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Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 October - 25 November 1986
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
According to OECD 474 - Limit test - section 34: When administration occurs for 14 days or more, the limit dose is 1000 mg/kg body weight/day. For administration periods of less than 14 days, the limit dose is 2000 mg/kg/body weight/day.
This test carried out in 1986 used 5000 bw as limit dose.
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
Piperazine phosphate CAS 1951-97-9 / EC 217-775-8 was used.
Piperazine phosphate (also referred to as ASCAREX P) was a white crystalline powder, batch WS 84/023.
The sample was 99.6% pure.
It was received on 25th April, 1986 and stored at room temperature in the dark.

Test animals

Details on species / strain selection:
Mice (and rats) are the preferred species for this test.
Details on test animals or test system and environmental conditions:
More than 35 male and 35 female out—bred CD-1 mice aged approximately 4 weeks, were obtained from Charles River (UK) Ltd., Margate, Kent. They were housed in groups of no more than 5 animals of the same sex in polypropylene cages with solid floors and wire mesh lids. Wood shavings to a depth of approximately one half inch were included in each cage. Bottled mains tapwater and B&K certified rat and mouse diet were provided ad libitum. Neither of these were known to contain any biological or chemical entity which might interfere with the conduct of the study.
After a short period of acclimatisation 35 healthy male and 35 healthy female mice were ear-tagged sequentially and randomised, using a table of random numbers, to groups of S with approximately equal group weights.
At the time of treatment the age of the mice was approximately 5 weeks. The weight range for males was 21 — 27 g and for females 20 - 24 g.

All cages were washed in detergent, rinsed and dried before use. The room in which the cages were placed is designed to maintain a temperature of 22-23 deg C and a relative humidity of 50+/-10%. This room is also designed to be illuminated by fluorescent light for 12 hours out of each 24 hour cycle and to receive at least 15 fresh air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Carboxymethylcellulose in distilled water
Details on exposure:
The vehicle in the study was 1% (w/v) carboxymethyl cellulose made up in distilled water. Piperazine phosphate was suspended in this at a concentration of 200 mg/ml. For the range finder, dilutions of 180 and 160 mg/ml were made from this stock concentration. Periodically during dosing, the suspensions were gently inverted.
Cyclophosphamide (CPA) was dissolved in water at 3.2 mg/ml to serve as the positive control. All solutions/suspensions were freshly prepared for use.

In an initial range finding study, groups of 2 male and 2 female mice were treated with the 3 concentrations of piperazine phosphate at a dose volume of 25 ml/kg giving doses of 5000, 4500 and 4000 mg/kg. One male mouse treated with 4000 mg/kg was found dead after three days. Why this occurred when higher doses were survived is not clear. Survival at the higher doses was considered justification for choosing 5000 mg/kg as the treatment for the main study.

Animals were weighed before dosing and the volume of vehicle, test agent or positive control solution to be administered was calculated based on a dose volume of 25 ml/kg. Fifteen male and 15 female mice each received piperazine phosphate orally at 5000 mg/kg, and 15 male and 15 female mice received 1% (w/v) carboxymethyl cellulose as the negative control, also orally at the same dose volume. Five male and 5 female mice received cyclophosphamide (CPA) orally at 80 mg/kg (3.2 mg/ml and a dose volume of 25 ml/kg).
Duration of treatment / exposure:
Single oral dose
Frequency of treatment:
Single oral dose
Post exposure period:
Test article and vehicle treated mice were killed in groups of 5 males and 5 females after 24, 48 and 72 hours, and CPA-treated mice were killed after 48 hours. Mice were killed by asphyxiation with carbon dioxide following the same sequence used for dosing.
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 mice /sex per sacrifice time point
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide oral administration 80 mg/kg to 5 male and 5 female mice.


Tissues and cell types examined:
One femur from each animal was exposed, removed, cleaned of adherent muscle and connective tissue so that the shank could be removed from the ends by using fine clippers or scissors. Disposable centrifuge tubes containing 1 ml of foetal bovine serum (PBS) were labelled with the animal numbers. Some of the PBS was drawn into a 1 ml syringe with a fresh needle, and this was used to aspirate the contents of the femur into the centrifuge tube.

