2-piperazin-1-ylethylamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Aminoethylpiperazine (AEP)

IUPAC Name:

Aminoethylpiperazine (AEP)

Details on test material:

- Name of test material (as cited in study report): Aminoethylpiperazine (AEP)
- Physical state: liquid
- Analytical purity:98.4% by gas chromatography
- Impurities (identity and concentrations):0.051% water by Karl Fischer coulometric titration, and 0.76% diethylenetriamine (DETA) and 0.09% aminoethylethanolamine (AEEA) by gas chromatography.
- Composition of test material, percentage of components:
- Purity test date: 7 July 2009
- Lot/batch No.:XG03081150
- Stability under test conditions:The test substance is considered to be stable under the storage conditions.
- Storage condition of test material:Ambient

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Laboratories, Inc.
- Age at study initiation: 91-101 days
- Weight at study initiation: Males 300-500 gm; Females 200-300 gm
- Housing:All animals were individually housed in clean suspended wire mesh cages in an environmentally controlled room during the acclimation period and continuing until mating. Following successful mating, the females were housed individually in a plastic cage containing ground
corncob nesting material (Bed O'Cobs) and remained in these cages until euthanasia on lactation day 4.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Actual mean daily temperature ranged from 70.5°F to 71.4°F (21.4°C to 21.9°C).
- Humidity (%): Mean daily relative humidity ranged from 38.2% to 52.0% during the study.
- Air changes (per hr):10 room air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark photoperiod

IN-LIFE DATES: From: 5 March 2010 To: 27 April 2010

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

VEHICLE: Water
The test substance was administered as a constant concentration (mg/ml) in reverse osmosis-treated drinking water.

The test item formulations were prepared approximately weekly as single formulations for each dosage level; the pH of each formulation was adjusted to 9.0 ± 0.1 with 1 N HCl (prepared using 37% hydrochloric acid, NF; lot nos. YT0470 and YW0968, exp. date: 5 June 2012 and 30 November 2012, respectively, received from Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ). The test item formulations were transferred into 10-L plastic carboys for administration and stored at room temperature. The test item formulations were stirred continuously throughout the preparation and sampling.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical concentrations were within 98.6 to 110% of target doses.

The analyzed drinking water formulations were stable for 14 days of room temperature storage.

Duration of treatment / exposure:

Males/Females: At least 28-Days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 2000 or 8000 ppm
Basis:
nominal in water

No. of animals per sex per dose:

12

Control animals:

yes

Details on study design:

- Dose selection rationale:Due the steep dose response curve (in the range-finding/palatability study) between 10000 ppm, which exceeded the
maximum tolerated dose, and 7500 ppm which resulted in a slight, transient reduction in body weights, food consumption, and water consumption, the high-dose level of 8000 ppm for the definitive study was chosen.

Males: Individual body weights were recorded twice weekly, beginning one week prior to test substance administration, on the first day of dosing and twice weekly thereafter until euthanasia. Individual food consumption and water consumption were recorded twice weekly, beginning one week prior to test substance administration, on the first day of dosing and twice weekly thereafter until animals were paired for breeding. Following evidence of mating, males continued to have individual food consumption and water consumption recorded twice weekly thereafter until euthanasia.

Females: Individual body weights were recorded twice weekly, beginning one week prior to test substance administration, on the first day of dosing and twice weekly thereafter until evidence of copulation was observed. Individual food consumption and water consumption were recorded twice weekly, beginning one week prior to test substance administration, on the first day of dosing and twice weekly thereafter until animals were paired for breeding. Females with no evidence of mating had body weights, food consumption, and water consumption recorded twice weekly upon completion of the breeding period through euthanasia.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
A detailed physical examination was conducted weekly on each animal and on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly

FOOD CONSUMPTION:
- Time schedule for examinations: Twice weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Individual water consumption were recorded twice weekly, beginning one week prior to test substance administration, on the first day of dosing and twice weekly thereafter until animals were paired for breeding.

