2-piperazin-1-ylethylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: 2e: Meets generally accepted scientific standards, well-documented and acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The Ames test was conducted using strains TA98, TA100, TA1535, TA1537, and TA1538

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-piperazin-1-ylethylamine

EC Number:

205-411-0

EC Name:

2-piperazin-1-ylethylamine

Cas Number:

140-31-8

Molecular formula:

C6H15N3

IUPAC Name:

2-piperazin-1-ylethanamine

Details on test material:

- Name of test material (as cited in study report): N-Aminoethylpiperazine
- Analytical purity: 99.37%
- Purity test date: no data
- Lot/batch No.: 36-ARD-31-3; Sample #: 49-427
- Storage condition of test material: room temperature

Method

Target gene:

The indicator strains used for this test are all histidine requiring (his-) bacteria which carry a mutation in the histidine locus.

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and TA1538)

Details on mammalian cell type (if applicable):

Properly maintained: yes
Indicator organisms are stored at -80°C. Working cultures are prepared monthly by inoculating nutrient broth from the frozen cultures and incubating with agitation overnight. Bacteria are then plated onto Vogel-Bonner Medium E agar plates (master plates) with an excess of histidine and biotin (required because of the lipopolysaccharide deficiency). After incubation for 24 hours, the strains are checked for their genetic markers to verify their identity and purity.

For testing, the broth cultures are prepared by inoculating from the master plates or directly from frozen permanent stock cultures into nutrient broth and incubated overnight (8 to 10 hours) with agitation. The broth cultures are kept on ice during the day of testing. Fresh cultures are made each day of testing.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver homogenate

Test concentrations with justification for top dose:

0.01, 0.03, 0.1, 0.3, 1.0 mg/plate

Preliminary test concentrations: 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 5.0, 10.0, 30.0, 98.5 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water; BRRC #49-27; CAS #7732-18-5

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine; BRRC #44-71; CAS #99-56-9, 9-aminoacridine; BRRC #44-233; CAS #90-45-9, sodium azide; BRRC #44-72; CAS 4126628-22-8, 2-aminoanthracene; BRRC #44-67; CAS #613-13-8

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Sample Preparation: For definitive testing, an initial stock solution of the test substance was prepared by mixing AEP in water to achieve a concentration of 100 mg/ml. All subsequent dilutions were made in the same solvent. Dilutions of the test substance were made fresh each day of testing. All dilutions for the mutagenicity tests were analyzed gravimetrically to determine actual concentrations.

Evaluation criteria:

The spontaneous reversion for the solvent controls should be within this laboratory's historical range. The positive controls should demonstrate that the test systems are responsive with known mutagens. A test chemical is considered to be a bacterial mutagen if the number of revertant colonies is at least twice the solvent control for at least one dose level and there is evidence of a dose-related increase in the number of revertant colonies. If a test chemical produces a marginal or weak response that cannot be reproduced in a second test, the test result will be considered negative. If there is no evidence of a dose-related increase in the number of revertant colonies and the number of revertant colonies is not twice the solvent control, then the test chemical is not considered to be a bacterial mutagen.

Statistics:

Not applicable

Results and discussion

Test results

Test results 1

Species / strain:

other: Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and TA1538)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The highest dose level of AEP (1.0 mg/plate), produced evidence of cytotoxicity with all five strains.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

