and Escherichia coli strain: WP2uvrA, WP2uvrA/pKM101

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix

Test concentrations with justification for top dose:

9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: see remarks

Remarks:

2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, 2-amino anthracene, bleomycin, pyruvic aldehyde

Details on test system and experimental conditions:

The test substance was dissolved in 0.1 mL of distilled water. If the test was performed with metabolic activation 0.5 mL of S9mix was added, else 0.1M phosphate buffer (pH 7.4) was added. 0.1 mL of tester strain suspension was added and the mixture incubated for 20 min at 37 °C. Afterwards it was mixed with 2 ml of molten Top Agar and poured out onto the minimal agar plates. The plates were incubated for 48 hours at 37 °C and the number of revertants were counted.
The cytotoxicity was evaluated by examining the growth of the background lawn under a microscope. The background growth is the result of the addition of small amounts of histidine/tryptophan to the agar. Cytotoxic compounds lead to a decrease in number of these micro colonies and in increase of their diameter.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

For the concentrations 2500 and 5000 µg/plate cytotoxicity was observed

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A chemical is considered mutagenic when a dose-related increase in revertant colony count is observed and the number of revertant per plate is more than twice than that of the solvent control (reproducible).

Remarks on result:

other: Salmonella typhimurium strains: TA 98, TA 100, TA 102, TA 104 TA 1535, TA 1537, TA 1538 and Escherichia coli strain: WP2uvrA, WP2uvrA/pKM101

Any other information on results incl. tables

1st experiment:

Concentration μg/plate	Mean number of revertants / plate
	TA98		TA100		TA1535			TA1537		WP2uvrA
	S9-	S9+	S9-	S9+	S9-	S9+		S9-	S9+	S9-	S9+
solvent control: Water	32	35	147	161	7	11		9	15	36	44
9.77	38	41	140	166	8	10		12	9	34	43
19.5	27	30	166	179	7	7		12	19	38	48
39.1	25	37	149	163	8	11		11	15	27	45
78.1	37	34	143	166	7	9		9	16	34	39
156	33	39	155	165	8	9		9	14	31	44
313	29	29	158	175	9	11		12	16	38	39
625	31	32	157	164	7	10		12	18	36	42
1250	38	25	114*	147	5*	12		14	13	43	42
2500	5*	11*	3*	7*	3*	7*		4*	11*	25*	38*
5000	7*	13*	0*	5*	0*	5*		6*	7*	5*	20*
Positive Control	AF2	2AA	AF2	2AA	NaN3	2AA		9AA	2AA	AF2	2AA
	413	441	742	830	386	251		227	148	201	1209

2nd experiment:

Concentration μg/plate	Mean number of revertants / plate
	TA98		TA100		TA1535			TA1537		WP2uvrA
	S9-	S9+	S9-	S9+	S9-	S9+		S9-	S9+	S9-	S9+
solvent control: Water	29	31	137	159	13	16		7	13	35	50
19.5					16
39.1	32	29	137	147	15	16			19		47
78.1	26	26	143	160	15	16		7	16	41	50
156	28	29	163	150	13	15		8	15	42	51
313	25	32	138	154	13	12		6	13	41	58
625	23	32	166	143	15	12		7	17	43	42
1250	44	26	173	151	9*	16		9	14	39	47
2500	16*	25*	66*	101*		10*		12	13*	39	39*
5000								8*		27*
Positive Control	AF2	2AA	AF2	2AA	NaN3	2AA		9AA	2AA	AF2	2AA
	424	455	802	1031	582	243		379	175	245	1502

Concentration μg/plate	Mean number of revertants / plate
	TA102			TA104		WP2uvrA/pKM101
	S9-		S9+	S9-	S9+	S9-	S9+
solvent control: Water	286		364	275	306	112	158
78.1	292		343	241	306	100	122
156	283		341	285	308	101	125
313	274		340	266	333	105	141
625	239		351	277	312	108	143
1250	244		317	268	315	105	125
2500	111*		187*	182*	256*	30*	109*
5000	0*		0*	125*	211*	0*	92*
Positive Control	BLM		2AA	PA	2AA	AF2	2AA
	682		1278	2145	1429	871	859

*: Cytotoxicity was observed

AF2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

2AA: 2-amino anthracene

9AA: 9 -aminoacridine

NaN3: sodium azide

BLM: bleomycin
PA: pyruvic aldehyde

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test substance did not induce gene mutations.

Therefore, it is considered not to be mutagenic in this in vitro bacterial mutagenicity test.

Executive summary:

The substance N,N'-bis(3-aminopropyl)piperazine (CAS,7209-38-3) was tested in the Bacterial Reverse Mutation Assay.

The assay was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 104, TA 1535, TA 1537 and the Escherichia coli strains WP2uvrA, WP2uvrA/pKM101 with and without metabolic activation by S9-mix.

Solutions of the test substance were prepared in distilled water. The following

concentrations were tested: 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/plate

The direct plate incorporation assay was utilized.

The following results were obtained:

Cytotoxic effects were noted at the concentration of 2500 and 5000 µg/plate.

For non of the tested strains and concentrations (with and without metabolic activation) an enhanced revertant rate were observed.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test substance did not induce gene mutations.

Therefore, N,N'-bis(3-aminopropyl)piperazine (CAS: 7209 -38 -3) is considered not to be mutagenic in this in vitro bacterial mutagenicity test.