OPHTHALMOSCOPIC EXAMINATION: As part of the Functional Observation Battery which included a pupil response

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the scheduled necropsies (study day 28, males and lactation day 4, females).
- Anaesthetic used for blood collection: Yes - isoflurane
- Animals fasted: All males were fasted overnight prior to blood collection.
- How many animals:All animals (with the exception of the female that failed to deliver)
- Blood for hematology was collected from the retro-orbital sinus following isoflurane anesthesia.
- Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing EDTA (hematology) or sodium citrate (clotting determinations).
- Parameters examined
Total leukocyte count (WBC)
Erythrocyte count (RBC)
Hemoglobin (HGB)
Hematocrit (HCT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelet count (PLATELET)
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)
Reticulocyte count Percent (RETIC)
Absolute (RETIC ABSOLUTE)
Mean Platelet Volume (MPV)
Red cell distribution width (RDW)
Hemoglobin Distribution Width (HDW)
Differential leukocyte count –
Percent and absolute
-Neutrophil (NEU)
-Lymphocyte (LYMPH)
-Monocyte (MONO)
-Eosinophil (EOS)
-Basophil (BASO)
-Large unstained cell (LUC)
Platelet estimate
Red cell morphology (RBC Morphology)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the scheduled necropsies (study day 28, males and lactation day 4, females).
- Animals fasted: All males were fasted overnight prior to blood collection.
- How many animals:All animals (with the exception of the female that failed to deliver)
- Blood for serum chemistry was collected from the retro-orbital sinus following isoflurane anesthesia.
- Blood was collected into tubes containing no anticoagulant (serum chemistry).
- Parameters examined
Albumin
Total protein
Globulin [by calculation]
Albumin/globulin ratio (A/G Ratio) [by calculation]
Total bilirubin (Total Bili)
Urea nitrogen
Creatinine
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma glutamyltransferase (GGT)
Glucose
Total cholesterol (Cholesterol)
Calcium
Chloride
Phosphorus
Potassium
Sodium
Triglycerides (Triglyceride)
Bile acids

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:Females:Prior to dosing and on lactation day 4. Males:Prior to dosing and following approximately 28 days of dose administration
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity / body temperature

OTHER:

Sacrifice and pathology:

UNSCHEDULED DEATHS
Gross necropsies were performed on the males and females that were euthanized (by carbon dioxide inhalation) in extremis or found dead during the course of the study. A gross necropsy was performed. The animals were then discarded.

SCHEDULED EUTHANASIA
All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period. Females that delivered were euthanized on lactation day 4; the numbers of former implantation sites and corpora lutea were recorded. Females that failed to deliver were euthanized on post-cohabitation day 25 (females with no evidence of mating). Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964). Necropsy included examination of the external surface, all orifices and the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.

At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin (except as noted):
Adrenal glands (2)
Aorta Axillary
Bone with marrow (sternebrae)
Bone marrow smear
Brain
Cerebrum level 1
Cerebrum level 2
Cerebellum with medulla/pons
Coagulating glands
Eyes with optic nerve (2)b
Gastrointestinal tract
- Esophagus
- Stomach
- Duodenum
- Jejunum
- Ileum
- Cecum
- Colon
- Rectum
Heart
Kidneys (2)
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed by inflation with fixative)
Lymph node
- Axillary
- Mesenteric
- Mandibular
Ovaries and oviducts (2)
Pancreas
Peripheral nerve (sciatic)
Pituitary gland
Prostate gland
Salivary gland [mandibular (2)]
Seminal vesicles (2)
Skeletal muscle (rectus femoris)
Skin with mammary glandc
Spinal cord (cervical)
Spleen
Testes with epididymidesd (2)
Thymus gland
Thyroids [with parathyroids, if present (2)]
Trachea
Urinary bladder
Uterus with vagina
All gross lesions (all groups)

The following organs were weighed from all F0 animals at the scheduled necropsies:
Adrenal glands
Brain
Epididymidesa
Heart
Kidneys
Liver
Ovaries with oviducts
Spleen
Testes
Thymus gland
Thyroids with parathyroids

MICROSCOPIC EXAMINATIONS
After fixation, protocol-specified tissues were trimmed. Trimmed tissues were processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides, and stained with hematoxylin and eosin, with the exception of the testes and epididymides which were stained with PAS and hematoxylin to allow for a detailed histopathological examination.

