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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 March - 9 July 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: FDA 1986, Toxicological principles for Safety Assessment of Direct Food Additives and Color Additives Used in Food
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Piperazine
EC Number:
203-808-3
EC Name:
Piperazine
Cas Number:
110-85-0
Molecular formula:
C4H10N2
IUPAC Name:
piperazine
Specific details on test material used for the study:
Name of test material used: Piperazine dihydrochloride CAS 142-64-3, EC 205-551-2
PURITY: 52.25% as piperazine base
DESCRIPTION: White to off-white crystalline powder
DATE RECEIVED: 25 February 1998
STORAGE: Room temperature

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
- Age at study initiation: 6 weeks
- Weight at study initiation: M: 205 g F 156 g
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 36 - 74%
- Photoperiod (hrs dark / hrs light): 12 h cycle

Animals were doubly housed in elevated, stainless steel, wire mesh cages during the first week of. the acclimation period and individually housed thereafter.
Certified Rodent Diet, No. 5002; (Meal) (PMI Nutrition International, St. Louis, Missouri) was available without restriction. Fresh food was presented weekly.
Analysis of each feed lot used during this study was performed by the manufacturer. Results are maintained on file at the Testing Facility.
Water was available without restriction via an automated watering system (Elizabethtown Water Company, Westfield, New Jersey).

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
Appropriate amounts of the test material were mixed with Certified Rodent Diet No. 5002 to achieve the desired concentrations. Method of preparation, i.e., type of mixer, length of mixing time, etc., were developed based on physical characteristics ofthe test material and size of batches required. Control animals received untreated diets. Fresh diets were prepared once weekly.
Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Certified Rodent Dier, no 5002
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine homogeneity, stability, and concentration of the test article with carriers under the conditions of this study were performed by the Testing Facility.
Prior to initiation of the study, batches of low-concentration and high-concentration diet were prepared. Three samples each from the top, middle and bottom portion of each mixture were taken for analysis.
Triplicate samples ofthe low- and high-concentration diets were assayed 4, 7, and 14 days after preparation.
All three dietary levels were assayed weekly for the 4 weeks (one sample per concentration was taken and 3 subsamples were analyzed). Subsequent assays were performed at monthly intervals for the remainder of the study.
Mean analytical concentrations (% of nomimal were):
94.4-95.3 (low dose)
98.1-98.4 (mid dose)
102.5-102.9 (high dose)
Duration of treatment / exposure:
90 days
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
actual ingested: 401 mg/kg bw (M), 398 mg/kg bw (F)
Dose / conc.:
1 200 mg/kg bw/day (nominal)
Remarks:
actual ingested: 1203 mg/kg bw (M), 1197 mg/kg bw (F)
Dose / conc.:
2 400 mg/kg bw/day (nominal)
Remarks:
actual ingested: 2393 mg/kg bw (M), 2396 mg/kg bw (F)
No. of animals per sex per dose:
20
Control animals:
yes, plain diet

