2-piperazin-1-ylethylamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

One OECD 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was conducted in rats administered aminoethyl piperazine (AEP) in the drinking water at 500, 2000 and 8000 ppm.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

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

Route of administration:

oral: drinking water

Vehicle:

water

Details on 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.

Details on mating procedure:

The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages.

For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.

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

Details on study schedule:

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.

Dose / conc.:

500 ppm

Remarks:

nominal in water

Dose / conc.:

2 000 ppm

Remarks:

nominal in water

Dose / conc.:

8 000 ppm

Remarks:

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.

Parental animals: Observations and examinations:

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

PARTURITION
All females were allowed to deliver naturally and rear their young to PND 4. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.

Oestrous cyclicity (parental animals):

No data.

Sperm parameters (parental animals):

No data. (Only examination focused on whether sperm were present in vaginal lavage fluid.)

Litter observations:

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.

Postmortem examinations (parental animals):

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.

Postmortem examinations (offspring):

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

Reproductive indices:

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

Where N = PND 0-1 and 1-4

Offspring viability indices:

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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 (87 g to 122 g) with reduced feed (≤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 and/or red material around the right eye.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One 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 feed 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.

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 with corresponding reduced food consumption was noted during the first week of treatment (study days 0-6) in fem

Food consumption and compound intake (if feeding study):

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 with corresponding reduced food consumption was noted during the first week of treatment (study days 0-6) in fem

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

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 the pre-mating period (study days 0-13). 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. Differences from the control group were slight and not statistically significant.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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-related response. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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-related response. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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 pr etest 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.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: Lower mean water consumption observed in the high dose group.

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

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.

Dose descriptor:

NOAEC

Effect level:

8 000 mg/L drinking water

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The general physical condition of all F1 pups in this study were unaffected by test item administration. Pups (litters) that were found dead numbered 1(1), 0(0), 1(1), and 1(1) in the control, 500, 2000, and 8000 ppm groups, respectively. Four (4), 4(2), 4(4), and 4(3) pups (litters) in these same respective groups were missing and presumed to have been cannibalized.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean male and female pup body weights and body weight changes in the 500, 2000, and 8000 ppm groups were unaffected by test item administration during PND 1-4. Differences from the control group were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

F1 LITTER DATA
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
The mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 500, 2000, and 8000 ppm groups were unaffected by test item administration. Differences from the control group were slight and not statistically significant.

GENERAL PHYSICAL CONDITION
The general physical condition of all F1 pups in this study were unaffected by test item administration. Pups (litters) that were found dead numbered 1(1), 0(0), 1(1), and 1(1) in the control, 500, 2000, and 8000 ppm groups, respectively. Four (4), 4(2), 4(4), and 4(3) pups (litters) in these same respective groups were missing and presumed to have been cannibalized.

OFFSPRING BODY WEIGHTS
Mean male and female pup body weights and body weight changes in the 500, 2000, and 8000 ppm groups were unaffected by test item administration during PND 1-4. Differences from the control group were slight and not statistically significant.

NECROPSIES OF PUPS FOUND DEAD
The numbers of pups (litters) found dead during PND 0-4 numbered 1(1), 0(0), 1(1), and 1(1) in the control, 500, 2000, and 8000 ppm groups, respectively. Aside from the absence of milk in the stomach, no other internal findings were noted.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

8 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: In the absence of effects on the general physical condition of the F1 pups, the NOEL for neonatal toxicity was 8000 ppm.

Reproductive effects observed:

not specified

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

Table 2 Results of Reproductive Performance

	Dosage Level (ppm)				WIL HC^a
Parameters	0	500	2000	8000	Mean (Range)
Male Mating Index	100	100	100	85.7	96.7 (84 -100)
Female Mating Index	100	100	100	90	98.2 (86.7 -100)
Male Fertility Index	100	100	100	85.7	91.0 (60.0 -100)
Female Fertility Index	100	100	100	90	93.2 (60.0 - 100)
Male Copulation Index	100	100	100	100	94.4 (71.4 - 100)
Female Conception Index	100	100	100	100	94.9 (65.2 - 100)
Pre-Coital Interval (days)	3.0	3.6	4.1	2.4	3.0 (1.8 -5.5)

a = WIL historical control data

Conclusions:

There were no test item-related effects on reproductive performance, gestation length, parturition, and the mean numbers of corpora lutea, implantation sites, and unaccounted-for sites at any dosage level. Mean numbers of pups born, live litter size, percentage of males per litter, and postnatal survival were unaffected by test item administration. No test item-related effects were noted on the general physical condition of the F1 pups at any dosage level.