Microscopic examination was performed on all tissues listed from all animals in the control and 8000 ppm groups at the scheduled necropsies and from the males and females that died or were euthanized in extremis. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section, or other reasons as appropriate.

Other examinations:

LITTER VIABILITY AND DEATHS
Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring dying were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. The carcass of each pup was then discarded.

CLINICAL OBSERVATIONS
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nursing behavior were recorded.

BODY WEIGHTS
Pups were individually weighed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by dose group. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods a given pup was not weighed were left blank or designated as “NA” on the individual
report tables.

SEX DETERMINATION
Pups were individually sexed on PND 0 and 4.

CALCULATION OF LITTER PARAMETERS
Litter parameters were defined as follows:
Mean Live Litter Size = Total No. of Viable Pups on PND 0/No. of Litters with Viable Pups PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) =
Sum of (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter)/No. of Litters Per Group x 100

Postnatal Survival for All Other Intervals (% Per Litter) =
Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)/No. of Litters Per Group x 100

Where N = PND 0-1 and 1-4

SCHEDULED EUTHANASIA
On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded.

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.

Analyses
were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group by sex.

Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Mean parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites and corpora lutea, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relative organ weights, clinical pathology values (except gamma glutamyltransferase), and pre-coital intervals were
subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences between the control and test item-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett’s test (Dunnett, 1964) was used to compare the test item-treated groups to the control group. FOB parameters (sensory observations) that yield scalar or descriptive data and histopathological findings in the test item-treated groups were compared to the control group using Fisher’s Exact test (Steel and Torrie, 1980).

Continued below

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Increased mortaility in 8000 ppm group

Mortality:

mortality observed, treatment-related

Description (incidence):

Increased mortaility in 8000 ppm group

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean male body weight in 8000 ppm group was up to 11.0% lower than the control group during the treatment period. Lower mean body weight gain was noted during the first week of treatment (study days 0-6) in female rats in 8000 ppm group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 8000 ppm male and female rats reduced feed consumption was observed.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Lower mean water consumption observed in the high dose group.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Only examined as part of FOB

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

OTHER FINDINGS: In a previous range-finding/palatability study (WIL-768001, Kuhl, Draft), groups of three male and female Crl:CD(SD) rats were administered 0, 750, 1875, 3750, 7500,10000, or 15000 ppm AEP in pH 9.0 drinking water for 14 days. The 15000 and 10000 ppm groups exceeded the maximum tolerated dose after seven days of treatment, as evidenced by significant body weight losses (~20-30%) that was driven by feed and water consumption of less than 5g/animal/day. Based on these findings, animals in these dose groups were euthanized for humane reasons on study day (SD) 7. Animals in the 7500 ppm group had slight, but not statistically identified, effects on body weightlbody weight gain that were likely driven by lower feed and water consumption upon initiation of treatment (SD 1-4). This effect was transient and recovered by SD 7. There were no effects at concentrations of 3750 ppm AEP or lower.

F0 GENERATION
CLINICAL OBSERVATIONS AND SURVIVAL
In the 8000 ppm group, 5 males (nos. 67248, 67254, 67256, 67258, and 67273) and 1 female (no. 67217) were euthanized in extremis between study days 7-13. The moribundity of these animals occurred following body weight losses (46 g to 122 g) with reduced food (≤19 g/day) and water consumption (≤22 g/day) from study day 0 through the day of death/euthanasia. Clinical findings noted for these animals on the days prior to
or on the day of death/euthanasia were limited to decreased defecation, red material around the eye or nose, and/or hair loss on the forelimb. In addition, 1 female (no. 67216) was found dead on study day 20. Upon microscopic examination, the cause of death for this female was determined to be hydrocephalus, which was presumed to be an incidental finding that was unrelated to administration of the test item. The cause of moribundity could not be determined microscopically for any the animals euthanized in extremis because no significant internal findings were observed. However, the moribundity of these animals occurred in the presence of test item-related reductions in body weight and food and water consumption noted at this same dosage level, and therefore was considered to be test item-related. All other animals survived to the scheduled necropsies.