Details of tissue and slide preparation:
The tubes were centrifuged at 1250 x 'g' for 2-3 minutes, the serum was carefully decanted to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of h labelled slides. The end of a clean slide was placed across the drop, and a smear made by drawing the clean slide along the labelled slide.
Slides were allowed to air-dry and were stored for at least 48 hours before staining according to the modification of Gollapudi and Kamra). Two slides from each set of four were taken (the others were kept in reserve in case they were needed) and fixed for 5 minutes in absolute methanol. After rinsing several times in water, the slides were then stained for 10 minutes in Gurr's Giemsa R66 stain diluted 1:6 (v/v) in distilled water. Slides were rinsed, and allowed to dry thoroughly before clearing in Xylene for 3 minutes. When dry, slides were finally mounted with coverslips.
Evaluation criteria:
The slides from each kill time (excluding the positive controls at 48 hours) were grouped according to sex and handed to a person not connected with the scoring of the slides to be coded. Sticky labels were used to cover any identification marks on the slides so that the cytogeneticists could only see the study reference number, the time of killing, the sex of the mouse and the code.
Starting at one corner of the slide, and following a ziz-zag pattern which avoids crossing the same area more than once, a series of random fields were observed under high power. Initially the relative proportions of poly-chromatic erythrocytes (PCE), seen as pale blue or blue/grey enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller orange-stained enucleate cells, were determined using tally counters on a total of approximately 400 cells counted. Any PCE or NCE containing micronuclei were recorded on raw data sheets. For each animal, counting continued until 500 PCE had been totalled in CPA-treated animals, and 1000 PCE had been totalled in all other animals.
The frequency of micronucleated PCE was readily determined from these counts. The frequency of micronucleated NCE was determined by calculating the number of thousands of NCE that would have been observed for a given number of PCB counted, and dividing the number of micronucleated NCE actually counted by this number.
In order to calculate the mean PCE/NCE ratio per group, the number of NCE that would have been observed per 1000 PCE scored is calculated for each animal. The values for the individual animals in each group are then totalled and 5000 PCE (total scored per group) divided by this number as follows: Mean ratio PCE/NCE = Total PCE counted for 5 animals / Estimate of total NCE counted for 5 animals
To evaluate the results statistically, numbers of micronucleated PCE and NCE were compared between groups of piperazine phosphate or CPA-treated mice and vehicle controls by using the formula:
chi-squared = Sigma ((O-E) - 0.5)^2 / E
where 0 = observed total number of micronucleated cells per total counted
and E = expected total number of micronucleated cells per total counted.

Results and discussion

Test results
Vehicle controls validity:
Positive controls validity:
Additional information on results:
Mice treated with the positive control chemical CPA generally exhibited much lower PCE/NCE ratios indicating bone marrow toxicity. In all of the CPA-treated mice, numbers of micronucleated PCE clearly exceeded those seen in vehicle controls, such that the mean frequency (34.0/1000) was approximately 24x the mean frequency in the concurrent control. Chi-squared analysis on the total counts revealed the increased numbers of micronuclei to be significant.
One male treated 5000 mg/kg with piperazine phosphate died before sampling was possible. This observation, which is consistent with one death at 4000 mg/kg in the range-finder, indicated that it would probably not have been practical to administer the test agent at a higher dose. This small reduction in the number of animals available for analysis does not affect the validity of the assay.
Mice treated with piperazine phosphate exhibited PCE/NCE ratios which were similar to vehicle controls at all sampling times. The numbers of micronucleated PCB and NCE were also similar to those seen in controls and were not significantly different by
chi-squared analysis.

Applicant's summary and conclusion

It is concluded that piperazine phosphate was not able to induce micronuclei in the polychromatic or normochromatic erythrocytes of the bone marrow of mice when administered at 5000 mg/kg, a dose at which lethality occurred.
Executive summary:

Piperazine phosphate was assayed in an in vivo micronucleus test at a dose of 5000 mg/kg, in groups of 5 male and 5 female mice killed 24, 48 and 72 hours after treatment.

In an initial toxicity range-finder study the test agent, suspended in 1% (w/v) carboxymethyl cellulose, was administered to mice orally. Two male and 2 female mice each received the test article at a dose of 5000, 4500 and 4000 mg/kg. No lethality was observed at 5000 mg/kg. For the micronucleus test piperazine phosphate was suspended as described and administered at this dose.

The negative control in the study was 1% (w/v) carboxymethyl cellulose made up in distilled water and also administered orally. Groups of 5 male and 5 female mice treated with this suspension were killed and sampled after 24, 48 and 72 hours. Cyclophosphamide (CPA), dissolved in water was administered orally at 80 mg/kg to one group of 5 male and 5 female mice which were killed after 48 hours.

In general, positive control animals exhibited toxicity in the bone marrow as seen by an increased proportion of normochromatic erythrocytes (NCE), and increased numbers of micronucleated polychromatic erythrocytes (PCE) and NCE such that the micronucleus frequency in the positive control group was significantly greater than in controls (p < 0.001).

Negative control mice exhibited normal ratios of PCB to NCE with group means for males and females ranging from 0.9 to 1.59, and normal frequencies of micronucleated PCE (mean 1.2 - 2.8/1000) and NCE (range 0.32 - 1.8/1000). Mice treated with piperazine phosphate exhibited ratios of PCB to NCE and frequencies of micronucleated PCB and NCE which were similar to controls. Group mean PCEzNCE ratios ranged from 1.16 to 2.04; mean frequencies of micronucleated PCE were 0.8 — 2.8 per 1000 and of micronucleated NCE, 0.9 - 2.85. There were no instances of statistically significant, treatment-related increases in micronucleus frequency for any of the animals receiving piperazine phosphate at any sampling time.

It is concluded that piperazine phosphate was not able to induce micronuclei in the polychromatic or normochromatic erythrocytes of the bone marrow of mice when administered at 5000 mg/kg, a dose at which lethality occurred.