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EC number: 205-411-0 | CAS number: 140-31-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Toxicity to reproduction
Administrative data
- 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
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 010
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- not specified
- Principles of method if other than guideline:
- not applicable
- GLP compliance:
- yes
- Limit test:
- no
Test material
- Reference substance name:
- Aminoethylpiperazine (AEP)
- IUPAC Name:
- Aminoethylpiperazine (AEP)
- Details on test material:
- - Name of test material (as cited in study report): Aminoethylpiperazine (AEP)
- Physical state: liquid
- Analytical purity:98.4% by gas chromatography
- Impurities (identity and concentrations):0.051% water by Karl Fischer coulometric titration, and 0.76% diethylenetriamine (DETA) and 0.09% aminoethylethanolamine (AEEA) by gas chromatography.
- Composition of test material, percentage of components:
- Purity test date: 7 July 2009
- Lot/batch No.:XG03081150
- Stability under test conditions:The test substance is considered to be stable under the storage conditions.
- Storage condition of test material:Ambient
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:Charles River Laboratories, Inc.
- Age at study initiation: 91-101 days
- Weight at study initiation: Males 300-500 gm; Females 200-300 gm
- Housing: All animals were individually housed in clean suspended wire mesh cages in an environmentally controlled room during the acclimation period and continuing until mating. Following successful mating, the females were housed individually in a plastic cage containing ground
corncob nesting material (Bed O'Cobs) and remained in these cages until euthanasia on lactation day 4.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Actual mean daily temperature ranged from 70.5°F to 71.4°F (21.4°C to 21.9°C).
- Humidity (%): Mean daily relative humidity ranged from 38.2% to 52.0% during the study.
- Air changes (per hr):10 room air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark photoperiod
IN-LIFE DATES: From: 5 March 2010 To: 27 April 2010
Administration / exposure
- Route of administration:
- oral: drinking water
- Vehicle:
- water
- Details on 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.
Doses / concentrationsopen allclose all
- 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.
Examinations
- 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
Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
- 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 / performance (P0)
- Reproductive function: oestrous cycle:
- not specified
- Reproductive function: sperm measures:
- not specified
- Reproductive performance:
- no effects observed
Details on results (P0)
CLINICAL OBSERVATIONS AND SURVIVAL
In the 8000 ppm group, 5 males (nos. 67248, 67254, 67256, 67258, and 67273) and 1 female (no. 67217) were euthanized in extremis between study days 7-13. The moribundity of these animals occurred following body weight losses (46 g to 122 g) with reduced food (≤19 g/day) and water consumption (≤22 g/day) from study day 0 through the day of death/euthanasia. Clinical findings noted for these animals on the days prior to
or on the day of death/euthanasia were limited to decreased defecation, red material around the eye or nose, and/or hair loss on the forelimb. In addition, 1 female (no. 67216) was found dead on study day 20. Upon microscopic examination, the cause of death for this female was determined to be hydrocephalus, which was presumed to be an incidental finding that was unrelated to administration of the test item. The cause of moribundity could not be determined microscopically for any the animals euthanized in extremis because no significant internal findings were observed. However, the moribundity of these animals occurred in the presence of test item-related reductions in body weight and food and water consumption noted at this same dosage level, and therefore was considered to be test item-related. All other animals survived to the scheduled necropsies.
For animals that survived to the scheduled necropsy, clinical findings were limited to hair loss on the limbs. These findings occurred infrequently and/or at similar frequencies in the control group, and were not attributed to test item administration.
BODY WEIGHTS
MALES
Test item-related mean body weight losses were noted in the 8000 ppm group males during study days 0-10 followed by a lower mean body weight gain during study days 10-13; differences from the control group were significant (p<0.01) during study days 0-3 and 3-6. Mean body weights in the 8000 ppm group were up to 11.0% lower compared to the control group during study days 0-13; differences from the control group were significant (p<0.05) on study day 6. These body weight effects were primarily due to the 5 males in this group euthanized in extremis during study days 7-13. Mean body weights and body weight gains for the surviving males were comparable to the control group during the remainder of the treatment period (study days 13-31). Due to the initial mean body weight losses and lower mean body weight gain in the 8000 ppm group, a significantly (p<0.05) lower mean body weight gain was noted for the overall pre-mating period (study days 0-13) and a lower (not statistically significant) mean body weight gain was noted when the entire generation (study days 0-31) was evaluated compared to the control group.
