Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 April - 24 December 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
RF study: The test material was administered orally, by dietary inclusion, to groups of mated female rats throughout the gestation and lactation phase of the reproductive cycle. Following weaning, selected offspring were exposed to the test material until six weeks of age. The dose levels were 5000, 25000 and 50000 ppm of Piperazine dihydrochloride in the diet with a similar size control group receiving untreated diet. Groups of 10 female F0 generation rats were paired to produce F1 litters. At weaning of the
offspring, animals were selected at random to form the F1 generation. The F1 animals were treated for a minimum of 3 weeks and then the study was terminated. Parental animals were observed twice daily for clinical signs of toxicity. Mated females were
weighed and food consumption recorded on specific days post coitum and postpartum. The offspring were observed daily for clinical signs of toxicity. The litter signs and individual pup bodyweights were recorded on specific days post partum. Post mortem macroscopic examinations were performed on all adults and offspring, including decedents.
Adults: At 50000 ppm there was severe toxicity observed which included adult mortality and clinical signs of toxicity including piloerection and hunched posture for all animals. Bodyweight gain and food consumption were significantly reduced compared to controls. At necropsy effects on the upper gastrointestinal tract, notably gastric ulceration, were observed. At 25000 ppm there was one mortality and clinical signs of toxicity but at a lower incidence than at 50000 ppm. There was a reduction in bodyweight gain and food consumption. At 5000 ppm all parameters were comparable to control values. Minor clinical signs were considered to be an adaptation to the test material.
Litters: At 50000 ppm no offspring survived until weaning. At 25000 ppm there was an overall slight reduction in offspring viability from birth to weaning. There was a reduction in offspring bodyweight gain particularly during the last week of lactation. At 5000 ppm offspring viability and growth were comparable to control values.
Selected F1 Offspring: There were no significant findings for selected offspring up to six weeks of age.
Administration of Piperazine dihydrochloride at 25000 ppm and 5000 ppm represent the maximum high dose and low dose levels, respectively, recommended for a subsequent two generation reproduction study in the rat.

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:
Sponsor's identification: Ascarex D
Chemical name: Piperazine Dihydrochloride was used (CAS 142-64-3 / EC 205-551-2)
Batch number: 373033
Date received: 24 December 1993
Description: off-white solid
Storage conditions: room temperature
The test material was prepared as a dietary admixture, weekly by preweighing an aliquot of the test material into a suitable container. The aliquot of test material was mixed with the appropriate quantity of untreated diet. The test material/untreated diet
was mixed together for 30 minutes using a Hobart mixer. The test material/diet mixture was transferred to a suitable container with a colour coded label and date of preparation. This process was repeated for each dose level.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen for this study as it represents the species historically used for safety evaluation studies and is specified by the principle regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
A total of 140 male and 142 female Sprague-Dawley CD strain rats were ordered from Charles River (U K) Limited, Margate, Kent. On arrival the animals were examined and found to be in good health. The animals were ordered in the age range of
approximately 4 weeks and 8 weeks, at arrival, for males and females respectively.The animals were acclimatised to the laboratory conditions for at least 17 days before the start of the study.

The animals were housed in air-conditioned animal rooms, providing at least 15 air changes per hour. The room was maintained to operate within a temperature range of 19 and 25°C and a relative humidity of 40 and 70%, with these conditions monitored on a daily basis. On isolated occasions the temperature and/or humidity was outside specified limits. It was concluded that these variations did not adversely affect the outcome of this study. The lighting within the room was controlled to allow 12 hours of continuous light within a 24-hour period.

Upon arrival, and subsequently through the maturation periods for F0 and F1 adults, the animals were housed in groups of four in polypropylene cages with stainless steel grid floors and tops suspended over paper-lined polypropylene trays.

Following evidence of successful mating, the males were returned to their original cages and the females were housed, individually, in polypropylene cages with solid floors and stainless steel tops. Mated females were given softwood chips as bedding
throughout gestation and lactation.

