2-piperazin-1-ylethylamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

According to OECD 414 guidelines. Contingent on the outcome of the study in the rat.

GLP compliance:

yes

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

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): Aminoethylpiperazine
- Physical state: Clear Colourless Liquid
- Analytical purity: 98%
- Lot/batch No.: 3C27081150
- Expiration date of the lot/batch: 30Mar 2016
- Storage condition of test material: Ambient in the dark

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK
- Age at study initiation: 4-5 months old
- Weight at study initiation: 2.5 and 4 kg.
- Fasting period before study:
- Housing: Females were housed individually in appropriately sized stainless steel cages with a ‘Noryl’ dual level interior and perforated floor. Beneath each cage was a suspended tray containing absorbent paper. Bedding material was provided with a certificate of analysis for significant contaminants. An analytical certificate for each batch of bedding used was retained at Charles River Laboratories, Edinburgh. Animals were allowed a period of exercise in a separate floor pen.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 3 to 5 days before the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17°C to 18°C
- Humidity (%): 35% to 61%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): A 12 hour light/12 hour dark cycle was maintained, except when interrupted for designated procedures.

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Test item dosing formulations were prepared based on a method established at the Test Facility under Study No. 432775 at appropriate concentrations to meet dosage level requirements. The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 4C, and dispensed daily. All formulations were adjusted to pH9 with HCl. The dosing formulations were removed from the refrigerator and were stirred for at least 30 minutes before dosing. The dosing formulation was also stirred continuously during dosing. Details of the preparation and dispensing of the test item have been retained in the Study Records.
- Storage temperature of food: The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 4C, and dispensed daily

VEHICLE
The control item, Milli-Q water, was prepared weekly, stored in a refrigerator set to maintain 4C, and dispensed daily. The prepared control item was removed from the refrigerator and stirred for at least 30 minutes before dosing. The control item was also stirred continuously during dosing. Details of the preparation and dispensing of the control item have been retained in the Study Records.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by Gas Chromatography with Flame Ionisation using a validated analytical procedure .
Concentration and Homogeneity Analysis:
Samples for Analysis: Duplicate top, middle, and bottom samples (duplicate middle only for Groups 1 and 3); sent for analysis as noted in Section 8.6.3 (see Appendix 1 for deviations and other events).
Backup Samples: Triplicate top, middle, and bottom samples (triplicate middle only for Groups 1 and 3); maintained at the Test Facility (see Appendix 1 for deviations and other events).
Sampling Containers: Appropriate sized glass containers.
Sample Volume: Group 1: 0.5 mL (by weight) for analysis and backup samples. Groups 2 to 4: 0.1 mL (by weight) for analysis and backup samples.
Storage Conditions:2-8ºC
Acceptance Criteria: For concentration, the criteria for acceptability were the mean sample concentration results within or equal to ± 10% of theoretical concentration. For homogeneity, the criteria for acceptability will be a relative standard deviation (RSD) of concentrations of £ 10% for each group

Stability Analysis: Stability analyses performed previously in conjunction with Charles River Study No.432775 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study

Duration of treatment / exposure:

Day 6 to Day 28 of gestation

Frequency of treatment:

once daily

No. of animals per sex per dose:

24 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were decided after evaluation of existing toxicity data including a Preliminary Developmental Toxicity Study of Aminoethylpiperazine in the rabbit (Charles River Study Number 497029) where administration at 150 mg/kg/day resulted in slight reductions in food consumption and fetal weight
- Other: At study assignment, each animal was identified using a subcutaneously implanted electronic cylindrical, ‘glass-sealed’ TROVAN microchip.
For each mated female, records were supplied of its parentage (mother and father) and the identity of the inseminating male was also supplied

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, once at the start and once towards the end of the working day throughout the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Day 4 of gestation, then daily from Day 6 to 29 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 4 of gestation, then daily from Day 6 to 29 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was quantitatively measured. In the event that food consumption for an animal was low, hay consumption was monitored to assess the welfare of the animals. In addition, when food consumption was low animals were given some time in an “exercise area”, at the technicians discretion. The full details were recorded and retained within the study data.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #29

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:

Fetal examinations:

- External examinations: Yes: Fetuses were examined for external abnormalities including macroscopic examination of the eyes, cranial bones, brain, nasal passage and tongue following removal of the skin. Late embryonic deaths and dead fetuses were examined for external abnormalities to the extent possible
- Soft tissue examinations: Yes: Prior to fixation, the fetuses were sexed and examined by open dissection for abnormalities of the thoracic and abdominal viscera. For half of the fetuses there was macroscopic examination of the eyes and cranial bones, following removal of the skin from these areas; the cranium was sectioned once through the coronal suture to allow inspection of the brain in that region. For the remaining fetuses, the head was removed from the spine (as close to the head as possible) and placed in Bouins fluid for subsequent serial sectioning and evaluation (see Appendix 1 for deviations and other events).
The internal structure of the heart and kidneys of all fetuses was examined.
The thoracic and abdominal viscera were then discarded and the fetuses were fixed in methylated ethyl alcohol
- Skeletal examinations: Yes: All of the eviscerated carcasses were then macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, then the fetuses cleared with aqueous glycerol solutions. All the preparations were then examined for the presence of skeletal abnormalities and for the extent of ossification.
- Head examinations: Yes:The heads fixed in Bouins fluid were examined for soft tissue abnormalities using a free hand sectioning technique derived from that of Wilson et al. 1965.