Mean male body weights and body weight gains in the 500 and 2000 ppm groups were similar to the control group throughout the treatment period. Differences from the control group were slight and not statistically significant.
FEMALES
PRE-MATING
A test item-related, significant (p<0.01) mean body weight loss followed by an absence of body weight gain were noted in the 8000 ppm group during the first week of the treatment period (study days 0-6). As a result, mean body weight in this group was 5.0% lower (not statistically significant) compared to the control group on study day 6. During the remainder of the pre-mating period (study days 6-13), mean body weight gain in the 8000 ppm group was similar to the control group. The mean body weight loss and absence of body weight gain noted in the 8000 ppm group during the first week of treatment were of sufficient magnitude to result in a significantly (p<0.05) lower mean body weight gain when the entire pre-mating period (study days 0-13) was evaluated, and thus were considered to be adverse.
Mean female body weights and body weight gains in the 500 and 2000 ppm groups were similar to the control group during the pre-mating period. Differences from the control group were slight and not statistically significant.
GESTATION
Slightly lower (not statistically significant) mean body weight gains were noted in the 2000 and 8000 ppm groups generally throughout gestation compared to the control group. As a result, significantly (p<0.05 or p<0.01) lower mean body weight gains were noted in these groups when the overall gestation treatment period (gestation day 0-20) was evaluated, and mean body weight in the 8000 ppm group was 5.8% lower (not statistically significant) than the control group on gestation day 20. Conversely, the lower mean body weight gains noted in the 2000 ppm group were not of sufficient magnitude to affect mean body weight, and therefore were not considered to be test item-related.
Mean body weights and body weight gains in the 500 ppm group were generally similar to those in the control group throughout gestation. Differences from the control group were slight and not statistically significant.
LACTATION
Mean maternal body weight gains were unaffected by test item administration during lactation days 1-4. However, mean body weights in the 8000 ppm group were up to 6.6% lower (not statistically significant) than the control group during lactation days 1-4 as a result of the lower mean body weights noted in this group during the pre-mating period and gestation.
Mean body weights in the 500 and 2000 ppm groups were unaffected by test item administration during lactation days 1-4. Differences from the control group were slight and not statistically significant.
FOOD CONSUMPTION
MALES
Test item-related lower mean male food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 8000 ppm group during the pre-mating period (study days 0-13); differences from the control group were significant (p<0.05 or p<0.01). The lower mean food consumption corresponded to the overall lower mean body weight gain noted in this group during the pre-mating period and was primarily due to the 5 males in this group euthanized in extremis during study days 7-13. Mean food consumption in this group was similar to the control group during study days 27-30.
Mean male food consumption in the 500 and 2000 ppm groups was similar to the control group during the pre-mating period (study days 0-13). Differences from the control group were slight and not statistically significant.
FEMALES
PRE-MATING
Test item-related lower mean female food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 8000 ppm group during the first week of treatment (study days 0-6); differences from the control group were significant (p<0.01) and corresponded to a period of mean body weight loss. Mean food consumption in this group was similar to the control group during study days 6-13.
Mean food consumption in the 500 and 2000 ppm groups was unaffected by test item administration during the pre-mating period (study days 0-13). Prior to the initiation of treatment (study days -7 to -3), significantly (p<0.01) higher mean food consumption was noted in the 2000 ppm group. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.
GESTATION
Mean food consumption in the 500, 2000, and 8000 ppm groups was unaffected by test item administration during gestation. Significantly (p<0.05) higher mean food consumption (g/animal/day value only) was noted in the 500 ppm group during gestation days 0-4. However, in the absence of an exposure-related response, the increased mean food consumption was not considered to be treatment-related. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.