Throughout the study the animals were given powdered diet (SQC Rat and Mouse Breeder Diet No. 3, Expanded Ground, Special Diets Services Limited, Witham, Essex, UK) ad Iibitum. Each batch of diet is analysed for contaminants. Mains water was
supplied from polycarbonate bottles attached to the cage. The water is periodically analysed for contaminants. It was concluded that the level of contaminants did not affect the purpose or integrity of the study.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test material was administered by direct dietary inclusion at a constant dietary concentration throughout. Control animals were given untreated diet.
Details on mating procedure:
During the mating periods for F0 and F1 adults the animals were transferred to a similar type cage on a one male to one female per cage basis.
After their respective maturation periods, the F0 and F1 generation adults were paired on a 1 male to 1 female basis for a period of up to 21 days. Following pairing, the polypropylene trays beneath each cage were checked each morning for the presence of ejected copulation plugs. Additionally each female was checked for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating. Mated females were then separated from the male and housed individually during the period of gestation and lactation. The males were returned to their original holding cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the start of the study a procedure was developed to prepare the test material for dose administration. These preparations were analysed for achieved concentration, stability and homogeneity.
During the study, samples of the test material/diet mixes were taken weekly for analysis of achieved concentration of the preparations.
Duration of treatment / exposure:
Prior to pairing for each generation, the test material was administered in the diet to the F0 generation males and females for 73 and 17 days respectively; F1 males and females received treated diet for 80 days. Subsequently exposure to diets continued
throughout the breeding, gestation and lactation periods for both generations.
Frequency of treatment:
Daily via the diet.
Details on study schedule:
Seven days before the start of dosing the animals to be used for the F0 generation were assigned to treatment groups using a randomisation procedure based on stratified bodyweight to ensure comparable group mean bodyweights for each treatment group. The weight range for males and females at allocation was 202 to 273g and 232 to 286g respectively.
At or around weaning, offspring from the F0 generation matings were selected to form the F1 generation. Within each dose group, offspring were selected from those litters nearest the median weaning date. One male and one female were selected at random using the random number tables. Where insufficient litters were available to provide offspring for the F1 generation, a second male and female were selected from appropriate litters.
- F0 generation animals were dosed with their respective treated diets for 73 and 17 days for males and females respectively.
- F0 males and females were paired within their respective dose groups for up to 21 days.
- Following evidence of mating, the animals were separated and males returned to their holding cages.
- The pregnant females were allowed to deliver their offspring. The offspring were observed for grt and development during lactation.
- At or around weaning on day 21 postpartum the offspring were selected at random to form the F1 generation. The remaining offspring were killed and examined macroscopically post mortem.
- The adult F0 animals were killed and examined macroscopically post mortem. Selected tissues and organs were weighed and/or retained in fixative. Tissues and organs from the high dose and control animals were processed and
subsequently examined microscopically by a pathologist.
- F1 animals were dosed with their respective treated diets for 80 days. All animals were observed for sexual development.
- F1 males and females were paired within their respective dose groups for up to 21 days.
- Following evidence of mating the animals were separated and males returned to their holding cages.
- Pregnant F1 females were allowed to deliver their offspring. The offspring were observed for growth and development.
- At or around Day 21 F1 males and females and their offspring were killed and examined macroscopically post mortem with tissues from adult F1 animals preserved for microscopic examination. Additionally, implantation sites were
counted on all F1 females.
- Selected tissues from high dose and control F1 adults were processed and examined microscopically.
Doses / concentrationsopen allclose all
Dose / conc.:
5 000 mg/kg diet
Dose / conc.:
12 000 mg/kg diet
Remarks:
calculated as piperazine base
Dose / conc.:
25 000 mg/kg diet
Remarks:
calculated as piperazine base
No. of animals per sex per dose:
32 males and 32 females per group in the F0 generation, 28 males and 28 females per gorup in the F1 generation.
Control animals:
yes, plain diet
Details on study design:
Based on the levels in the diet, the animals were administrated piperazine dihydrochloride at 0, 250, 600 and 1250 mg/kg bw/day ( corresponding to 0, 125, 300 and 625 mg/kg bw/day piperazine base) throughout maturation, mating, gestation and lactation phases for two successive generations.
Positive control:
Not used.