other: Salmonella typhimurium (TA98, TA100, TA1537, and TA1538)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Positive and dose-related increases in numbers of revertant colonies were observed only with strain TA1535. A maximum increase of approximately 2.9 times the concurrent control value was observed with the highest dose level of AEP tested.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In preliminary tests to determine cytotoxicity, ten concentrations of AEP ranging from 0.01 to 98.5 mg/plate were tested with or without the presence of an S9 metabolic activation system. Cytotoxicity was defined as either a reduction in the number of revertant colonies or an inhibition of growth of the background lawn. Dose levels ranging from 5 to 98.5 mg/plate produced complete absence of growth of the background lawn in the test without S9. Lower dose levels of 1.0 and 3.0 mg/plate produced cytotoxicity evident as sparse growth of the background lawn and decreased relative numbers of revertant colonies to approximately 29% and 20% of the control value, respectively. In the preliminary test performed with activation, dose levels of 30 and 98.5 mg/plate produced absence of growth of the background lawn. A lower dose of 10 mg/plate produced cytotoxicity evident by sparse growth of the background lawn and reduced the relative number of revertant colonies to approximately half the concurrent control value. Based on the results of these preliminary toxicity tests, a concentration range from 0.01 to 1.0 mg/plate was tested without S9 and from 0.1 to 10 mg/plate was tested in the presence of S9 in definitive mutagenicity experiments using triplicate cultures at each of 5 dose levels.


MAIN STUDY: In tests performed in the presence of a rat-liver S9 metabolic activation system, only strain TA1535 showed positive and dose related increases in numbers of revertant colonies with a maximum response of approximately 3-fold above the concurrent control value.

All five bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without S9 metabolic activation. Negative (solvent) controls were also tested with each strain. With the exception of strain TA1535 tested without S9, and strain TA1537 tested with S9, the average numbers of spontaneous revertants were within the historical 95% confidence range at this laboratory. The average spontaneous reversion frequency for strain TA1535 tested without S9 was 14 revertant colonies, which was only 2 colonies lower than the lower limit of the 95% confidence interval. In contrast, the average spontaneous reversion frequency for strain TA1537 tested with S9 was 14 revertant colonies per plate, which was 2 colonies higher than the upper limit of the 95% confidence range for this strain. These deviations are not considered to be sufficient to compromise the validity of the test results with these strains. All positive and negative controls were tested concurrently with the test chemical. Concurrent sterility testing showed that the S9 mix, PBS, the test chemical and the solvent control agent was sterile.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Results of the Salmonella Mutagenicity Assay

Without Activation

Test Agent	Dose/plate	Mean	S.D.
Strain TA98
Solvent:	100 mg	26	4.0
4 -NPD	0.01 mg	1055	65.7
AEP	0.01 mg	22	3.2
	0.03 mg	23	5.3
	0.1 mg	32	5.2
	0.3 mg	28 (T)	-
	1 mg	S?T	-
Strain TA100
Solvent	100 mg	94	1.0
NaN3:	0.01 mg	2511	76.3
AEP	0.01 mg	104	18.4
	0.03 mg	106	5.7
	0.1 mg	107	1.2
	0.3 mg	87 (S)	25.5
	1 mg	S	-
Strain TA1535
Solvent	100 mg	14	3.6
NaN3:	0.01 mg	2275	64.1
AEP	0.01 mg	19	0.6
	0.03 mg	18	4.5
	0.1 mg	20	5.3
	0.3 mg	12 (S)	0.7
	1 mg	S/T	-
Strain TA1537
Solvent	100 mg	8	1.2
9 -AA	0.06 mg	111	14.6
AEP	0.01 mg	11	3.5
	0.03 mg	10	1.5
	0.1 mg	9	1.5
	0.3 mg	4 (T)	1.4
	1 mg	S/T	-
Strain TA1538
Solvent	100 mg	11	3.0
4 -NPD	0.01 mg	1301	46.5
AEP	0.01 mg	10	1.7
	0.03 mg	13	1.7
	0.1 mg	9	4.0
	0.3 mg	10 (T)	0.7
	1 mg	T	-

T: Toxic: Clearing of background lawn, or average number of colonies <1/2 solvent control value.

