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REACH

2-piperazin-1-ylethylamine

EC number: 205-411-0 | CAS number: 140-31-8

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Six in vitro key studies were identified for aminoethylpiperazine. This includes 4 negative Ames studies in all strains and conditions, a negative in vitro UDS study, and two negative in vitro mammalian cell mutagenicity assays.

Link to relevant study records

Referenceopen all close all

Reference 1

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

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

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 / 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

Untreated negative controls:

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

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

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

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'.

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

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.

Reference 2

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

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 and TA1537.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

TA1535 and TA100: hisG46; TA1537: hisC3076; TA98: hisD3052

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
Cultures of the histidine-dependent strains fo Salmonella typhimurium were derived from cultures provided by Prof. Bruce Ames, Aniveristy of California USA.

Metabolic activation:

with and without

Metabolic activation system:

S-9: Young male CD rat liver microsomal preparation. Aroclor 1254 was administered as a single i.p. injection

Test concentrations with justification for top dose:

5000, 1580, 500, 158 and 50 µg

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water and DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine and sodium azide

Remarks:

All positive control compounds were prepared as solutions in DMSO, except sodium azide, which was dissolved in purified water.

Details on test system and experimental conditions:

Pour-plate assay for mutagenesis: An aliquot (0.1 ml) of each concentration of NAEP was placed in a sterile tube. Molten, histidine-deficient top-agar (2 ml) and bacterial suspension (0.1 ml), maintained at 45 ºC, was then added. The tubes were mixed by inversion and 0.5 ml rat liver microsomal preparation (S-9 mix) was added where appropriate. The tubes were again inverted to mix thoroughly and the contents poured onto plates containing solidifeid minimal medium (20 ml).

Further plates were prepared without inclusion of the test rganisms to verify the sterility of the S-9 mix and the test material. A control series of plates was prepared to confirm the inability of purified water (0.1 ml) to induce reversion in the bacterial strains, and to provide a measure of the spontaneous mutation rates.

Aliquots (0.1 ml) of a 10E-6 dilution of culture were spread on the surface of plates of complete medium to measure the viability and cell density of each culture.

All plates were prepared in triplicate, allowed to solidify and incubated at 37 ºC for 2 days. After incubation, numbers of revertant colonies were counted, either manually or with a Biotran II automatice colony counter. Total colonies on nutrient plates were counted in the same way. Growth of the baackground lawn of non-revertant cells on minimal plates was verified.

Results obtained with all strains were confirmed in a second, independent experiment.

Evaluation criteria:

A compound is considered to have mutagenic potential if it induces a dose-related increase in revertants over the concurrent solvent or vehicle controls, and if this increase reaches at least a doubling of the control values. If an increase of 2-fold or greater is recorded only at the highest tested dose (or the highest not showing clear toxicity) and this increase is reporducible, this too would be considered a positive result. Such a positive result may be observed under one or more sets of test conditions (i.e. in one or more tester strains, with or without s-9 mix).

Statistics:

Mean values and standard deviations of colony counts

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Refer to Table below

MAIN STUDY
Sterility checks, spontaneous reversion rate and viability checks: The absence of colonies on NAEP and S-9 mix sterility check plates indicates that these preparations were free of significant microbial contamination.

The total colony counts on plates confirmed the viability and high cell density of the cultures of the individual organisms. The counts recorded on appropriate negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains and the absence of effects on these rates of purified water inclusion.

Mutagenic activity of positive control chemical: Appropriate positive control chemicals (with S-9 mix where required) induced marked increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S-9 mix.

Action of NAEP: No increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to NAEP at levels from 50 to 5000 µg per plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Preliminary toxicity test

TA98

Test material (µg/plate)	Background lawn			Revertant colonies
	Plate A	Plate B	Plate C	Plate A	Plate B	Plate C

2500 (sterility check)	A	A	A	0	0	0
5000	P	P	P	18	19	22
500	P	P	P	27	18	26
50	P	P	P	29	24	21
5	P	P	P	15	16	26

2500	P	P	P	30	15	23
250	P	P	P	18	14	24
25	P	P	P	28	19	18
2.5	P	P	P	16	19	19

0	P	P	P	23	22	18
0(0.2 ml solvent)	P	P	P	24	22	23

A=Absent; P=Present

No visible thinning of the background lawn of non-revertant cells was obtained following exposure to NAEP. A top exposure level of 5 mg per plate was therefore selected for use in the main tests.

Table 2 Results of Mean Revertant Colony Counts with Salmonella typhimurium

		Mean + S.D.
Test Material	S-9 mix: + present; - absent	TA98	TA98 (second test)	TA100	TA100 (second test)	TA1535	TA1535 (second test)	TA1537	TA1537 (second test)
None: sterility check	+	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0
AEP (5000); sterility check	-	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0	0 + 0
AEP (5000)	+	31 + 9	30 + 4	174 + 11	121 + 12	10 + 2	6 + 3	12 + 1	5 + 4
AEP (1580)	+	32 + 9	26 + 5	176 + 27	122 + 26	8 + 1	8 + 4	14 + 1	9 + 5
AEP (500)	+	33 + 4	30 + 3	177 + 15	122 + 22	11 + 2	7 + 1	13 + 1	6 + 1
AEP (158)	+	29 + 4	30 + 1	197 + 14	122 + 8	12 + 1	9 + 1	9 + 2	7 + 3
AEP (50)	+	25 + 7	36 + 5	179 + 24	90 + 11	12 + 4	11 + 3	10 + 2	7 + 1
Purified water (0.1 ml)	+	35 + 10	33 + 4	130 + 14	123 + 9	11 + 2	10 + 1	12 + 2	9 + 2
AEP (5000)	-	23 + 3	21 + 2	152 + 20	102 + 9	7 + 3	10 + 3	14 + 3	7 + 2
AEP (1580)	-	19 + 9	23 + 2	145 + 20	116 + 12	9 + 1	12 + 1	11 + 2	6 + 3
AEP (500)	-	25 + 7	21 + 7	154 + 15	108 + 13	9 + 1	10 + 3	8 + 2	8 + 2
AEP (158)	-	18 + 2	21 + 3	153 + 20	99 + 6	9 + 2	8 + 2	10 + 3	9 + 1
AEP (50)	-	16 + 1	22 + 4	169 + 12	93 + 4	9 + 1	9 + 3	14 + 1	8 + 2
Purified water (0.1 ml)	-	23 + 3	30 + 4	166 +14	99 + 4	10 + 2	11 + 3	16 + 2	11 + 3
Benzo[a]pyrene (5)	-	31 + 5	25 + 3	149 + 12	91 + 10	-	-	15 + 2	9 + 1
Benzo[a]pyrene (5)	+	179 + 46	331 + 64	461 + 84	976 + 66	-	-	105 + 7	154 + 4
2 -Nitrofluorene (1)	-	69 + 16	124 + 25	-	-	-	-	-	-
Sodium azide (2)	-	-	-	474 + 43	870 + 89	860 + 213	739 + 68	-	-
2 -Aminoanthracene (2)	-	-	-	-	-	10 + 2	11 + 1	-	-
2 -Aminoanthracene (2)	+	-	-	-	-	116 + 23	134 + 26	-	-
9 -Aminoacridine (80)	-	-	-	-	-	-	-	208 + 10	201 + 18
None; 10(-6) dilution of bacterial culture only; plated on nutrient agar		195 + 31	204 + 10	251 + 11	274 + 29	258 + 13	261 +33	187 + 5	158 + 9

Conclusions:

NAEP was devoid of mutagenic activity under the conditions of the test.