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.

Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels.

Mean numbers of corpora lutea and unaccounted-for sites, mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 500, 2000, and 8000 ppm groups were similar to the control group values. Mean pup body weights and body weight gains at all dosage levels were unaffected by dose administration. No test item-related clinical findings or macroscopic findings for F1 pups that were found dead were noted at any dosage level.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

598 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

An OECD 422 study was conducted in Crl:CD(SD) rats where AEP was administered via the drinking water at 500, 2000, and 8000 ppm. The test substance was given to males two weeks prior to mating, breeding, and until necropsy. Females were exposed to the test material 14 days prior to mating and through lactation day 4. Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and parturition were unaffected by the treatment. Mean numbers of corpora lutea and pups born, live litter size, the percentage of males at birth, and postnatal survival of all treatment groups were similar to controls.

At necropsy, a lower mean final body weight was noted in the 8000 ppm females. No other changes in clinical pathology, gross necropsy observations, or organ weight changes were associated with AEP.

Therefore, a dose level of 8000 ppm (598 mg/kg/day) was considered to be the NOAEL for reproductive toxicity of AEP.

Effects on developmental toxicity

Description of key information

Three studies are referenced for developmental toxicity for AEP.  A Rat and Rabbit OECD 414 study was conducted by oral administration.  Lastly, OECD 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was conducted in rats administered in drinking water.  

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

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

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.

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

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).

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.

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

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.

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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. 

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.

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate
- Age at study initiation: ca 9-10 weeks old
- Weight at study initiation: 222-298 g
- Housing: Animals were housed 2 per cage in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings which were provided with a certificate of analysis for significant contaminants. An analytical certificate for each batch of bedding used has been retained at Charles River, Edinburgh.
- Diet: SDS VRF-1 breeder diet was provided ad libitum throughout the study, except during designated procedures.The feed is analysed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are retained at Charles
River, Edinburgh. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): The animals had access to water ad libitum from the public supply from polycarbonate water bottles which were changed as necessary throughout the course of the study. The water used by Charles River Edinburgh is analysed at regular intervals for dissolved materials, heavy metals, pesticide residues, pH, nitrates and nitrites. Microbiological screening is also conducted. An analytical certificate for each analysis is retained at Charles
River, Edinburgh. The water used is considered not to contain any additional substances, in sufficient concentration, to have any influence on the outcome of the study.
- Acclimation period: Animals were allowed to acclimatise to the Charles River rodent toxicology accommodation from arrival until dosing on Day 6 of gestation (3-5 days).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 44% to 77%
- 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): 12-hour light/12-hour dark cycle

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4°C, and dispensed daily. All formulations were adjusted to pH 9 with HCl. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulations were also stirred continuously during dosing. Any residual volumes were discarded.

VEHICLE
The control item, Milli-Q water, was prepared weekly, stored in a refrigerator set to maintain 4°C and dispensed daily for administration to Group 1 control animals. The 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. Any residual volumes were discarded.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All samples to be analysed were transferred to the analytical laboratory at the Test Facility and analysed within the established stability period. Any residual/retained analytical samples have been discarded. Analyses were performed by Gas Chromatography with Flame Ionisation using a validated analytical procedure.

Duplicate top, middle and bottom sets of samples (duplicate middle only for control) at volumes of 0.5 mL (by weight) for Group 1 and 0.1 mL (by weight) for Groups 2 – 4 and triplicate top, middle and bottom (triplicate middle samples only for control) sets of back up samples (of the same volumes) were taken from formulations prepared for use on Day 1 and Week 2. The analytical samples were transferred to the analytical laboratory for analysis, stored in a refrigerator set to maintain 4°C and were analysed within the established stability period. Concentration results were accepted if mean sample concentration results were
within or equal to ± 10%. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentrations of ≤ 10% for each group.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

The test and control items were administered to the appropriate animals by once daily oral gavage from Days 6-19 of gestation

Frequency of treatment:

Daily

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral gavage route of administration was selected for this study as this was defined as an appropriate method to define prenatal development toxicity and may be a possible route of human exposure.
The dose levels were selected in agreement with the Sponsor following review of the data from a preliminary prenatal developmental toxicity study using the same test item (Charles River Study No. 497008) and the recommended limit dose of 1000 mg/kg/day from the regulatory guidelines (OECD Guideline 414).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, once in the morning and once as late as possible during the working day, throughout the study, animals were observed for general health/mortality and moribundity. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, from the start of dosing animals were removed from their cage for examination. Prior to dosing and regularly throughout the day, all the animals were examined for reaction to treatment with particular attention being paid to the animals during and for the first hour after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed once during pretrial on Day 5 of gestation and daily during the dosing period from Day 6 – 20 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was quantitatively measured for all animals daily from Day 6 of gestation (first measured quantity given on Day 5 of gestation).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was monitored by visual inspection of the water bottles on a regular basis
throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: All adult animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

OTHER:

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

Fetal examinations:

- External examinations: Yes: Fetuses were examined for external abnormalities. Late embryonic deaths were examined for external abnormalities to the extent possible. Each implant was classified as being live, or a dead fetus (dead full term fetus that showed no sign of maceration), or a late embryonic death (macerated tissue identifiable as an embryo fetus, with recognizable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placentae), or an early embryonic death (discrete, formless, discoloured tissue mass attached to the internal uterine wall; may be of varying size).

- Visceral examinations: Yes: Half of the viable fetuses from each uterus were fixed in methylated ethyl alcohol. Following initial fixation fetuses were examined internally for sex and eviscerated; the viscera were not examined from fetuses prior to disposal. The remaining half of viable fetuses from each uterus were fixed in Bouin's fluid. The fetuses fixed in Bouin's fluid were examined for soft tissue abnormalities and sex using a freehand sectioning technique derived from that of Wilson.

- Skeletal examinations: Yes:The eviscerated carcasses were macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, then the fetuses cleared with aqueous glycerol solutions. These preparations were examined for the presence of skeletal abnormalities and for the extent of ossification.

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.
Where required to assist interpretations, tests were applied to determine the statistical significance of observed differences between Control and groups receiving test item. 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 nonparametric ANOVA were used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical Observations: All animals dosed at 300 and 1000 mg/kg/day had evidence of yellow staining of the cage shavings for the majority of the study. There were no other clinical observations that were considered to be related to test item administration.

Body Weights and Body Weight Changes: At 1000 mg/kg/day approximately 50% of animals had a slight body weight loss after the first day of dosing. This was recovered by Day 2 of dosing (Day 7 of gestation); however, the overall body weight gains for the dosing period were reduced compared to controls. At dose levels up to 300 mg/kg/day the group mean body weight gains were similar to the control group for the duration of the study.

Feed Consumption: At 1000 mg/kg/day, when compared with Controls, there was a slight reduction in group mean food consumption for the majority of the dosing period with a maximum daily reduction of -28%. This was most notable during the start and end of the dosing period. At dose levels up to 300 mg/kg/day the group mean food consumption performance was similar to the control group for the duration of the study.

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The type and distribution of all fetal abnormalities and variations, including those indicating the extent of skeletal ossification were similar in all groups and did not indicate any association with test item administration.
There were no treatment related differences in fetal weights, group mean gravid uterine weight or any of the pregnancy performance parameters evaluated. Any slight intergroup differences are considered to be due to natural variation and are considered to be unrelated to test item administration.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Administration of Aminoethylpiperazine at 1000 mg/kg/day was associated with slight reductions in food consumption and body weight gain along with yellow staining of the cage shavings at 300 and 1000 mg/kg /day. At dose levels up to 1000 mg/kg/day there were no test item-related effects on gross
pathology findings, pregnancy performance parameters or fetal weights, fetal abnormalities and variants.
In conclusion, under the conditions of this study, the maternal no observed effect level (NOEL) was considered to be 100 mg/kg/day based on yellow staining of the cage shavings at 300 mg/kg/day, and the maternal no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg/day. There was no effect on embryofetal development or survival detected at any dose level; therefore, the embryofetal no observed effect level (NOEL) was considered to be 1000 mg/kg/day.