LACTATION
Mean food consumption in the 500, 2000, and 8000 ppm groups was unaffected by test item administration during lactation days 1-4. Differences from the control group were slight and not statistically significant.
WATER CONSUMPTION
MALES
Mean water consumption, evaluated as g/animal/day and g/kg/day, in the 8000 ppm group males was lower than the control group during study days 0-10. Differences from the control group were generally significant (p<0.01) and corresponded to a test item-related lower mean body weight gain noted during the same interval. In addition, a significant (p<0.01) decrease in mean water consumption was noted in the 8000 ppm group during study days 27-31.
Mean water consumption in the 500 and 2000 ppm group was similar to that in the control group throughout the study. Lower (p<0.05) mean water consumption was also noted in the 2000 ppm group compared to the control group during study days 27-31. Due to the lack of a concurrent effect on mean body weight gain during this interval, the decreased mean water consumption in this group was not considered to be test item-related. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.
FEMALES
PRE-MATING
Lower mean water consumption, evaluated as g/animal/day and g/kg/day, was noted in the 8000 ppm group during study days 0-10; differences from the control group were significant (p<0.01) during study days 0-3 and corresponded to a test-item-related mean body weight loss during the first week of dose administration. Mean water consumption in this group was similar to the control group during study days 10-13.
Mean water consumption in the 500 and 2000 ppm groups was unaffected by test item administration during the pre-mating period (study days 0-13). Increased mean water consumption was noted in both groups during study days 10-13; differences from the control group were generally significant (p<0.05 or p<0.01). However, in the absence of a similar effect in the high-dose group, the increased mean water consumption noted in
the 500 and 2000 ppm group was not considered to be test item-related. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.
GESTATION
Mean maternal water consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test item administration during gestation. Differences between the control, 500, 2000, and 8000 ppm groups were slight and not statistically significant.
LACTATION
Mean maternal water consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test item administration during lactation days 1-4. Differences between the control, 500, 2000, and 8000 ppm groups were slight and not statistically significant.
TEST ITEM CONSUMPTION
The average quantities of aminoethylpiperazine consumed during the F0 generation are presented below (Table 1). Values for the entire lactation period in all groups were elevated, as is commonly seen in nursing animals.
FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
SENSORY OBSERVATIONS
Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study day 27 (males) or on lactation day 4 (females).
NEUROMUSCULAR OBSERVATIONS
Neuromuscular parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study day 27 (males) or on lactation day 4 (females).
PHYSIOLOGICAL OBSERVATIONS
A lower (6.7%) mean body weight was noted for the 8000 ppm group females compared to the control group at the physiological observations on lactation day 4; the difference from the control group was significant (p=0.002). The lower mean body weight corresponded to the lower mean body weight recorded on lactation day 4. Mean body temperature was unaffected by test item administration at all dosage levels. There were no other statistically significant differences for the test item-treated groups when compared to the control group on study day 27 (males) or lactation day 4 (females).
LOCOMOTOR ACTIVITY
Locomotor activity patterns (total activity) in F0 animals were unaffected by test item administration at all concentrations when evaluated on study day 27 (males) and lactation day 4 (females). Values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values with the following exceptions. Mean total activity counts for females in the 500 and 8000 ppm groups at the lactation day 4 evaluation were higher for the 6 subintervals as well as the overall 60-minute test session; differences from the control group achieved significance (p≤0.003) for these groups when the overall 60-minute test session was evaluated for total motor activity counts by a repeated measures analysis. However, no dose-related trend was apparent and the increase in motor activity was primarily attributed to individual females in the 500 and 8000 ppm groups with atypically high total motor activity values. In addition, mean total motor activity counts in the 500 and 8000 ppm groups on lactation day 4 were generally similar to pretest values during the first subinterval (0-10 minutes) and differences between the pretest and lactation evaluations were limited to greater habituation on lactation day 4. Therefore, the increased mean total activity counts for females in the 500 and 8000 ppm groups were not considered to be test item-related.
No remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated at study day 27 (males) and lactation day 4 (females).
CLINICAL PATHOLOGY
HEMATOLOGY
There were no test item-related alterations in hematology and coagulation parameters. A significantly (p<0.05) lower mean corpuscular hemoglobin (MCH) value was noted in the 2000 ppm group males. This group mean difference was not considered to be test item-related because the value did not show a dose- or time-related response. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.
SERUM CHEMISTRY
There were no test item-related effects on serum chemistry parameters. Significantly (p<0.05) higher mean glucose and triglyceride values were noted in the 500 ppm group males and females, respectively. These group mean differences were not considered to be test item-related because the values did not show a dose- or time-related response. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.
REPRODUCTIVE PERFORMANCE
F0 male and female reproductive parameters are presented in Table 2.
No test item-related effects on reproductive performance were observed at any exposure level. No statistically significant differences were noted between the control and test item-treated groups. One mating pair in the 8000 ppm group did not produce a litter. The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. None of these differences were statistically significant.
GESTATION LENGTH AND PARTURITION
Mean gestation lengths in the 500, 2000, and 8000 ppm groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.
ANATOMIC PATHOLOGY
MACROSCOPIC EXAMINATIONS
In the 8000 ppm group, 5 males and 1 female were euthanized in extremis between study days 7-13, and 1 female was found dead study day 20. All other animals survived to the scheduled necropsies. No test item-related internal findings were observed at any dosage level in males and females that died, were euthanized in extremis, failed to deliver, or at the scheduled necropsies. Macroscopic findings observed in the test item-treated groups, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related. The mean numbers of unaccounted-for sites, implantation sites, and corpora lutea in the 500, 2000, and 8000 ppm groups were similar to the control group values.
ORGAN WEIGHTS
The mean final body weight for the 8000 ppm group females was 6.4% lower (not statistically significant) than the control group females. This lower body weight was considered an adverse test item-related effect. A significantly (p<0.05) higher mean relative (to body weight) thyroid/parathyroid weight was noted in the 8000 ppm group females when compared to the control group; however, this change was attributed to the test item-related decrease in mean body weight and was not considered to be a direct effect of the test item. In addition, lower mean absolute and relative (to body and brain weight) spleen weights were noted in the 8000 ppm group females compared to the control group. The differences were significant (p<0.05 or p<0.01), but the splenic weight differences were not considered test item-related given the lack of microscopic changes consistent with cell loss. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.
MICROSCOPIC EXAMINATIONS
There were no test item-related histologic changes. Five males and 1 female in the 8000 ppm group were euthanized in extremis between study days 7-13; a specific cause of death was not determined microscopically for these animals. In addition, female no. 67216 was found dead on study day 20. Microscopically, all ventricles of the brain were markedly dilated (hydrocephalus) and were lined by hyperplastic and hypertrophic ependymal cells. The surrounding neuropil contained increased numbers of cells consistent with gliosis and chronic inflammation. The ependymal and surrounding neuropil changes were most evident in the lateral and third ventricles. A specific etiology for the ventricular changes in the brain was not determined. The cause of death for this rat was hydrocephalus, which was presumed to be an incidental finding that was unrelated to administration of the test item.
Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test item. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Effect levels (P0)
- 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'
Results: F1 generation
General toxicity (F1)
- 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
Developmental neurotoxicity (F1)
- Behaviour (functional findings):
- not specified
Developmental immunotoxicity (F1)
- Developmental immunotoxicity:
- not specified
Details on results (F1)
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.
Effect levels (F1)
- 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.
Overall reproductive toxicity
- Reproductive effects observed:
- not specified
Any other information on results incl. tables
Table 1 Mean Calculated F0 Test Item Consumption mg/kg/day
Males | Females | |||||
Target Exposure Level | Prior to Mating | After Mating | TWAa | 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 HCa | ||||
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
Applicant's summary and conclusion
- 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.
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