Executive summary:

N-(2 -aminoethyl)-Piperazine (NAEP) was examined for mutagenic activity in four histidine-dependent auxotrophs of Salmonella typhimurium, strains TA98, TA100, TA1535 and TA1537, using pour-plate assays. The procedures used complied with OECD Guideline for Testing of Chemicals No. 471 (issued 1983). Results obtained with all strains were confirmed in a second, independent experiment.

The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed following a preliminary toxicity test in strain TA98, and included solvent (purified water) control with and without S-9 mix.

No increases in reversion to prototrophy were obtained with any of the four bacterial strain at the NAEP levels tested, either in the presence or absence of S-9 mix.

Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine and sodium azide when examined under similar conditions.

Therefore, it was concluded that NAEP was devoid of mutagenic activity under the conditions of the test.

Reference 3

Endpoint:: in vitro gene mutation study in mammalian cells
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
Remarks:: On 21-02-2020, ECHA communicated a final decision on compliance check (Decision number: CCH-D-2114500801-64-01/F) and in it deemed that the CHO HGPRT test was not regarded as scientifically robust and therefore considered as unreliable. An in vitro TK gene mutation study in mammalian cells (according to OECD 490; GLP) was therefore conducted to fulfil the information requirements set out in Annex VIII, Section 8.4.3.
Qualifier:: no guideline available
Principles of method if other than guideline:: 1. CHO Test:
A. Dose Selection - Appropriate concentrations of N-(2-Aminoethyl)piperazine for testing were determined by measurements of cytotoxicity to CHO cells of seven concentrations tested both in the presence and absence of a liver S9 metabolic activation system. Selection of a maximum concentration for testing depended upon an estimate of a dose level which would permit survival of at least 10% of the treated cells. Sterile water (H20) was used as the solvent and solvent control.
To simplify tables and to allow comparisons between different tests, concentrations of N-(2-Aminoethyl)piperazine are given in terms of volume percentages x 10^-2 to eliminate zeros in the lower concentration values.
B. Mutation - CHO cells were exposed for 5 hours to a minimum of five concentrations of N-(2-Aminoethyl)piperazine both with and without the addition of an S9 metabolic activation system. Dilutions of N-(2-Aminoethyl)piperazine for testing were prepared by either direct addition of various aliquots of the test agent into the cell culture medium or by making sequential one half dilutions of the stock solution for the maximum concentrations using sterile H20. The surviving fraction was determined at 20 t o 24 hours after treatment and the mutant fraction was determined after a 10-day period to allow "expression" of the mutant phenotype. Only the data from the top five concentrations which allowed sufficient cell survival for assessment of survival and induction of mutants are usually presented.

2. SCE Test:
Production of SCE's following exposure to various concentrations of N-(2-Aminoethyl)piperazine was studied in CHO cells both with and without the incorporation of an S9 metabolic activation system. Selection of a maximum dose level which would permit survival of at least 50% of the treated cells was based on the prescreening test for cytotoxicity performed as part of the CHO Mutation test. Dilutions of N-(2-Aminoethyl)piperazine for testing were prepared either by direct addition of various aliquots into the culture medium or by making sequential one-half dilutions of the stock solution for the maximum dose level using sterile H20. For determination of direct mutagenic action, CHO cells were exposed to N-(2-Aminoethyl) piperazine and appropriate controls for 5 hours without S9 activation. Indirect mutagenic action, requiring metabolic activation by liver S9 homogenate, was studied with a 2-hour exposure period. Bromodeoxyuridine (BrdU) required to differentiate between the individual "sister " chromatids by SCE staining , was present at a concentration of 3 ug/ml in the growth medium during treatment and during the culture period following exposure. A total of 20 cells/dose level and 5 dose levels, tested either with or without metabolic activation, were microscopically examined.

3. UDS Test:
Production of primary DNA damage in rat liver cells (hepatocytes), was studied at a minimum of six dose levels which spanned a 1000-fold range of concentrations. Cells were treated with N-(2-Aminoethyl)piperazine for 2 hours i n culture medium containing 3H-thymidine, hydroxyurea and appropriate dilutions of N-(2-Aminoethyl)piperazine prepared i n DMSO. Determination of UDS activity was performed by analyses of incorporation of 3H-thymidine into isolated hepatocyte nuclei or into DNA (precipitated from aliquots of the isolated nuclei) using a Searle Analytic Model 81 or Packard Model 2650 scintillation spectrometer.

4. Controls - Positive, negative and solvent controls were tested concurrently with the test sample to assure the sensitivity of the test system and the concurrence of the results to previous test performance. For the CHO and SCE assays, dimethylnitrosamine (DMN)-CAS #62-75-9 and ethylmethanesulfonate (EMS)-CAS #62-50-0 were used as positive control agents to assure the sensitivity of the test system for detecting indirect and direct-acting mutagens, respectively. Deionized water-CAS #7732-18-5, sterilized by membrane filtration and cell culture medium were used as the solvent and negative controls, respectively.
In the UDS assay, DMN and 4-nitroquinoline oxide (4-NQ0)-CAS #56-57-5 were used as positive controls representing indirect- or direct-acting mutagens, respectively. DMSO was used as the solvent and the solvent control.
GLP compliance:: yes
Type of assay:: in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:: HGPRT gene
Species / strain / cell type:: Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):: CHO cells used in these studies were obtained from Abraham Hsie at Oak Ridge National Laboratory with the designation CHO-Kl-BH4-Dl
(or simply CHO for report purposes).
Metabolic activation:: with and without
Metabolic activation system:: S9 liver homogenate, prepared from Arochlor 1254- induced, Sprague-Dawley male rats.
Test concentrations with justification for top dose:: CHO Mutation Assay (with and without): (1.25, 2.5, 5.0, 10.0, 20.0, 40.0) x10E-2 % v/v
Vehicle / solvent:: - Vehicle(s)/solvent(s) used: deionized water [CHO assays]
Untreated negative controls:: yes
Negative solvent / vehicle controls:: yes
Positive controls:: yes
Positive control substance:: other: For the CHO assay, dimethylnitrosamine (DMN)-CAS #62-75-9 and ethylmethanesulfonate (EMS)-CAS #62-50-0 were used as positive control agents to assure the sensitivity of the test system for detecting indirect and direct-acting mutagens, respectively.
Details on test system and experimental conditions:: Refer to the method section below.
Evaluation criteria:: Not applicable
Statistics:: Data from the SCE and UDS tests were analyzed by appropriate parametric tests following Standard Operating Procedures for statistical analyses at the Bushy Run Research Center. Data from the CHO test do not follow a normal distribution according to experience with historical controls. Thus, the Student's t-test was used after transformation of the mutation frequencies (MF) according to the method of Irr and Snee (MF + 1)0.15 ( Irr, J. D. and R. Snee, Proceedings of the Cold Spring Harbor-Banbury Conference,II (1979), 263-274).

Rounding of data to either two decimal places or to the appropriate number of significant figures was performed for presentation on tables. Although statistically significant decreases in mutation indices can occur because of cytotoxic responses, only statistically significant increases in responses above control values are indicated on Tables for simplicity. The degree of statistical significance is denoted by: a: 0.05 > p > 0.01, b: 0.01 > p > 0.001, or c: p < 0.001. No superscript (or NS) indicates p > 0.05.
Species / strain:: Chinese hamster Ovary (CHO)
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:: RANGE-FINDING/SCREENING STUDIES: Preliminary experiments were performed to select an appropriate range of test concentrations in which the maximum concentration would allow survival of approximately 10% of the treated cells. A maximum concentration of 100 x 10E-2% (by volume) was chosen for the highest dose level and a total of eight concentrations of N-(2-Aminoethyl)piperazine were tested in subsequent mutagenesis experiments. Concentrations of 40 x 10E-2% and higher produced > 99% cytotoxicity and could not be analyzed for mutation induction.