Examinations

Parental animals: Observations and examinations:
All animals were checked twice daily during the normal working week and once daily on weekends and public holidays.
All animals were observed twice daily for clinical signs of toxicity and reported weekly.
During the maturation and mating period the F0 and F1 generation adults were weighed weekly. Following mating the F0 and F1 males were weighed weekly until termination. F0 and F1 generation females showing evidence of mating were weighed
on days 1, 4, 7, 14 and 21 post coitum. F0 and F1 generation females with a live litter were weighed on days 1, 4, 7, 14 and 21 postpartum.
During the maturation periods food consumption was recorded for each cage of F0 and F1 generation adults. For F0 and F1 generation females showing evidence of mating, food consumption was recorded for the periods covering days 1 to 7, 7 to 14 and 14 to 21 post coitum. For F0 and F1 generation females with live litters, food consumption was recorded for the periods covering days 1 to 7 and 7 to 14 post partum.
Calculated weekly during the maturation period for the F0 and F1 generations: Food Conversion Ratio = Group mean bodyweight gain (g/day) during week / Group mean food consumption (g/rat/day)

Each pregnant F0 and F1 female was observed at 0830, 1230 and 1630 hours at or around the period of expected parturition. At weekends, observations were carried out at 0830 and 1230 hours only. The following was recorded for each female.
i) Date of mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition

Calculated weekly during the maturation period for the F0 and F1 generations: Chemical Intake = Dose level (ppm) x group mean food consumption (g/rat/day) / Group mean bodyweight for week
Litter observations:
At the observation of completion of parturition, the number of live and dead offspring was recorded. The date and time of Day 1 post partum litter observations were recorded.
Individual offspring weights were recorded on days 1, 4, 7, 14, 21 post partum.
The number of offspring was recorded daily, up to weaning, and reported for days 1, 4, 7, 14 and 21 postpartum. Offspring sex was recorded for days 1 and 21 post partum.
The clinical condition of individual offspring was observed daily and any findings recorded.
Live offspring were observed for the following landmarks of development.
- Detachment of pinna - as noted by the separation of the edges and subsequent unfolding of both pinnae.
- Tooth Eruption - as noted by the eruption of the'upper incisors through the gum.
- Eye Opening - as noted by the separation of the upper and lower eyelids of both eyes.
Offspring Reflexological Assessment - Live offspring were assessed for reflexological response to various stimuli as follows:
- Surface Righting Reflex - on day 1 postpartum, offspring were tested for their ability to turn over to a normal resting position when placed on their back on a flat surface.
- Mid-air Righting Reflex - on day 17 postpartum, offspring were tested for their ability to turn their body to a normal upright position, in mid-air, when dropped from a standard height above a bed of sawdust.
- Startle Reflex - on day 21 postpartum, auditory function is assessed by the reaction of offspring to a short auditory stimulus.
- Pupil Reflex - on day 21 postpartum, offspring pupil reaction to light, shone on each eye, was assessed.
- F1 Sexual Development - All F1 males and females were observed for sexual development. For females the appearance of vaginal opening (separation of labia) was recorded; for males the separation of the prepuce from the glans penis was recorded.
Postmortem examinations (parental animals):
All adult animals, killed in extremis or found dead during the course of the study, were examined macroscopically for internal and external abnormalities. All significant abnormalities were retained in fixative for possible further study. All offspring that died, or were killed in extremis during the lactation period, were examined macroscopically internally and externally.
Following successful weaning of the offspring from the F0 and F1 matings, all the surviving adults, including non-fertile animals, were killed by carbon dioxide asphyxiation. All animals were examined macroscopically for both internal and external abnormalities.
The uteri of all F1 females at terminal necropsy were stained with 10% ammonium polysulphide solution and the number of implantation sites visible was recorded.
The following list of tissues and organs, from all adult F0 generation animals at terminal necropsy were preserved in fixative: Ovaries, Seminal Vesicles, Uterus, Prostate, Cervix, Coagulating gland, Vagina, Pituitary gland, Testes, Epididymides, and significant abnormalities.
All the above tissues and organs were preserved in 10% formalin except the testes which were retained in Bouin's fluid for 48 hours and then transferred to 70% industrial methylated spirits.
The tissues from high dose and control animals were processed and embedded in paraffin wax B.P. (mp 56°C). Sections of the tissues were taken at 5 pm thickness, mounted on glass slides and stained with haematoxylin and eosin.
The sections of tissues from control and high dose animals were examined microscopically by a pathologist.
Postmortem examinations (offspring):
Unselected offspring from the F0 mating and all F1 offspring were killed by carbon dioxide asphyxiation. All offspring were examined macroscopically for internal and external abnormalities. Any significant abnormalities were retained in the appropriate fixative.
Statistics:
The following parameters were analysed statistically, where appropriate using the test methods outlined below:
- Adult male and female bodyweight during the maturation, gestation and lactation periods, adult male food consumption, female food consumption during maturation, gestation and lactation, litter size, litter weight, individual offspring bodyweight: values were analysed by F-max test to establish homogeneity of group variances followed by one way analysis of variance. Where significant differences were seen, pairwise comparison of control values against treated group values was performed
using Students 't' test.
- Gestation length, offspring sex ratios, landmarks of offspring physical development and reflexological responses and group mean pre-coital length and female implantation sites: iindividual values were compared using the Kruskall Wallis non-parametric rank sum test. Where significant differences were seen, pairwise comparison of control values against treated group values was performed using the Mann-Whitney U-test.
- Mating, Pregnancy and Parturition lndices: probability values were established using the Fisher Exact probability test.
- Offspring Viability lndices: probability values were established using Chi-squared analysis.