For animals that survived to the scheduled necropsy, clinical findings were limited to hair loss on the limbs. These findings occurred infrequently and/or at similar frequencies in the control group, and were not attributed to test item administration.

BODY WEIGHTS
MALES
Test item-related mean body weight losses were noted in the 8000 ppm group males during study days 0-10 followed by a lower mean body weight gain during study days 10-13; differences from the control group were significant (p<0.01) during study days 0-3 and 3-6. Mean body weights in the 8000 ppm group were up to 11.0% lower compared to the control group during study days 0-13; differences from the control group were significant (p<0.05) on study day 6. These body weight effects were primarily due to the 5 males in this group euthanized in extremis during study days 7-13. Mean body weights and body weight gains for the surviving males were comparable to the control group during the remainder of the treatment period (study days 13-31). Due to the initial mean body weight losses and lower mean body weight gain in the 8000 ppm group, a significantly (p<0.05) lower mean body weight gain was noted for the overall pre-mating period (study days 0-13) and a lower (not statistically significant) mean body weight gain was noted when the entire generation (study days 0-31) was evaluated compared to the control group.

Mean male body weights and body weight gains in the 500 and 2000 ppm groups were similar to the control group throughout the treatment period. Differences from the control group were slight and not statistically significant.

FEMALES
PRE-MATING
A test item-related, significant (p<0.01) mean body weight loss followed by an absence of body weight gain were noted in the 8000 ppm group during the first week of the treatment period (study days 0-6). As a result, mean body weight in this group was 5.0% lower (not statistically significant) compared to the control group on study day 6. During the remainder of the pre-mating period (study days 6-13), mean body weight gain in the 8000 ppm group was similar to the control group. The mean body weight loss and absence of body weight gain noted in the 8000 ppm group during the first week of treatment were of sufficient magnitude to result in a significantly (p<0.05) lower mean body weight gain when the entire pre-mating period (study days 0-13) was evaluated, and thus were considered to be adverse.

Mean female body weights and body weight gains in the 500 and 2000 ppm groups were similar to the control group during the pre-mating period. Differences from the control group were slight and not statistically significant.

GESTATION
Slightly lower (not statistically significant) mean body weight gains were noted in the 2000 and 8000 ppm groups generally throughout gestation compared to the control group. As a result, significantly (p<0.05 or p<0.01) lower mean body weight gains were noted in these groups when the overall gestation treatment period (gestation day 0-20) was evaluated, and mean body weight in the 8000 ppm group was 5.8% lower (not statistically significant) than the control group on gestation day 20. Conversely, the lower mean body weight gains noted in the 2000 ppm group were not of sufficient magnitude to affect mean body weight, and therefore were not considered to be test item-related.

Mean body weights and body weight gains in the 500 ppm group were generally similar to those in the control group throughout gestation. Differences from the control group were slight and not statistically significant.

LACTATION
Mean maternal body weight gains were unaffected by test item administration during lactation days 1-4. However, mean body weights in the 8000 ppm group were up to 6.6% lower (not statistically significant) than the control group during lactation days 1-4 as a result of the lower mean body weights noted in this group during the pre-mating period and gestation.

Mean body weights in the 500 and 2000 ppm groups were unaffected by test item administration during lactation days 1-4. Differences from the control group were slight and not statistically significant.

FOOD CONSUMPTION
MALES
Test item-related lower mean male food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 8000 ppm group during the pre-mating period (study days 0-13); differences from the control group were significant (p<0.05 or p<0.01). The lower mean food consumption corresponded to the overall lower mean body weight gain noted in this group during the pre-mating period and was primarily due to the 5 males in this group euthanized in extremis during study days 7-13. Mean food consumption in this group was similar to the control group during study days 27-30.

Mean male food consumption in the 500 and 2000 ppm groups was similar to the control group during the pre-mating period (study days 0-13). Differences from the control group were slight and not statistically significant.