CHO Mutation Test: N-(2-Aminoethyl)piperazine produced a statistically significant increase in the frequency of mutations of CHO cells at only a few, non-consecutive concentrations between 40 x10E-2% to 1.25 x 10E-2 (by volume) in tests with and without the incorporation of a liver S9 metabolic activation system. The lack of a dose-related effect of treatment on the induction of mutations indicated that N-(2-Aminoethy1)piperazine was not active in producing gene mutations in CHO cells within the range of concentrations tested.

CHO Mutation Test-Cytotoxicity:A steep dose-response effect with the test agent was observed from the > 99% cell killing obtained with the highest concentration (40 x 10-2%) of N-(2-Aminoethy1)piperazine , in tests with or without S9 a comparison to the markedly lower cytotoxicity obtained at only s lightly lower dose-levels (e.g. 20 x 10E-2%). Concentrations of 100 x 10E-2% and 60 x 10E-2% were also tested but essentially all cells were killed by the treatment.

CHO Mutation Test -Mutation:N-(2-Aminoethy1)piperazine did not produce a statistically significant, dose-related increase in the frequency of
mutants/10E6 viable cells over the 32-fold range of concentrations tested either with or without the presence of an S9 metabolic activation system. A few non-consecutive dose levels of N-(2-Aminoethyl)piperazine produced increases in the mutation frequency which were statistically significant from the concurrent solvent control. However, these values were not considered to be biologically significant because occasional increases of similar magnitude have been obtained in previous tests with solvent or negative controls. The test was interpreted as a negative indication for potential mutagenic action of the test chemical in the CHO mutation test.

Mutation frequencies for the solvent controls for tests both with and without S9 activation were in an acceptable and low range based upon experience with historical control values. Statistically significant increases in the mutation frequencies were obtained for the DMN and EMS positive controls for both experiments and these values were within the expected range of values observed in historical control data.

SCE Test: N-(2-Aminoethyl)piperazine produced a highly statistically significant and dose-related effect on the frequency of SCE in CHO cells in tests both with and without the incorporation of an S9 metabolic activation system. An overall range of concentrations between 40 x 10E-2 % to 1.25 x 10E-2% (by volume) was used. The test results indicated that N-(2-Aminoethyl)piperazine was an active mutagenic agent for producing SCE in vitro. Determinations of SCE Induction.

1. A highly statistically significant increase in the SCE frequency was produced by three of five dose levels of N-(2-Aminoethy1)piperazine tested for direct action in the absence of a metabolic activation system. Treatment with the test chemical produced a dose-related increase in the number of SCE, a result which is generally considered to indicate a biologically significant effect. The test without S9 activation was considered a positive indication for potential direct mutagenic action of N-(2-Aminoethy1) piperazine.

The number of SCE produced by the concurrent EMS positive control was highly statistically significant from the concurrent solvent control and these data indicated an appropriate sensitivity of the test system comparable to our historical positive control data. The numbers of SCE obtained with the solvent and media controls were also in an acceptable range of values included in the variability encountered in our historical control values for this test.

2. A statistically significant increase in the SCE values was observed with four of five of the tested concentrations of N-(2-Aminoethy1)piperazine. The data also indicated a dose-response trend in the SCE frequency. The positive effects observed in this experiment were consistent with the findings in the test without addition of S9 and N-(2-Aminoethy1)piperazine was considered as an active mutagenic agent in the induction of SCE in vitro.

The SCE values for the negative and solvent controls in the test with S9 activation were in an acceptable range of variability as encountered in previous experiments with this test system. Highly statistically significant numbers of SCE were produced by the DMN positive control which indicated that the metabolic activation system was suitably active.

UDS Test: N-(2-Aminoethyl)piperazine did not produce a dose-related effect on the amount of UDS activity in evaluations of concentrations between 100 x 10E-2% to 0.1 x 10E-2% (by volume). Positive effects were observed at only a single dose level but this isolated effect was not considered to be biologically significant. N-(2-Aminoethyl)piperazine was considered to be inactive in the present test with the hepatocyte test system.
Conclusions:: Aminoethylpiperazine was negative in the in vitro CHO HGPRT assay with and without metabolic activation.
Executive summary:: N-(2-Aminoethy1)piperazine was evaluated for potential mutagenic activity with a battery of three -in -vitro tests, which were: the Chinese Hamster Ovary (CHO) Mutation test, the Sister Chromatid Exchange (SCE) test and an assay for induction of Unscheduled DNA Synthesis (UDS) in rat liver cells. The results indicated that N-(2- Aminoethy1)piperazine produced a statistically significant and dose-related mutagenic effect in the SCE tests with and without metabolic activation, but a definite positive effect was not obtained in the CHO or UDS tests. The lack of confirmation of mutagenic activity in at least two of the three tets performed indicated that the results for N-(2-Aminoethy1)piperazine should be considered an unconfirmed indication of a positive effect. Determination of the significance and repeatability of this result would require additional testing using other mutagenesis test systems or using higher but narrower concentrations ranges in the CHO and UDS test systems. N-(2-Aminoethy1)- piperazine could not be definitely classified as an active or inactive mutagenic agent because of the conflicting results obtained in these separate tests.

Reference 4

Endpoint:: in vitro DNA damage and/or repair study
Remarks:: Type of genotoxicity: DNA damage and/or repair
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
Qualifier:: no guideline available
Principles of method if other than guideline:: Study examined the ability of aminoethylpiperazine to affect sister chromatid exchange in CHO cells.
GLP compliance:: yes
Type of assay:: sister chromatid exchange assay in mammalian cells
Target gene:: Not applicable
Species / strain / cell type:: other: CHO; hepatocytes
Details on mammalian cell type (if applicable):: Chinese hamster ovary (CHO) cells were obtained from Abraham Hsie at Oak Ridge National Laboratory with the designation CHO-K1-
BH4-Dl (referred to simply as CHO for report purposes).
Metabolic activation:: with and without
Metabolic activation system:: S9 liver homogenate
Test concentrations with justification for top dose:: SCE Assay (with and without): (1.25, 2.5, 5.0, 10.0, 20.0, 40.0) x10E-2 % v/v
Vehicle / solvent:: - Vehicle(s)/solvent(s) used: deionized water [CHO SCE assay]
Untreated negative controls:: yes
Negative solvent / vehicle controls:: yes
Positive controls:: yes
Positive control substance:: other: For the CHO SCE assay, dimethylnitrosamine (DMN)-CAS #62-75-9 and ethylmethanesulfonate (EMS)-CAS #62-50-0 were used as positive control agents to assure the sensitivity of the test system for detecting indirect and direct-acting mutagens, respectively.
Details on test system and experimental conditions:: Refer to the method section below.
Evaluation criteria:: Not applicable
Statistics:: Data from the SCE tests were analyzed by appropriate parametric tests following Standard Operating Procedures for statistical analyses at the Bushy Run Research Center. Data from the CHO test do not follow a normal distribution according to experience with historical controls. Thus, the Student's t-test was used after transformation of the mutation frequencies (MF) according to the method of Irr and Snee (MF + 1)0.15 ( Irr, J. D. and R. Snee, Proceedings of the Cold Spring Harbor-Banbury Conference,II (1979), 263-274).