Histopathology:
- Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater
Reproductive indices:
The following parameters were calculated from individual data during the mating period for the F0 and F1 generations.
Pre-coitallnterval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
Fertility lndices; for each group the following were calculated:
- Mating Index (%) = Number of animals mated x 100 / Number of animals paired
- Pregnancy Index (%) = Number of pregnant females x 100 / Number of animals mated
Gestation Length: Calculated as the number of days of gestation including the days for observation of mating and the start of parturition. Where the start of parturition occurred overnight, the total was adjusted by subtracting half a day to the completion day
Gestation and Parturition Index; the following was calculated for each group:
- Parturition Index (%) = Number of females delivering live pups x 100 / Number of pregnant females
Offspring viability indices:
The following indices were calculated for each group from individual data:
- Live Birth Index (%) = Number of pups alive on Day 1 x 100 / Number of pups born
- Viability index 1 (%) = Number of pups alive on Day 4 x 100 / Number of pups alive on Day 1
- Viability Index 2 (%) = Number of pups alive on Day 7 x 100 / Number of pups alive on Day 4
- Viability Index 3 (%) = Number of pups alive on Day 14 x 100 / Number of pups alive on Day 7
- Viability Index 4 (%) = Number of pups alive on Day 21 x 100 / Number of pups alive on Day 14
- Viability Index 5 (%) = Number of viable pups at weaning x 100 / Number of pups on Day 1
Sex Ratio; group mean values were calculated from each litter value on Day 1 and 21 using the following formula: Number of male pups x 100 / Number of pups of determined sex

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
For all females at all dose levels, excluding controls, the most significant clinical sign was bright yellow stained urine on bedding. This was only observed when females were transferred to cages with a sawdust bedding.
There were no other significant clinical signs observed for either males or females at any dose level.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 25000 ppm one female, number 250, was found dead on Day 19 post partum. There was no significant clinical history and no abnormalities were observed at macroscopic necropsy or histopathology.
At 12000 ppm and 5000 ppm there were no mortalities.