S: Sparse growth of background lawn; counts not included in calculation of mean and standard deviation

4 -NPD: 4 -Nitro-o-phenylenediamine

9 -AA: 9 -Aminoacridine

NaN3: Sodium azide

2 -AA: 2 -aminoanthracene

Table 2 Results of the Salmonella Mutagenicity Assay

With Activation

Test Agent	Dose/plate	Average	S.D.
Strain TA98
Solvent	100 mg	27	12.0
2 -AA	0.01 mg	562	134.4
AEP	0.1 mg	34	16.1
	0.3 mg	41	13.5
	1 mg	37	9.5
	3 mg	31	11.1
	10 mg	T	-
Strain TA100
Solvent	100 mg	90	7.2
2 -AA	0.01 mg	563	166.2
AEP	0.1 mg	105	6.6
	0.3 mg	94	15.0
	1 mg	99	12.7
	3 mg	90	5.6
	10 mg	66 (S/T)	-
Strain TA1535
Solvent	100 mg	10	4.4
2 -AA	0.01 mg	141	147.0
AEP	0.1 mg	9	4.6
	0.3 mg	9	1.2
	1 mg	19	1.7
	3 mg	16	3.1
	10 mg	29 (S)	-
Strain TA1537
Solvent	100 mg	14	0.6
2 -AA	0.01 mg	183	128.5
AEP	0.1 mg	8	2.0
	0.3 mg	16	2.0
	1 mg	12	4.6
	3 mg	8 (S)	1.4
	10 mg	S/T	-
Strain TA1538
Solvent	100 mg	26	4.6
2 -AA	0.01 mg	226	22.5
AEP	0.1 mg	26	7.8
	0.3 mg	28	1.5
	1 mg	25	4.0
	3 mg	18 (S)	4.9
	10 mg	S/T	-

T: Toxic: Clearing of background lawn, or average number of colonies <1/2 solvent control value.

S: Sparse growth of background lawn; counts not included in calculation mean and standard deviation.

4 -NPD: 4 -Nitro-o-phenylenediamine

9 -AA: 9 -Aminoacridine

NaN3: Sodium azide

2 -AA: 2 -Aminoanthracene

Applicant's summary and conclusion

Conclusions:

AEP produced weakly positive and dose-dependent mutagenic responses in only one of the five strains of Salmonella typhimurim tested with S9. None of the five strains tested without a rat-liver metabolic activation system showed evidence of mutagenic activity. Therefore, under the conditions of this assay, AEP was considered to be weakly mutagenic in the Salmonella/ microsome mutagenicity assay in the presence of metabolic activation.

Executive summary:

N-Arninoethylpiperazine (AEP) was tested for potential mutagenic activity using the Salmonella/microsome bacterial mutagenicity assay (Ames test). Test doses for the Ames test were chosen from data obtained in a preliminary study with strain TA100 performed both with and without a rat- liver S9 activation system. In tests without S9, dose levels of 1.0 and 3.0 mg/plate allowed only sparse growth of the background lawn and reduced the numbers of revertant colonies to less than half the control level. In contrast, tests with S9 activation allowed confluent growth of the background lawn at dose levels ranging from 0.01 to 5.0 mg/plate. A higher dose of 10.0 mg/plate allowed only sparse growth of the background lawn and produced a significant decrease in relative numbers of revertant colonies. Based on these results, five doses ranging from 0.01 to 1.0 mg/plate were tested in the definitive test without

S9 and a higher range of 0.1 to 10 mg/plate were tested in the presence of the S9 metabolic activation system. These concentrations were tested with five different strains of Salmonella tvphimurium (TA98, TA100, TA1535, TA1537, and TA1538) using triplicate cultures at each dose level for each strain.

In the test without S9, no evidence of positive or dose-related mutagenic activity was observed with any of the five strains. In tests performed in the presence of a rat-liver S9 metabolic activation system, only strain TA1535 showed positive and dose related increases in numbers of revertant colonies with a maximum response of approximately 3-fold above the concurrent control value. Thus, AEP was considered to be weakly mutagenic in the presence of a liver S9 metabolic activation system in this in vitro bacterial assay.