Rounding of data to either two decimal places or to the appropriate number of significant figures was performed for presentation on tables. Although statistically significant decreases in mutation indices can occur because of cytotoxic responses, only statistically significant increases in responses above control values are indicated on Tables for simplicity. The degree of statistical significance is denoted by: a: 0.05 > p > 0.01, b: 0.01 > p > 0.001, or c: p < 0.001. No superscript (or NS) indicates p > 0.05.
Species / strain:: Chinese hamster Ovary (CHO)
Metabolic activation:: with and without
Genotoxicity:: not specified
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Additional information on results:: RANGE-FINDING/SCREENING STUDIES: Preliminary experiments were performed to select an appropriate range of test concentrations in which the maximum concentration would allow survival of approximately 10% of the treated cells. A maximum concentration of 100 x 10E-2% (by volume) was chosen for the highest dose level and a total of eight concentrations of N-(2-Aminoethyl)piperazine were tested in subsequent mutagenesis experiments. Concentrations of 40 x 10E-2% and higher produced > 99% cytotoxicity and could not be analyzed for mutation induction.

SCE Test: N-(2-Aminoethyl)piperazine produced a highly statistically significant and dose-related effect on the frequency of SCE in CHO cells in tests both with and without the incorporation of an S9 metabolic activation system. An overall range of concentrations between 40 x 10E-2 % to 1.25 x 10E-2% (by volume) was used. The test results indicated that N-(2-Aminoethyl)piperazine was an active mutagenic agent for producing SCE in vitro. Determinations of SCE Induction.

1. A highly statistically significant increase in the SCE frequency was produced by three of five dose levels of N-(2-Aminoethy1)piperazine tested for direct action in the absence of a metabolic activation system. Treatment with the test chemical produced a dose-related increase in the number of SCE, a result which is generally considered to indicate a biologically significant effect. The test without S9 activation was considered a positive indication for potential direct mutagenic action of N-(2-Aminoethy1) piperazine.

The number of SCE produced by the concurrent EMS positive control was highly statistically significant from the concurrent solvent control and these data indicated an appropriate sensitivity of the test system comparable to our historical positive control data. The numbers of SCE obtained with the solvent and media controls were also in an acceptable range of values included in the variability encountered in our historical control values for this test.

2. A statistically significant increase in the SCE values was observed with four of five of the tested concentrations of N-(2-Aminoethy1)piperazine. The data also indicated a dose-response trend in the SCE frequency. The positive effects observed in this experiment were consistent with the findings in the test without addition of S9 and N-(2-Aminoethy1)piperazine was considered as an active mutagenic agent in the induction of SCE in vitro.

The SCE values for the negative and solvent controls in the test with S9 activation were in an acceptable range of variability as encountered in previous experiments with this test system. Highly statistically significant numbers of SCE were produced by the DMN positive control which indicated that the metabolic activation system was suitably active.
Remarks on result:: other: all strains/cell types tested
Remarks:: Migrated from field 'Test system'.
Conclusions:: N-(2-Aminoethy1)piperazine was evaluated for potential mutagenic activity with the Chinese Hamster Ovary (CHO) Sister Chromatid Exchange (SCE) test. The results indicated that N-(2-Aminoethy1)piperazine produced a statistically significant and dose-related mutagenic effect in the SCE tests with and without metabolic activation.

Reference 5

Endpoint:: in vitro DNA damage and/or repair study
Remarks:: Type of genotoxicity: DNA damage and/or repair
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
Qualifier:: no guideline available
Principles of method if other than guideline:: The ability of aminoethylpiperazine to affect unscheduled DNA synthesis was examined in rat hepatocytes.
GLP compliance:: yes
Type of assay:: DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:: Test measures uptake of 3H-Thymidine
Species / strain / cell type:: hepatocytes:
Details on mammalian cell type (if applicable):: Hepatocytes were obtained from Hilltop-Wistar albino rats
Metabolic activation:: without
Test concentrations with justification for top dose:: UDS: (0.1, 1.0, 3.0, 10.0, 30.0, 100)x 10E-3%
Vehicle / solvent:: - Vehicle(s)/solvent(s) used: DMSO was used as the solvent and the solvent control. [UDS assays]
Untreated negative controls:: yes
Negative solvent / vehicle controls:: yes
Positive controls:: yes
Positive control substance:: other: DMN and 4-nitroquinoline oxide (4-NQ0)-CAS #56-57-5 were used as positive controls representing indirect- or direct-acting mutagens, respectively.
Details on test system and experimental conditions:: Refer to the method section below.
Evaluation criteria:: Not applicable
Statistics:: Data from the UDS tests were analyzed by appropriate parametric tests following Standard Operating Procedures for statistical analyses at the Bushy Run Research Center. Data from the CHO test do not follow a normal distribution according to experience with historical controls. Thus, the Student's t-test was used after transformation of the mutation frequencies (MF) according to the method of Irr and Snee (MF + 1)0.15 ( Irr, J. D. and R. Snee, Proceedings of the Cold Spring Harbor-Banbury Conference,II (1979), 263-274).

Rounding of data to either two decimal places or to the appropriate number of significant figures was performed for presentation on tables. Although statistically significant decreases in mutation indices can occur because of cytotoxic responses, only statistically significant increases in responses above control values are indicated on Tables for simplicity. The degree of statistical significance is denoted by: a: 0.05 > p > 0.01, b: 0.01 > p > 0.001, or c: p < 0.001. No superscript (or NS) indicates p > 0.05.
Species / strain:: hepatocytes: from Rat Liver
Metabolic activation:: without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: not specified
Positive controls validity:: valid
Additional information on results:: UDS Test: N-(2-Aminoethyl)piperazine did not produce a dose-related effect on the amount of UDS activity in evaluations of concentrations between 100 x 10E-2% to 0.1 x 10E-2% (by volume). Positive effects were observed at only a single dose level but this isolated effect was not considered to be biologically significant. N-(2-Aminoethyl)piperazine was considered to be inactive in the present test with the hepatocyte test system.
Conclusions:: Aminoethylpiperazine was negative in the unscheduled DNA synthesis (UDS) in rat liver cells.
Executive summary:: N-(2-Aminoethy1)piperazine was evaluated for potential mutagenic activity in the Unscheduled DNA Synthesis (UDS) in rat liver cells. The results indicated that N-(2-Aminoethy1)piperazine was negative in the UDS tests.

Reference 6

Endpoint:: in vitro gene mutation study in mammalian cells
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 17 Aug 2020 - 08 Dec 2020
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:: adopted 29 July 2016
Deviations:: no
GLP compliance:: yes
Type of assay:: in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:: TK gene
Species / strain / cell type:: mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):: CELLS USED
- Type and source of cells: L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells; American Type Culture Collection, (ATCC, Manassas, USA, 2001)
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD 490)

For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 100 µM hypoxanthine (Sigma), 0.2 µM aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 16 µM thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
- Periodically checked for karyotype stability: no information
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:

Horse serum: Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: 3 h exposure: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium); 24 h exposure:
Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium). Due to a shortage of the R5 stock, R10 was used as exposure medium in the first experiment instead of R5. Since, the mutation frequency found in the solvent control and positive control cultures was within the acceptability criteria of this assay, the excess amount of horse serum present had no effect on the results of the study.
Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 µg/mL trifluorothymidine (TFT) (Sigma).
Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).