Statistics:

Means and standard deviations were calculated for body weight, food consumption, pregnancy data and fetal weights and group incidence data was calculated for fetal abnormalities and variations.
All statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Body weight and food consumption, data were analysed for homogeneity of variance using the ‘F Max' test. If the group variances appear homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F test is significant. If the variances are heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remain heterogeneous, then a Kruskal-Wallis non-parametric ANOVA were used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical Observations: Administration at 150 mg/kg/day was associated with intermittent abnormal faecal output in 5/24 animals including small, decreased, abnormal colour and liquid faeces. Periods of decreased or small faeces in individual animals in the 150 mg/kg/day group were associated with extended periods of greatly reduced food consumption). Also at this dose level one animal was found to have red staining of the cage tray paper over Days 26-27 of gestation.
At dose levels up to and including 75 mg/kg/day there were no clinical observations that were considered to be test item-related. Minor observations such as sparse hair, fur staining, scab(s) etc were found at a similar incidence throughout the dose groups including controls, therefore, were considered to be incidental and not related to test item administration.

Body Weights Body Weight Changes: Administration at 150 mg/kg/day was associated with a slight reduction in group mean body weight gain throughout the dosing period (-17%), when compared to controls. When the body weight gain over Days 6-29 of gestation is adjusted for gravid uterine weight all groups show a body weight loss; however, it is noted that the group mean body weight loss at 150 mg/kg/day is slightly higher than controls.
The group mean body weight gain at dose levels up to and including 75 mg/kg/day was similar to control throughout the dosing period.

Food Consumption: Administration at 150 mg/kg/day was associated with a reduction in food consumption throughout the majority of the dosing period, when compared to controls. This was most notable over Days 7-21 of gestation where the daily group mean food consumption was found to be reduced by up to -38%.
The group mean food consumption at dose levels up to and including 75 mg/kg/day was similar to control throughout the dosing period.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal Abnormalities and Variants: When compared to controls, administration at 150 mg/kg/day was associated with an increase in the number of fetuses with unossified/incompletely ossified bones including skull bone(s), hyoid, odontoid process, cervical centrum, pubis(es), epiphyses, metacarpals and phalanges. The incidence of all other skeletal abnormalities and variations were similar to controls in all dose groups, including the number of ribs.
At dose levels up to and including 150 mg/kg/day there were no effects on the number or type of fetal abnormalities or variations, with the exception of at 150 mg/kg/day where there was a slight increase in fetuses noted as being small.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Pregnancy Performance, Gravid Uterine and Fetal Weights:

Administration at 150 mg/kg/day was associated with an increase in dead implants (early deaths, late deaths and dead fetuses) with a corresponding reduction in live implants, when compared to total implants and controls. The percentage of dead implants increased from 8% in controls to 20% at 150 mg/kg/day, resulting in an increase in post-implantation loss at this dose level. A slight decrease in fetal weight and gravid uterine weight was also noted at this dose level, with a 7% and 11% reduction in the mean litter mean fetal weight and mean gravid uterine weight, respectively, compared to controls. 

There were no similar test item-related effects on implant survival, fetal or gravid uterine weights at dose levels up to and including 75 mg/kg/day.

At dose levels up to and including 150 mg/kg/day there were no test item-related effects on pregnancy frequency, number of corpora lutea and implants or the pre-implantation loss. 

Applicant's summary and conclusion

Conclusions:

The reduced body weight gain observed in animals administered Aminoethylpiperazine at 150 mg/kg/day corresponded with a reduction in food consumption throughout the majority of the dosing period along with an associated delay in fetal development, as observed by the reduced fetal weights and decreased bone ossification at this dose level.
Also at 150 mg/kg/day, the decrease in live implants correlated with an increase in dead implants on Day 29 of gestation and of the surviving fetuses, several fetuses were noted as being small at necropsy. With the exception of the decreased embryofetal survival, delayed ossification and reduced fetal weight, there were no other fetal abnormalities and variations that could be positively associated with treatment at this dose level.

Executive summary:

The objective of this study was to detect effects ofAminoethylpiperazineon embryofetal development in the rabbit following oral administration of the test item from Day 6 to 28 of gestation. The study also provided information on any maternal toxicity.

The study design was as follows:

Text Table 1: Experimental design

Group No.	Test Item	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	No. of Animals (Females
1	Control	0	5	0	24
2	AEP	25	5	5	24
3	AEP	75	5	15	24
4	AEP	150	5	30	24

Vehicle/control was Milli-Q water.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight changes, food consumption, gross necropsy findings, fetal weights and examinations and gravid uterus weights.

Administration ofAminoethylpiperazine at 150 mg/kg/day was associated with abnormal faecal output (small, decreased, abnormal colour and liquid faeces), reductions in food consumption and body weight gain along with a decrease in embryofetal survival, decrease in fetal and gravid uterine weight and a decrease in fetal bone ossification. 

At dose levels up to 75 mg/kg/day there were no treatment related effects noted in any of the parameters examined including clinical observations, body weight gain, food consumption, gross pathology findings, pregnancy performance parameters, fetal and gravid uterine weights, fetal abnormalities and variants.

In conclusion, under the conditions of this study, the maternal and fetal no observed adverse effect level (NOAEL) and no observed effect level (NOEL) were considered to be 75 mg/kg/day.