FEMALES
PRE-MATING
Test item-related lower mean female food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 8000 ppm group during the first week of treatment (study days 0-6); differences from the control group were significant (p<0.01) and corresponded to a period of mean body weight loss. Mean food consumption in this group was similar to the control group during study days 6-13.

Mean food consumption in the 500 and 2000 ppm groups was unaffected by test item administration during the pre-mating period (study days 0-13). Prior to the initiation of treatment (study days -7 to -3), significantly (p<0.01) higher mean food consumption was noted in the 2000 ppm group. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

GESTATION
Mean food consumption in the 500, 2000, and 8000 ppm groups was unaffected by test item administration during gestation. Significantly (p<0.05) higher mean food consumption (g/animal/day value only) was noted in the 500 ppm group during gestation days 0-4. However, in the absence of an exposure-related response, the increased mean food consumption was not considered to be treatment-related. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

LACTATION
Mean food consumption in the 500, 2000, and 8000 ppm groups was unaffected by test item administration during lactation days 1-4. Differences from the control group were slight and not statistically significant.

WATER CONSUMPTION
MALES
Mean water consumption, evaluated as g/animal/day and g/kg/day, in the 8000 ppm group males was lower than the control group during study days 0-10. Differences from the control group were generally significant (p<0.01) and corresponded to a test item-related lower mean body weight gain noted during the same interval. In addition, a significant (p<0.01) decrease in mean water consumption was noted in the 8000 ppm group during study days 27-31.

Mean water consumption in the 500 and 2000 ppm group was similar to that in the control group throughout the study. Lower (p<0.05) mean water consumption was also noted in the 2000 ppm group compared to the control group during study days 27-31. Due to the lack of a concurrent effect on mean body weight gain during this interval, the decreased mean water consumption in this group was not considered to be test item-related. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

FEMALES
PRE-MATING
Lower mean water consumption, evaluated as g/animal/day and g/kg/day, was noted in the 8000 ppm group during study days 0-10; differences from the control group were significant (p<0.01) during study days 0-3 and corresponded to a test-item-related mean body weight loss during the first week of dose administration. Mean water consumption in this group was similar to the control group during study days 10-13.

Mean water consumption in the 500 and 2000 ppm groups was unaffected by test item administration during the pre-mating period (study days 0-13). Increased mean water consumption was noted in both groups during study days 10-13; differences from the control group were generally significant (p<0.05 or p<0.01). However, in the absence of a similar effect in the high-dose group, the increased mean water consumption noted in
the 500 and 2000 ppm group was not considered to be test item-related. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

GESTATION
Mean maternal water consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test item administration during gestation. Differences between the control, 500, 2000, and 8000 ppm groups were slight and not statistically significant.

LACTATION
Mean maternal water consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test item administration during lactation days 1-4. Differences between the control, 500, 2000, and 8000 ppm groups were slight and not statistically significant.

TEST ITEM CONSUMPTION
The average quantities of aminoethylpiperazine consumed during the F0 generation are presented below (Table 1). Values for the entire lactation period in all groups were elevated, as is commonly seen in nursing animals.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
SENSORY OBSERVATIONS
Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study day 27 (males) or on lactation day 4 (females).

NEUROMUSCULAR OBSERVATIONS
Neuromuscular parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study day 27 (males) or on lactation day 4 (females).

PHYSIOLOGICAL OBSERVATIONS
A lower (6.7%) mean body weight was noted for the 8000 ppm group females compared to the control group at the physiological observations on lactation day 4; the difference from the control group was significant (p=0.002). The lower mean body weight corresponded to the lower mean body weight recorded on lactation day 4. Mean body temperature was unaffected by test item administration at all dosage levels. There were no other statistically significant differences for the test item-treated groups when compared to the control group on study day 27 (males) or lactation day 4 (females).