All incubations were carried out in a humid atmosphere (80 - 100%, actual range 41 - 98%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.2 – 37.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.
Additional strain / cell type characteristics:: other: no information
Metabolic activation:: with and without
Metabolic activation system:: Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life technologies). The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: The concentration of the S9-fraction in the exposure medium was 4% (v/v)
Test concentrations with justification for top dose:: Experiment 1 (without pH adjustment; 3 h; +/-S9):
- 50, 100, 200, 400, 800, 900, 950, 1000, 1050, 1100, 1150, 1200 and 1292 μg/mL
Dose levels selected to measure mutation frequencies at the TK-locus were:
- 100, 200, 400, 900, 1000, 1050, 1100 and 1200 μg/mL

The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control.

Experiment 1A (with pH adjustment; 3 h; +/-S9):
- 50, 100, 200, 400, 800, 900, 950, 1000, 1050, 1100, 1150, 1200 and 1292 μg/mL
Dose levels selected to measure mutation frequencies at the TK-locus were:
- -S9: 100, 200, 400, 800, 1000, 1100, 1200 and 1292 μg/mL
- +S9: 400, 900, 1000, 1050, 1100, 1150, 1200 and 1292 μg/mL

Experiment 2 (with pH adjustment; 24 h; -S9):
- 50, 100, 200, 400, 800, 900, 950, 1000, 1050, 1100, 1150, 1200 and 1292 μg/mL
Dose levels selected to measure mutation frequencies at the TK-locus were:
- 200, 400, 800, 900, 1000, 1100, 1200 and 1292 μg/mL

In the additional verification experiment (Experiment 1A) and the second experiment (Experiment 2), the highest tested concentration was 1292 µg/mL exposure medium. This concentration was equal to 0.01M.
Vehicle / solvent:: - Vehicle(s)/solvent(s) used: exposure medium

- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item formed a clear pink solution in exposure medium. Test item concentrations were used within 1.5 hours after preparation.
Untreated negative controls:: no
Negative solvent / vehicle controls:: yes
True negative controls:: no
Positive controls:: yes
Positive control substance:: cyclophosphamide; methylmethanesulfonate
Details on test system and experimental conditions:: NUMBER OF REPLICATIONS:
- Number of cultures per concentration: solvent control in duplicate (5 plates each with 2000 cells/well); treatment groups single (5 plates with 2000 cells/well), positive controls single (10 plates with 1000 cells/well)
- Number of independent experiments :
Three experiments were performed during this study: Experiment 1: 3 h exposure +/- S9, without pH adjustment for the test item concentrations in exposure medium. Experiment 1A: 3 h exposure +/- S9, with pH adjustment for the test item concentrations in exposure medium. Experiment 2: 24 h exposure - S9, with pH adjustment for the test item concentrations in exposure medium.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: Per culture 8 x 10^6 cells (10^6 cells/mL for 3 h treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 h treatment)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: In the first experiment (Experiment 1 and 1A), cell cultures were exposed for 3 h to the test item in exposure medium (see deviation) in the absence and presence of S9-mix. In the second experiment (Experiment 2), cell cultures were exposed to the test item in exposure medium for 24 h in the absence of S9-mix.
- Harvest time after the end of treatment: The cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium (R10-medium) and in 20 mL growth medium (R10-medium) for the 3 h and 24 h treatment, respectively. The cells in the final suspension were counted with the coulter particle counter.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (where possible) were subcultured every day in order to maintain log phase growth.
- Selection time: The microtiter plates were incubated for 11 or 12 days .
- Fixation time: After the incubation period, the plates for the TFT-selection were stained for 1.5 - 2 h, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well.
- Method used: microwell plates
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure. 5 µg/mL trifluorothymidine; 11 or 12 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: A total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates (see deviation), each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). In the first experiment in one of the solvent controls, a total number of 384 wells (four 96-well plates) was used for determination of the mutation frequency instead of 480 wells (five 96-well plates) as specified in the study plan. Due to a technical error, only four of the five plates were counted. Since the mutation frequency of 147 per 10^6 survivors was within the acceptability criteria range for the solvent control (≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors), this deviation had no effect on the results of the study.
- Criteria for small (slow growing) and large (fast growing) colonies: Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Lessseverely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method:
Dose-range finding test: Suspension growth (SG)
Mutagenicity tests: Relative suspension growth (RSG), cloning efficiency (CEday2), relative cloning efficiency (RCE), relative total growth (RTG); please refer to any other information on materials and methods for calcualtions
- Any supplementary information relevant to cytotoxicity: For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium. The microtiter plates were incubated for 11 or 12 days.

METHODS FOR MEASUREMENTS OF GENOTOXICIY : The plates were scored with the naked eye or with the microscope.
Rationale for test conditions:: According to OECD 490.
Evaluation criteria:: In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range. The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the microwell version of the assay the GEF is 126 x 10^-6.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Statistics:: No statistics included.
: Key result
Species / strain:: mouse lymphoma L5178Y cells
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: no cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: not applicable
True negative controls validity:: not applicable
Positive controls validity:: valid
Additional information on results:: TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: In the solubility experiment, the pH at concentrations of 1292, 1000 and 500 μg/mL was 7.958, 7.811 and 7.483 (solvent: 7.416). Experiment 1 was performed without pH adjustments of the test item concentrations in the exposure medium. To determine if the mutagenic effect observed in the first experiment was due to a pH effect, an additional verification experiment was performed with pH adjustments of the test item concentrations in the exposure medium. The pH of the test item concentrations in culture medium was adjusted using HCl. The pH of the final test system with test item was set to be comparable with the concurrent vehicle control group (at least pH vehicle control group ± 0.5). The amount of HCl to be added was determined in a separate pH determination test. These results (see Table pH measurement under Any other information on results) were obtained with 80 µL test item concentration added to 8.0 mL R5 medium. In addition,for the formulations used in the second experiment (Experiment 2), the pH of the test item concentrations in culture medium were adjusted. Volumes of HCl determined in the pH determination test were adjusted to the total volume used in the second experiment.
- Data on osmolality: In the solubility experiment, the osmolarity at 1292 μg/mL was 0.290 (solvent: 0.283).
- Precipitation and time of the determination: The test item did not precipitate in the exposure medium up to and including the concentration of 1292 μg/mL (= 10 mM).

RANGE-FINDING/SCREENING STUDIES:
In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 10^6 cells (10^6 cells/mL for 3 h treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 h treatment) with a number of test item concentrations increasing by approximately half log steps. The cell cultures for the 3 h treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 h treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test item was tested in the absence and presence of S9-mix. The highest tested concentration was 1292 µg/mL exposure medium. This concentration was equal to 0.01M. Since testing up to 0.01 M is recommended in the
guidelines, this concentration was used as the highest test item concentration in the doserange finding test. For the 3 h treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium (R10-medium). For the 24 h treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium (R10-medium). The cells in the final suspension were counted with the coulter particle counter. The surviving cells of the 3 h treatment were subcultured twice to determine cytotoxicity. After 24 h of subculturing, the cells were counted and subcultured again for another 24 h, after that the cells were counted. The surviving cells of the 24 h treatment were subcultured once. After 24 h of subculturing, the cells were counted. If less than 1.25 x 10^5 cells/mL were counted no subculture was performed. The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 h or only 24 h cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose-range for the mutagenicity tests.

