2-piperazin-1-ylethylamine

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Evaluation of the genotoxic potential of alkyleneamines.

Author:

Leung, H.W.

Year:

1994

Bibliographic source:

Mutation Research 320:31-43.

Report date:

1994

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Reference 3

Reference Type:

publication

Title:

No information

Author:

Slesinski, R.S., et al.

Year:

1984

Bibliographic source:

The Toxicologist 4:35

Report date:

1984

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

2-piperazin-1-ylethylamine

EC Number:

205-411-0

EC Name:

2-piperazin-1-ylethylamine

Cas Number:

140-31-8

Molecular formula:

C6H15N3

IUPAC Name:

2-piperazin-1-ylethanamine

Details on test material:

Purity of the test material was not reported. Solutions of the test material were pH adjusted prior to dosing animals.

Test animals

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.

Administration / exposure

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

Doses / concentrations

Remarks:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylenemelamine

Examinations

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.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:

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. In this study, no clastogenic effect was observed in the highest dose examined.

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.