Executive summary:

The objective of this study was to determine the potential maternal and developmental toxicity of Aminoethylpiperazine when given orally during the period of organogenesis to pregnant rats. Four groups of 24 female Sprague-Dawley rats were dosed orally by gavage on Days 6-19 of gestation (where Day 0 was detection of mating).

The following parameters and end points were evaluated in this study: clinical observations, body weights and body weight changes, food consumption, gross necropsy findings, gravid uterine weight, examination of pregnancies and fetal evaluations (external abnormalities, fetal weights, visceral and skeletal evaluations). Animals were killed on Day 20 of gestation.

There were 2 premature decedents in this study (Animal 37 on Day 15 of gestation at 100 mg/kg/day and Animal 91 on Day 18 of gestation at 1000 mg/kg/day). The cause of these deaths is unknown; however, there was no evidence to indicate any association with the dosing procedure or the test item and, therefore, both deaths are considered to be incidental.

Administration of Aminoethylpiperazine at 1000 mg/kg/day was associated with slight reductions in food consumption and body weight gain along with yellow staining of the cage shavings at 300 and 1000 mg/kg /day. At dose levels up to 1000 mg/kg/day there were no test item-related effects on gross pathology findings, pregnancy performance parameters or fetal weights, fetal abnormalities and variants.

In conclusion, under the conditions of this study, the maternal no observed effect level (NOEL) was considered to be 100 mg/kg/day based on yellow staining of the cage shavings at 300 mg/kg/day, and the maternal no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg/day. There was no effect on embryofetal development or survival detected at any dose level; therefore, the embryofetal no observed effect level (NOEL) was considered to be 1000 mg/kg/day.

Reference 3

Endpoint:

developmental toxicity

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

Qualifier:

according to guideline

Guideline:

other: OECD 422

Deviations:

not specified

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

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

Route of administration:

oral: drinking water

Vehicle:

water

Details on 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.

Details on mating procedure:

The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages.

Duration of treatment / exposure:

The vehicle and test drinking water were offered to males and females ad libitum for at least 14 consecutive days prior to mating. The males continued to be exposed to the test drinking water throughout mating and through the day of euthanasia. The females continued to be exposed to the test drinking water throughout mating, gestation, and lactation, through the day of euthanasia. Males were exposed for 32 consecutive days and females were exposed for 39-53 consecutive days. The test item was offered as a constant concentration (ppm) in the drinking water. Individual dosages were not adjusted based on body weights or water consumption.

Frequency of treatment:

Daily

Duration of test:

Males were exposed for 32 consecutive days and females were exposed for 39-53 consecutive days.

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.

Maternal 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.

Ovaries and uterine content:

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).

Fetal examinations:

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

Indices:

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

Historical control data:

Historical control data from WIL Laboratory was examined.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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 (87 g to 122 g) with reduced feed (≤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 and/or red material around the right eye. 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 feed 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.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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.
Females: 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.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Male: Test item-related lower mean male feed 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 feed 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 feed consumption in this group was similar to the control group during study days 27-30 (following the end of the mating period).
Female: Test item-related lower mean female feed 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 feed consumption in this group was similar to the control group during study days 6-13.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

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 the pre-mating period (study days 0-13). 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.
Females: 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<0.05 or p<0.01) during study days 0-3 and 3-6. The lower mean water consumption corresponded to a test item-related mean body weight loss or an absence of mean body weight gain during the first week of dose administration. Mean water consumption in this group was similar to the control group during study days 10-13.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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-related response. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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-related response. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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<0.01), 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.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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 (i.e., post-implantation loss), implantation sites, and corpora lutea in the 500, 2000, and 8000 ppm groups were similar to the control group values.

Neuropathological findings:

no effects observed

Description (incidence and severity):

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).

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Test substance intake: Lower mean water consumption observed in the high dose group.

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 500, 2000, and 8000 ppm groups were unaffected by test item administration. Differences from the control group were slight and not statistically significant.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

The general physical condition of all F1 pups in this study was unaffected by test item administration. Pups (litters) that were found dead numbered 1(1), 0(0), 1(1), and 1(1) in the control, 500, 2000, and 8000 ppm groups, respectively. Four (4), 4(2), 4(4), and 4(1) pups (litters) in these same respective groups were missing and presumed to have been cannibalized.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): 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.

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: reduced mean body weights, body weight gains, and food and water consumption in the 8000 ppm group females

Details on maternal toxic effects:
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.