In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 63 to 1292 µg/mL in the absence of S9-mix with 3 and 24 h treatment periods and in the presence of S9-mix with a 3 h treatment period. The dose-range finding test was performed without pH adjustments of the test item concentrations in the exposure medium.
3 h treatment:
In the absence of S9-mix, the relative suspension growth was 14% at the test item concentration of 1000 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentration of 1292 μg/mL. In the presence of S9-mix, the relative suspension growth was 37% at the test item concentration of 1000 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentration of 1292 μg/mL.
24 h treatment:
The relative suspension growth was 9% at the test item concentration of 1000 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentration of 1292 μg/mL.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Although the mutation frequency of the solvent control cultures in the first experiment (Experiment 1) in the absence of S9-mix were just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. Although in the first experiment (Experiment 1) in the absence of S9-mix the response of MMS was just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The suspension growth over the two-day expression period for cultures treated with exposure medium was between 16 and 27 (3 h treatment) and 81 and 124 (24 h treatment).

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
Experiment 1 (without pH adjustment; 3 h; +/-S9): In the absence of S9-mix, the relative total growth of the highest test item concentration (1200 µg/mL) with the 3 h treatment was 15% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the test item concentrations of 1100 and 1200 μg/mL with 3 h treatment was 19% and 9%, respectively, compared to the total growth of the solvent controls.

Experiment 1A (with pH adjustment; 3 h; +/-S9): No toxicity was observed in the absence and presence of S9-mix.

Experiment 2 (with pH adjustment; 24 h; -S9): No significant toxicity was observed.

- Genotoxicity results:
Experiment 1 (without pH adjustment; 3 h; +/-S9): After the 3 h treatment period, increases in the mutation frequency at the TK locus above the acceptability criteria for the solvent control (i.e. ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors) were observed. The increases in the absence of S9-mix, were just within the MF(controls) + GEF (i.e. 292 x 10^-6). The increase in the mutation frequency in the presence of S9-mix were up above the positive threshold of MF(controls) + GEF (i.e. above 273 x 10^-6) at the dose level of 1100 µg/mL (RTG 19%). Although the increase in the mutation frequency at the TK locus in the presence of S9-mix was only observed at a toxic dose level (RTG 19%), the mutation frequency at this concentration was above the GEF and the RTG of 19% is considered appropriate toxicity. Therefore, this increase is considered biologically relevant. The pH of the culture medium especially at the higher concentrations was elevated more than 1 pH unit over the pH of the solvent control. As stated in the OECD 490 Guideline changes in pH of the medium can produce artefactual positive results. To determine if the mutagenic effect observed in the first experiment in the presence of S9-mix was due to the pH of the 1100 µg/mL concentration being higher than the solvent control, an additional verification experiment was performed with pH adjustments of the test item concentrations in the culture medium (Experimetn 1A).

Experiment 1A (with pH adjustment; 3 h; +/-S9): No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. Thus, the increases in mutation frequency observed in Experiment 1 were not reproducible when the pH of the test item concentrations in the culture medium were adjusted comparable to the pH of the solvent control.

Experiment 2 (with pH adjustment; 24 h; -S9): No biologically relevant increase in the mutation frequency at the TK locus was observed after 24 hours of treatment with the test item. Thus, the negative result of Experiment 1A was confirmed in an independent experiment (Experiment 2) with modification in the duration of treatment (24 h treatment) and pH adjustment of the medium.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data (from experiments performed between June 2017 and June 2020)::
-S9 / 3 h treatment (Mean Mutation frequency per 10^6 survivors / Standard deviation / Number of observations / Lower Control Limit (95% Control Limit) / Upper Control Limit (95% Control Limits)): 888 / 329 / 113 / 244 / 1532
-S9 / 24 h treatment (Mean Mutation frequency per 10^6 survivors / Standard deviation / Number of observations / Lower Control Limit (95% Control Limit) / Upper Control Limit (95% Control Limits)): 775 / 253 / 101 / 279 / 1272
+S9 / 3 h treatment (Mean Mutation frequency per 10^6 survivors / Standard deviation / Number of observations / Lower Control Limit (95% Control Limit) / Upper Control Limit (95% Control Limits)): 1341 / 869 / 113 / -362 / 3044
- Negative solvent historical control data (from experiments performed between June 2017 and June 2020):
-S9 / 3 h treatment (Mean Mutation frequency per 10^6 survivors / Standard deviation / Number of observations / Lower Control Limit (95% Control Limit) / Upper Control Limit (95% Control Limits)): 102 / 30 / 114 / 43 / 160
-S9 / 24 h treatment (Mean Mutation frequency per 10^6 survivors / Standard deviation / Number of observations / Lower Control Limit (95% Control Limit) / Upper Control Limit (95% Control Limits)): 103 / 32 / 100 / 40 / 166
+S9 / 3 h treatment (Mean Mutation frequency per 10^6 survivors / Standard deviation / Number of observations / Lower Control Limit (95% Control Limit) / Upper Control Limit (95% Control Limits)): 101 / 29 / 113 / 44 / 158
Conclusions:: 2-piperazin-1-ylethylamine (N-AEP) is not mutagenic in the mouse lymphoma L5178Y test system after 3 and 24 h of treatment when pH of the culture medium is adjusted comparable to the pH of the solvent control.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

One in vivo key study (micronucleus test) was identified for aminoethylpiperazine with a negative outcome.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not applicable

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 1a: GLP guideline study

Qualifier:

no guideline available

Principles of method if other than guideline:

Followed EPA report 560/6-83-001

GLP compliance:

yes

Remarks:

Union Carbide Bushy Run Research Center, R.D. 4, Mellon road, Export, Pennsylvania 15632 USA

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

Five-week old, Swiss-Webster mice were used for all preliminary and definitive tests and were obtained from Charles Rivers Laboratories, Portage,
MI. Swiss-Webster mice were used because of this laboratory's toxicological experience with this strain. For preliminary range-finding tests to determine approximate toxicity, 30 male mice were obtained on February 3, 1987. For the toxicity tests, performed to select test concentrations for the definitive micronucleus determination, 64 male and 64 female mice were obtained on March 3, 1987. For the definitive micronucleus test, 44 male and 43 female mice were obtained on March 17, 1987. Following the protocol, only the animals used for the definitive micronucleus test were randomized by weight and animals outside a range of two standard deviations from the mean were not used. Upon arrival, animals were examined for general health status and were identified with unique BRRC animal numbers using monel ear tags. An acclimation period of 5 to 6 days prior to dosing was used for all studies. All animals used for these tests appeared healthy. For the definitive tests, animals were weighed and randomized on the day prior to dosing.

Five mice/sex/cage were housed in shoe-box type plastic cages, measuring 30 x 20 x 12.5 cm. Each cage was labeled with animal I.D. numbers, sex and treatment doses. Ab-Sorb-DriQD bedding (Garfield, HJ) was placed in the cages and changed weekly. Mice were fed ad libitum with a basic diet of Agway PROLAB@ Animal Diet (Rat/Mouse/Harnster 3000), Agway Inc., Syracuse, NY. Water was supplied by the Municipal Authority of Westmoreland County (Greensburg, PA) and was available libitum. Feed and water analyses are kept on file at the BRRC. The animal room temperature and humidity was contrblled and room lights were automatically timed for a 12-hr light/dark cycle. Room temperature and relative humidity yere monitored continuously by a Cole Parmer monitoring apparatus.

Route of administration:

intraperitoneal

Vehicle:

Water

Details on exposure:

The definitive toxicity study was conducted using 5 males and 5 females per dosage group. Animals were dosed with the test and control materials by intraperitoneal (i.p.) injection. I.P. injection is a typical route of dosing for this test system, and it was used because this route achieves accurate and rapid delivery of the test chemical. Toxicity was assessed by determining the incidence of deaths produced by dosages of AEP ranging from 434 to 900 mg/kg which was identified as an appropriate range in preliminary testing. The pooled LD50 value (700 mg/kg) for male and female Swiss-Webster mice was used to set dosages for the definitive micronucleus test because of the similarity of the respective male and female LD50s.