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly
BODY WEIGHT: Yes
- Time schedule for examinations: Twice pre-test, weekly during treatment and terminally after fasting..
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre test: day -5 and at termination day 92
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 0, test day 46, and at termination day 92 and 94
- How many animals: 10/sex/group
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 0, test day 46, and at termination day 93 and 94
- Animals fasted: Yes / No / No data
- How many animals: 10/sex/group
URINALYSIS: Yes
- Time schedule for collection of urine: Day -6, test day 43, and at termination day 87
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
ORGAN WEIGHTS: Yes (adrenal glands, brain, kidneys, liver, ovaries, spleen, testes, thymus, thyroid, parathyroid glands,
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
For each method, mean values of all dose groups were compared to the mean value for the control group at each time interval. Statistical evaluations were not performed when the standard deviation for the control group was 0. Dose groups were eliminated from statistical analysis if their standard deviation was 0.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
All animals were free of significant clinical signs.
Mortality:
no mortality observed
Description (incidence):
All animals survived for the duration of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dose-related, statistically significant, decreases in body weight gains, relative to control values, occurred in mid— and high-dose males and in all treated groups of females throughout most of the study. Mean weight values for the low- and mid-dose groups were generally within 5% of mean control values, while differences for the high-dose group were generally 10% or greater. The differences in the high-dose group are considered indicative of an adverse effect ofPiperazine Dihydrochloride administration.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption values of the mid- and high—dose males and females were statistically significantly increased, compared to the control values, throughout most of the study.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There was no indication of test article-related ocular abnormalities during the study.
Haematological findings:
no effects observed
Description (incidence and severity):
The mean hematology values of the test groups were generally comparable to control group values. The few test group values which were statistically significantly different from control values were within normal ranges and the differences were generally not dose-related; therefore, they are not considered biologically significant.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The mean clinical chemistry values of the test groups were generally comparable to control group values. The few test group values which were statistically significantly different from control values were within normal ranges and the differences were not dose-related; therefore, they are not considered biologically significant.
Endocrine findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
The mean urinalysis values ofthe test groups were generally comparable to control group values or exhibited normal variability.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Several statistically significant differences between organ weights and organ/body weight ratios for control and treated groups were seen. These were consistent with the low body weights in the treated groups and were not considered to represent direct effects of test article administration. A few statistically significant differences in organ weights relative to brain weights (which were similar for all groups) also occurred. The mean spleen/brain weight ratios of the high-dose males and females, the testes/brain weight ratio of the high-dose males, and the ovary/ and thymus/brain weight ratios of the high-dose females were slightly (statistically significantly) lower than the control values. Because these differences were slight and no changes were seen in the spleen, testes, ovaries and/or thymus upon microscopic examination, these differences are not considered biologically significant. The remaining mean organ weight values of the test groups were generally comparable to control group values.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic findings. Those seen occurred with comparable incidence and severity in rats from the treatment and control groups or they occurred sporadically. These incidental findings have been seen in rats of this strain and age used in other studies conducted in this facility.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no significant histopathological findings which could be attributed to administration of Piperazine Dihydrochloride.
There were a number of animals from both treated and control groups which had inflammatory changes in the lungs suggestive of a mild respiratory infection. These findings included perivascular lymphocytic infiltration, alveolar wall epithelialisation and alveolitis, and are occasionally seen in this age of rat maintained at these laboratories. They are not considered to be related to treatment with Piperazine Dihydrochloride. There was a slight difference in the incidence and severity of alveolar macrophage accumulation between control and treated groups but this difference was slight and cannot be reliably attributed to treatment.
Mammary acinar hyperplasia was seen in treated and control male rats. This is a common finding in rats of this age and is associated with hormonal changes that occur at maturity.
There was no evidence of alpha 2u-globulin nephropathy in the examined slides ofthe kidneys stained with hematoxylin and eosin.

Other microscopic findings occurred with comparable incidence and severity in rats from the treatment and control groups or they occurred sporadically; they were not considered to be treatment related. These incidental findings have been seen in rats of this strain and age used in other studies conducted in this facility.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 200 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Decrease in body weight gain at th next higher level tested
Dose descriptor:
NOAEL
Effect level:
627 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Recalculated to Piperazine base

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration, via dietary admixture, of Piperazine Dihydrochloride in rats, for a period of three months, produced decreased body weight gains at the 2400 mg/kg/day dose. The no observed effect level (NOEL), under the conditions of this study, was 1200 milligrams per kilogram of body weight per day. This value corresponds to 627 mg piperazine base per kg bw..
Executive summary:

This study was designed to assess the toxicity of Piperazine Dihydrochloride (CAS#: 142-64-3) when administered orally, via dietary admixture, to Sprague-Dawley CD® rats (20/sex/group) at doses of 400, 1200, and 2400 mg/kg/day for a period of at least 90 days. Control animals (20/sex) received untreated standard laboratory diet. ' '

Physical observations, ophthalmoscopic examinations, body weight and food consumption measurements were performed on all animals pretest and at selected intervals during the treatment period. Hematology, clinical chemistry, and urinalyses evaluations were performed on 10 males and 10 females prior to initiation ofthe study, and on 10 animals/sex/group following 1.5 and 3 months ofexposure to the test article. After at least 90 days of treatment, all animals were sacrificed, selected organs were weighed and organ/body weight and organ/brain weight ratios calculated. Complete macroscopic postmortem examinations were conducted on all animals. Histopathological evaluation of selected tissues were conducted on animals in the control and high-dose groups.

All animals survived and were free ofsignificant clinical signs throughout the study. The only treatnent-related effect seen was a statistically significant decrease in body weight gains, relative to control values, which occurred in high-dose males and females throughout most of the study. Differences in mean body weights of the control and high-dose groups were generally ten percent or greater. No effects of Piperazine Dihydrochloride administration were seen on food consumption, hematology, clinical chemistry, urinalysis or organ weight values. There were no significant macroscopic or microscopic changes which could be attributed to administration of Piperazine Dihydrochloride.

In conclusion, the oral administration, via dietary admixture, of Piperazine Dihydrochloride in rats, for a period of three months, produced decreased body weight gains at the 2400 mg/kg/day dose. The no observed effect level (NOEL), under the conditions of this study, was 1200 milligrams per kilogram of body weight per day (corresponding to 627 mg piperazin base per kg bw.