LOCOMOTOR ACTIVITY
Locomotor activity patterns (total activity) in F0 animals were unaffected by test item administration at all concentrations when evaluated on study day 27 (males) and lactation day 4 (females). Values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values with the following exceptions. Mean total activity counts for females in the 500 and 8000 ppm groups at the lactation day 4 evaluation were higher for the 6 subintervals as well as the overall 60-minute test session; differences from the control group achieved significance (p≤0.003) for these groups when the overall 60-minute test session was evaluated for total motor activity counts by a repeated measures analysis. However, no dose-related trend was apparent and the increase in motor activity was primarily attributed to individual females in the 500 and 8000 ppm groups with atypically high total motor activity values. In addition, mean total motor activity counts in the 500 and 8000 ppm groups on lactation day 4 were generally similar to pretest values during the first subinterval (0-10 minutes) and differences between the pretest and lactation evaluations were limited to greater habituation on lactation day 4. Therefore, the increased mean total activity counts for females in the 500 and 8000 ppm groups were not considered to be test item-related.

No remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated at study day 27 (males) and lactation day 4 (females).

CLINICAL PATHOLOGY
HEMATOLOGY
There were no test item-related alterations in hematology and coagulation parameters. A significantly (p<0.05) lower mean corpuscular hemoglobin (MCH) value was noted in the 2000 ppm group males. This group mean difference was not considered to be test item-related because the value did not show a dose- or time-related response. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

SERUM CHEMISTRY
There were no test item-related effects on serum chemistry parameters. Significantly (p<0.05) higher mean glucose and triglyceride values were noted in the 500 ppm group males and females, respectively. These group mean differences were not considered to be test item-related because the values did not show a dose- or time-related response. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

REPRODUCTIVE PERFORMANCE
F0 male and female reproductive parameters are presented in Table 2.

No test item-related effects on reproductive performance were observed at any exposure level. No statistically significant differences were noted between the control and test item-treated groups. One mating pair in the 8000 ppm group did not produce a litter. The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. None of these differences were statistically significant.

GESTATION LENGTH AND PARTURITION
Mean gestation lengths in the 500, 2000, and 8000 ppm groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.

ANATOMIC PATHOLOGY
MACROSCOPIC EXAMINATIONS
In the 8000 ppm group, 5 males and 1 female were euthanized in extremis between study days 7-13, and 1 female was found dead study day 20. All other animals survived to the scheduled necropsies. No test item-related internal findings were observed at any dosage level in males and females that died, were euthanized in extremis, failed to deliver, or at the scheduled necropsies. Macroscopic findings observed in the test item-treated groups, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related. The mean numbers of unaccounted-for sites, implantation sites, and corpora lutea in the 500, 2000, and 8000 ppm groups were similar to the control group values.

ORGAN WEIGHTS
The mean final body weight for the 8000 ppm group females was 6.4% lower (not statistically significant) than the control group females. This lower body weight was considered an adverse test item-related effect. A significantly (p<0.05) higher mean relative (to body weight) thyroid/parathyroid weight was noted in the 8000 ppm group females when compared to the control group; however, this change was attributed to the test item-related decrease in mean body weight and was not considered to be a direct effect of the test item. In addition, lower mean absolute and relative (to body and brain weight) spleen weights were noted in the 8000 ppm group females compared to the control group. The differences were significant (p<0.05 or p<0.01), but the splenic weight differences were not considered test item-related given the lack of microscopic changes consistent with cell loss. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

MICROSCOPIC EXAMINATIONS
There were no test item-related histologic changes. Five males and 1 female in the 8000 ppm group were euthanized in extremis between study days 7-13; a specific cause of death was not determined microscopically for these animals. In addition, female no. 67216 was found dead on study day 20. Microscopically, all ventricles of the brain were markedly dilated (hydrocephalus) and were lined by hyperplastic and hypertrophic ependymal cells. The surrounding neuropil contained increased numbers of cells consistent with gliosis and chronic inflammation. The ependymal and surrounding neuropil changes were most evident in the lateral and third ventricles. A specific etiology for the ventricular changes in the brain was not determined. The cause of death for this rat was hydrocephalus, which was presumed to be an incidental finding that was unrelated to administration of the test item.

Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test item. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Effect levels

Dose descriptor:

NOAEL

Effect level:

2 000 mg/L drinking water

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1 Mean Calculated F0 Test Item Consumption mg/kg/day

	Males			Females
Target Exposure Level	Prior to Mating	After Mating	TWA^a	Prior to Mating	Gestation	Lactation
500 ppm	41	37	40	61	57	83
2000 ppm	162	126	152	224	216	285
8000 ppm	416	404	409	598	899	1376

^aTime Weighted Average

Applicant's summary and conclusion

Conclusions:

Based on test item-related moribundity at 8000 ppm, reduced mean body weights, body weight gains, and food and water consumption in the 8000 ppm group males and females, the NOAEL for systemic toxicity was considered to be 2000 ppm.

Executive summary:

This study was designed to investigate the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to effect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development.

The test item, aminoethylpiperazine (AEP), was administered continuously in reverse osmosis-purified drinking water to 3 groups of Crl:CD(SD) rats, each group consisting of 12 males and 12 females. Exposure levels were 500, 2000, and 8000 ppm. A concurrent

control group of 12 rats/sex received the vehicle (reverse osmosis-purified drinking water) on a comparable regimen. Males and females were approximately 14 weeks of age at the beginning of test item administration. The test item was offered to males for a

minimum of 14 days prior to mating. Males continued to be exposed to the test item throughout mating and through the day of euthanasia. Females were exposed to the test item for a minimum of 14 days prior to mating through lactation day 4; the female that

failed to deliver was exposed to the test item through the day of euthanasia (post-cohabitation day 25). Males were exposed for 32 consecutive days and females were exposed for 39-53 consecutive days.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food and water consumption were recorded at appropriate intervals. FOB assessments and locomotor activity data were recorded for 12 animals/sex/group prior to the initiation of exposure and for 7-12 males/group following approximately 28 days of exposure and for 9-12 females/group on lactation day 4. All F0 females were allowed to deliver and rear their pups until lactation day 4.

F1 clinical observations and body weights were recorded on PND 1 and 4. Pups were necropsied on PND 4. Clinical pathology evaluations (hematology and serum chemistry) were performed on all available F0 animals (7-12/sex/group) at necropsy.

FOB assessments and locomotor activity and clinical pathology evaluations were not conducted for the female that failed to deliver. F0 males were euthanized following completion of the mating period. F0 females were euthanized on lactation day 4 for females that delivered and post-cohabitation day 25 for the female that failed to deliver. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups; gross lesions from all animals in all dosage groups were also examined microscopically.

Five males and 1 female in the 8000 ppm group were euthanized in extremis during the treatment period following body weight losses and reduced food and water consumption. The cause of moribundity of these animals could not be determined microscopically. In

addition, 1 female in the 8000 ppm group was found dead on study day 20; the cause of death for this female was determined to be an incidental finding (hydrocephalus) that was unrelated to administration of the test item. The moribundity observed in the 8000 ppm

group males was considered to be test item-related as it occurred in the presence of effects on body weight and food and water consumption noted at this same dosage level. All other animals survived to the scheduled necropsies.

In the 8000 ppm group males, test item-related lower mean body weight gains were noted when the overall pre-mating (study days 0-13) and treatment (study days 0-31) periods were evaluated; correspondingly lower mean food and water consumption were noted

during the pre-mating period. As a result, mean male body weight in this group was up to 11.0% lower than the control group during the treatment period. In addition, a test item-related lower mean body weight gain with corresponding reduced food and water

consumption was noted during the first week of treatment (study days 0-6) in the 8000 ppm group females, resulting in a lower (5.0%) mean body weight on study day 6. As a result of these body weight effects, lower mean body weights and/or body weight gains in the absence of effects on food and water consumption continued to be observed in the 8000 ppm group females throughout gestation and lactation.

Mean body weights, body weight changes, and food and water consumption were unaffected by test item administration in the 500 and 2000 ppm group males throughout the study and in the 500 and 2000 ppm group females during the pre-mating, gestation,

and lactation periods.

No test item-related effects were noted during the FOB assessments or locomotor activity evaluations at any dosage level.

At the scheduled necropsy, a test item-related lower mean final body weight was noted in the 8000 ppm group females. No other changes in clinical pathology parameters, gross necropsy observations, or organ weight changes associated with test item administration were observed.