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.

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.

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 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 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 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 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 lactation day 4 (females).

CLINICAL PATHOLOGY
HEMATOLOGY
There were no test item-related alterations in hematology and coagulation parameters. No 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 triglyceride values were noted in the 500 ppm group females. 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 group was 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.

Dose descriptor:

NOAEC

Effect level:

8 000 mg/L drinking water

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEC

Effect level:

2 000 mg/L drinking water

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEC

Effect level:

8 000 mg/L drinking water

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean male and female pup body weights and body weight changes in the 500, 2000, and 8000 ppm groups were unaffected by test item administration during PND 1-4. Differences from the control group were slight and not statistically significant.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The numbers of pups (litters) found dead during PND 0-4 numbered 1(1), 0(0), 1(1), and 0(0) in the control, 500, 2000, and 8000 ppm groups, respectively. Aside from the absence of milk in the stomach, no other internal findings were noted.

Changes in sex ratio:

not specified

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 500, 2000, and 8000 ppm groups were unaffected by test item administration. Differences from the control group were slight and not statistically significant.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

The mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 500, 2000, and 8000 ppm groups were unaffected by test item administration. Differences from the control group were slight and not statistically significant.

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
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.

Dose descriptor:

NOEL

Effect level:

8 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general physical condition of the F1 pups

Abnormalities:

not specified

Developmental effects observed:

not specified

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	40.1	61	57	83
2000 ppm	162	126	154	224	216	285
8000 ppm	416	404	409	598	899	1376

^aTime Weighted Average

Table 2 Results of Reproductive Performance

	Dosage Level (ppm)				WIL HC^a
Parameters	0	500	2000	8000	Mean (Range)
Male Mating Index	100	100	100	85.7	96.7 (84 -100)
Female Mating Index	100	100	100	90	98.2 (86.7 -100)
Male Fertility Index	100	100	100	85.7	91.0 (60.0 -100)
Female Fertility Index	100	100	100	90	93.2 (60.0 - 100)
Male Copulation Index	100	100	100	100	94.4 (71.4 - 100)
Female Conception Index	100	100	100	100	94.9 (65.2 - 100)
Pre-Coital Interval (days)	3.0	3.6	4.1	2.4	3.0 (1.8 -5.5)

a = WIL historical control data

Conclusions:

There were no test item-related effects on reproductive performance, gestation length, parturition, and the mean numbers of corpora lutea, implantation sites, and unaccounted-for sites at any dosage level. Mean numbers of pups born, live litter size, percentage of males per litter, and postnatal survival were unaffected by test item administration. No test item-related effects were noted on the general physical condition of the F1 pups at any dosage level.

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.

Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels.

Mean numbers of corpora lutea and unaccounted-for sites, mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 500, 2000, and 8000 ppm groups were similar to the control group values. Mean pup body weights and body weight gains at all dosage levels were unaffected by dose administration. No test item-related clinical findings or macroscopic findings for F1 pups that were found dead were noted at any dosage level.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Species:

rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In an OECD 414 study rats were exposed to Aminoethylpiperazine by Oral gavage and concluded the maternal no observed effect level (NOEL) was considered to be 100 mg/kg/day based on yellow staining of the cage shavings at 300 mg/kg/day, and the maternal no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg/day. There was no effect on embryofetal development or survival detected at any dose level; therefore, the embryofetal no observed effect level (NOEL) was considered to be 1000 mg/kg/day.

Additionally, AEP was tested in rabbits in an OECD 414 guideline study. Oral administration of the test item was from Day 6 to 28 of gestation. Aminoethylpiperazine 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. 

Lastly, An OECD 422 guideline study, "Combined Repeated Dose Toxicity Study with the Reproductive/Developmental Toxicity Screen Test, was conducted on Sprague Dawley rats at doses of 0, 500, 2000, and 8000 ppm via the drinking water. No teratological effects were seen at any dose level. Therefore the NOAEL for developmental effects was 8000 ppm (899 mg/kg/day).

Justification for classification or non-classification

The OECD 414 rabbit study indicates that AEP is a developmental toxicant due to post-implantation loss at the high dose group (150 mg/kg/day). Therefore AEP is classifiable as a Catagory 2 reproductive/developmental toxicant under GHS.