The PCE/NCE ratio was determined for the vehicle control animals and for the highest dose group in which at least 3 animals survived for 48 hr. Determination of the PCE/NCE ratio for only these groups of animals with partial mortality was performed to evaluate the possibility of bone marrow cytotoxicity from the test chemical.

The definitive micronucleus test employed a minimum of 5 male and 5 female mice/treatment group. Three additional animals were added to the highest dosage group because toxicity was expected to decrease survival. Animals were randomized separately by sex into various treatment and control groups using a computer generated randomization system. Mice with weights different from the group mean by over two standard deviations were not used for the study and were discarded. The body weights of the male mice dosed in the definitive study ranged from 24.5 g to 27.5 g and body weights of the female mice ranged from 20.2 g to 23.7 g.

Three dose levels of approximately 80%, 50% and 25% of the pooled LD50 value were evaluated for effects upon the incidence of micronuclei. Blood
samples were taken at 3 time periods at approximately 30, 48 and 72 hr after dosing. These sampling times have provided the maximum sensitivity
for detecting clastogenic effects for the peripheral blood micronucleus method .

Duration of treatment / exposure:

Mice are dosed once ip and observed for up to 72 hours.

Frequency of treatment:

Once

Post exposure period:

Animals were housed for

Remarks:

Doses / Concentrations:
175, 350 or 560 mg/kg
Basis:

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylenemelamine

Tissues and cell types examined:

Micronuclei in peripheral, polychromatic erythrocytes were examined

Details of tissue and slide preparation:

One or two blood smear slides were prepared for each animal per sampling time. Micronuclei in peripheral, polychromatic erythrocytes were stained with Gurr's R-66 Giemsa diluted in phosphate buffer. Slides were coded by animal number only and read blindly to prevent bias. A minimum of 1000 polychromatic erythrocytes was examined microscopically for each animal per sample time, unless cytotoxicity of the test material prevented this goal. The po1ychromatic:normochromatic erythrocyte ratio for approximately 1000 total cells was calculated and recorded and these data are summarized in the final report as an estimate of cytotoxicity of the test agent. Micronuclei were identified as darkly-stained, spherical, inclusions in polychromatic erythrocytes. Polychromatic erythrocytes were identified by the pale-bluish staining of the cytoplasm in contrast to the lack of blue stain for normochromatic erythrocytes.

Evaluation criteria:

See statistic section

Statistics:

Data were compared for significant differences from the vehicle control frequencies using the Fisher's Exact Test (Sokal and Rohlf, 1981). Data for
males and females sampled at 48 hr and 72 hr were combined for analyses because statistical tests showed that there was no significant difference in micronuclei frequencies between sexes. However, micronucleus frequencies from PCEs sampled 30 hr after treatment were analyzed separately by sex since AOV testing showed that there was a sex-related difference between the responses for male and female mice at this sample time. A positive result in the micronucleus test was concluded if at least one statistically significant (p I 0.01) increase above the vehicle control was observed with an indication of a dose-related effect (Linear Regression Analysis) of treatment. A test was considered to be inconclusive if only one dose produced effects statistically different from the control and a dose-effect relationship was apparent. A test result was considered to be negative if no statistically significant differences were apparent between the vehicle control and groups of animals treated with AEP.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Probe study: The LD50 dose for both sexes combined was approximately 700 mg/kg (634 to 777: 95% fiducial limits).

Definitive micronucleus study: did not produce positive increases in the incidence of micronuclei in peripheral blood polychromatic erythrocytes of the test animals sampled at either the 48 or 72 hour sample periods. One dose level (350 mg/kg), sampled 30 hours after injection, produced a single statistically significant increase in numbers of micronuclei in the female mice. Since there was no evidence of a statistically significant dose-related increase in numbers of micronuclei at this sample period, this test was considered to be inconclusive for determining the clastogenic activity under the criteria for evaluating results from this test system at BRRC. However, since the incidence of micronuclei was within the range of variability typically observed at BRRC, the single increase observed in this test is not likely to be of biological significance. Data from the positive and negative control groups of animals demonstrated the appropriate responses for the animals in the test system consistent with a valid test.

Table 1

Micronucleus Test: Summary of Frequencies

mg/kg	Sex	# PCE observed	% PCE with Micronucleus
		30 hr sample
0 -water	M	5000	0.42
	F	5000	0.14
175	M	5000	0.30
	F	5000	0.20
350	M	5000	0.50
	F	5000	0.40*
560	M	5000	0.28
	F	5000	0.18

0.3 mg/kg TEM	M	5000	2.40
	F	5000	1.70

		48 -hour sample
0 -water	C	10,000	0.21
175	C	10,000	0.33
350	C	10,000	0.24
560	C	10,000	0.13

		72 -hour sample
0 -water	C	10,000	0.18
175	C	10,000	0.30
350	C	10,000	0.18
560	C	10,000	0.22

C - Combined sexes
^aSignnificantly different from control, p<0.01.

Conclusions:

Executive summary:

N-Aminoethylpiperazine (AEP) was evaluated for potential clastogenic (chromosome-damaging) activity with the in vivo micronucleus test system employing both male and female Swiss-Webster mice. Test doses for the micronucleus test were chosen from data obtained in a preliminary toxicity study with mice. Five doses of AEP ranging from 434 mg/kg to 900 mg/kg were administered as a single intraperitoneal (i.p.) injection. The LD50 dose was calculated from the cumulative mortality observed during a three day period after dosing. To select dose levels for the definitive micronucleus test, a combined LD50 value of approximately 700 mg/kg (634 to 777 mg/kg; 95% fiducial limits) was calculated by pooling the total number of deaths for males and females.

For the definitive micronucleus test, doses of 175 mg/kg, 350 mg/kg and 560 mg/kg were tested with both male and female Swiss-Webster mice. Concurrent positive (triethylenemelamine) and negative (water) control agents, administered by i.p. injection, were used to demonstrate the reliability and sensitivity of the micronucleus test system. Results from the micronucleus determination demonstrated that AEP did not produce positive increases in the incidence of micronuclei in peripheral blood polychromatic erythrocytes of the test animals sampled at either the 48 or 72 hour sample periods. One dose level (350 mg/kg), sampled at the 30 hours interval after injection, produced a single statistically significant increase in numbers of micronuclei in the female mice. Since there was no evidence of a statistically significant, dose-related increase in numbers of micronuclei at this sample period, this test was considered to be inconclusive for determining the clastogenic activity of AEP under the criteria for evaluating results from this test system at BRRC. However, since the incidence of micronuclei was within the range of variability typically observed at BRRC, the single increase observed in this test is not likely to be of biological significance. Data from the positive and negative control groups of animals demonstrated the appropriate responses for the animals in the test system consistent with a valid test.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Additional information

In one key study aminoethyl piperazine (AEP) was evaluated for its mutagenic activity using the Salmonella/microsome bacterial mutagenicity assay with strains: TA98, TA100, TA1535, and TA1537. No evidence of mutagenicity was evident in any of the strains with or without metabolic activation. In a second key study aminoethyl piperazine (AEP) was evaluated for its mutagenic activity using the same strains as enumerated before and TA1538 in addition. No evidence of mutagenicity was evident in any of the strains without metabolic activation. With metabolic activation, none of the strains except TA1535 showed any mutagenic activity. It was noted that the TA1535 strain showed positive and dose related increases in the number of revertants with a maximum response of approximately 3 -fold and therefore considered weakly mutagenic. In 1994, a similar report analyzed the same data and concluded the AEP has no mutagenic activity with or without S9 activation.

Three supporting studies using the Ames assay to determine the mutagenic potential of AEP were negative with or without S9 activation.

Three additional in vitro tests were conducted to evaluate AEP's potential for mutagenic activity: Chinese Hamster Ovary (CHO) HGPRT test, Sister Chromatid Exchange (SCE), and Unscheduled DNA Synthesis (UDS). A statistically significant, dose related effect was observed in the SCE test both with and without metabolic activation. However, the CHO HGPRT and UDS tests were negative. However, on 21-02-2020, ECHA communicated a final decision on compliance check (Decision number: CCH-D-2114500801-64-01/F) and in it deemed that the CHO HGPRT test was not regarded as scientifically robust and therefore considered as unreliable. Therefore, anin vitrothymidine kinase (TK) gene mutation study in mammalian cells (according to OECD 490; GLP) using L5178Y mouse lymphoma cells was conducted to fulfil the information requirements set out in Annex VIII, Section 8.4.3. The test was performed in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period. In the first experiment (Experiment 1), the test item was tested up to concentrations of 1200 µg/mL in the absence and presence S9-mix, respectively. Increases in the mutation frequency (MF) at the TK locus above the acceptability criteria for the solvent control (i.e. ≥ 50 per 10⁶survivors and ≤ 170 per 10⁶survivors) were observed with and without S9-mix. However, only the increase in the MF in the presence of S9-mix was considered biologically relevant, although the increase in the MF was only observed at a toxic dose level (relative total growth, RTG, of 19%). In addition, the pH of the culture medium especially at the higher concentrations, was elevated more than 1 pH unit over the pH of the solvent control. In a second experiment (Experiment 1A; highest test concentration 1292 µg/mL – equal to 0.01M), conducted under the same conditions as Experiment 1, but the pH values of the test item concentrations in the culture medium were adjusted with 1 N HCl comparable to the pH values of the solvent control, the increases in the MF were not reproducible. This negative result was confirmed in an independent experiment (Experiment 2) with modification in the duration of treatment (24-hour treatment) and pH adjustment of the medium (highest test concentration 1292 µg/mL – equal to 0.01M). Therefore, it was concluded that AEP is not mutagenic in the mouse lymphoma L5178Y test system after 3 and 24 hours of treatment when pH of the culture medium was adjusted comparable to the pH of the solvent control.

The potential clastogenic activity of AEP was evaluated in a mouse micronucleus test. Mice were treated with 175, 350, and 560 mg/kg AEP. Only the 350 mg/kg dose level produced a single statistically significant increase in the numbers of micronuclei. Since there was no evidence of a statistically significant dose related increase in numbers of micronuclei at this sample period, the test was considered inconclusive. Also, the incidence of micronuclei was within the range of variability typically observed at the testing facility, the single increase observed in this test is not likely to be of biological significance.

Taken together all available in vitro and in vivo result referring to genotoxicity, it is concluded that there is no indication of a genotoxic hazard potential related to AEP.

Justification for classification or non-classification

The weight of evidence on the mutagenicity of AEP from in vitro and in vivo testing indicates that no classifiication for genotoxicity is required.

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Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Test Chemicals	Mutants/10(6) Viable Cells
Without S9
AEP (%, v/v)
0.40	Toxic
0.20	12.6^a
0.10	2.0
0.05	0
0.025	4.4
0.0125	10.7
Solvent control	1.8
Negative control	2.2
EMS (200 ug/ml)	295.1^c

With S9
AEP (%, v/v)
0.40	Toxic
0.20	23.1^a
0.10	0
0.05	18.7^b
0.025	11.0
0.0125	6.9
Solvent control	1.3
Negative control	8.4
DMN (3700 ug/ml)	270.6^c

Test Chemicals	Mean SCE/Chromosome + S.D.
AEP (%, v/v)
0.40	Toxic
0.20	0.997 + 0.211^c
0.10	0.933 + 0.217^c
0.05	0.614 + 0.119^c
0.025	0.542 + 0.175
0.0125	0.453 +0.198
Solvent control	0.465 + 0.141
Negative control	0.379 + 0.123
EMS (100 ug/ml)	1.458 + 0.412^c

Test Chemical	Mean number SCE/Chromosome + S.D.
AEP (%, v/v)
0.2	0.700 + 0.254^c
0.1	0.554 + 0.187^b
0.05	0.534 + 0.163^b
0.025	0.448 + 0.152
0.0125	0.470 + 0.109^a
Solvent control	0.380 + 0.160
Negative control	0.429 + 0.122
DMN (500 ug/ml)	1.599 + 0.654^c

Test Chemical	Concentration	Radioactivity in Nuclei Avg. DMP + S.D.
Solvent - DMSO	2%	6628 + 410
4 -NQO	3.0 ug/ml	27851 + 1022
	1.0 ug/ml	8196 + 2500
	0.3 ug/ml	7148 + 1630
DMN	1000 ug/ml	13191 +2439
	300 ug/ml	6675 + 721
	100 ug/ml	7887 + 1412

AEP	0.100%	3028 + 30
	0.030%	6186 + 35
	0.010%	6840 + 743
	0.003%	9664 + 1706
	0.001%	5621 + 286
	0.0001%	4811 + 3005

Concentrations	pH	pH after adjustment	µL 1 N HCl
Solvent	7.314	/	/
1292	8.920	7.397	160
1200	8.834	7.393	150
1150	8.750	7.397	140
1100	8.696	7.392	135
1050	8.676	7.377	135
1000	8.567	7.390	125
950	8.531	7.350	125
900	8.486	7.347	120
800	8.318	7.206	110
400	7.879	7.278	70
200	7.677	7.335	40
100	7.597	7.320	30
50	7.344	/	/

dose	RSG	CE day2	RCE	RTG	mutation frequency
dose	RSG	CE day2	RCE	RTG	per 10⁶survivors
(µg/mL)	(%)	(%)	(%)	(%)	total	(small	large)
without metabolic activation
3 h treatment
SC	100	78	100	100	170	(90	69)
SC	100	85	100	100	161	(81	69)
100	235	52	64	150	86	(49	35)
200	228	48	59	134	110	(49	58)
400	246	55	67	165	126	(45	77)
900	227	55	67	152	122	(41	77)
1000	167	37	46	77	220	(86	125)
1050	116	41	51	59	283	(97	170)
1100	26	84	103	27	270	(87	155)
1200	15	83	101	15	276	(118	127)
MMS	180	25	30	54	1917	(673	1027)
with metabolic activation
3 h treatment
SC	100	68	100	100	147	(81	60)
SC	100	97	100	100	147	(70	67)
100	102	75	91	93	153	(64	80)
200	94	95	116	109	132	(52	73)
400	96	71	87	83	192	(79	100)
900	74	99	121	89	149	(52	87)
1000	61	97	117	71	188	(75	96)
1050	46	101	122	56	209	(69	120)
1100	19	84	102	19	377	(130	190)
1200	7	94	114	9	245	(86	131)
CP	50	44	53	26	2944	(917	1145)

Registration Dossier

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Diss Factsheets

2-piperazin-1-ylethylamine

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

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Reference 1

Reference 2

Reference 3

Reference 4

Reference 5

Reference 6

Endpoint conclusion

Genetic toxicity in vivo

Description of key information

Link to relevant study records

Reference

Endpoint conclusion

Additional information

Justification for classification or non-classification

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