At 0 ppm three females (numbers 34, 40 and 43) were killed in extremis. Female 34 was killed in extremis on Day 15 postpartum. Previous clinical history included hunched posture, pilo-erection, tiptoe gait, distended abdomen and no faeces on
bedding. The clinical signs were present for two days prior to termination. At necropsy the small intestine was found to be distended with either liquid or compacted faecal material, with areas of thickened mucosa. Histopathology showed areas of ulcerated and necrotic intestinal tract. Female number 40 was killed in extremis on Day 10 post partum. The clinical signs immediately prior to termination were hunched posture, lethargy, pilo-erection, increased respiratory rate and pallor of the extremities. Macroscopic examination, postmortem, showed the principal macroscopic lesion was haemorrhagic areas on the lung with two of these areas having a pale/green core. Histopathology of the lung-showed bronchiolitis and extensive pneumonitis. Female number 43 was killed in extremis at/around the time of parturition. The clinical signs immediately prior to termination were hunched posture, lethargy, pilo-erection, tiptoe gait and pallor of the extremities. At macroscopic examination a large dead foetus was found within the uterus. Histopathology showed an ulcerated and necrotic area on the uterine endometrium.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Maturation:
At 25000 ppm there was a statistically significant reduction in male bodyweight gain compared to controls from the start of treatment (p<0.01) and subsequently from week 2 (p<0.001). The reduction in bodyweight gain was continued until week
7 of treatment and subsequently the difference between high dose and control males was maintained. Females prior to pairing showed a slight reduction in bodyweight gain from the start of treatment which resulted in a statistically significant difference (p<0.05) in group mean bodyweights at study week 11.
At 12000 ppm there was a reduction in male bodyweight gain compared to controls throughout maturation. This resulted in a statistically significant difference (p < 0.05) in group mean bodyweights compared to controls (weeks 3, 6, 7, 8, 9, 10) and
p<0.01 at week 11. Female bodyweight gain was comparable to controls.
At 5000 ppm there was a slight but statistically not significant reduction in male bodyweight gain compared to controls. There was no difference in female bodyweight gain compared to controls.

Gestation
At 25000 ppm there was a statistically significant difference in group mean bodyweights compared to controls during gestation (p<0.01 Day 1 post coitum, p<0.001 Days 4-21 post coitum).
At 12000 ppm there was a statistically significant, p < 0.05, difference in bodyweight at gestation Day 14 only.
At 5000 ppm there were no significant differences in bodyweights compared to controls.

Lactation:
At 25000 ppm there was a statistically significant difference in group mean bodyweights compared to controls (p<0.05 Day 1 post partum, p<0.001 Days 4-21 postpartum).
At 12000 ppm there was a slight but statistically not significant difference in group mean bodyweight compared to controls at Day 21 post partum.
At 5000 ppm there was no significant differences in group mean bodyweight compared to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Maturation:
There were no consistent differences in food consumption for males and females from all treated groups compared to control values. The isolated incidences of statistically significant differences were considered to be incidental.

Gestation:
At 25000 ppm there was a statistically significant (p<0.001) reduction in food consumption between Days 7 and 14 post coitum.
At 12000 ppm and 5000 ppm there were no significant differences in food consumption throughout gestation.

Lactation:
At 25000 ppm there was a statistically significant reduction in food consumption (p<0.01 Days 1-7 post partum, p<0.001 Days 7-14 post partum) compared to controls.
At 12000 ppm and 5000 ppm there were no significant differences compared to controls.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Maturation:
At 25000 ppm, the reduced bodyweight gain and the equivalence in food consumption compared to controls resulted in a reduced food conversion ratio during maturation for males and females.
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 significant abnormalities seen at histopathology.
All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered
to be without toxicological significance.
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 25000 ppm there was a reduction in the number of pregnancies. This was due to a combination of a reduced mating index and pregnancy index. Both parameters were reduced compared to controls but did not attain statistical significance. The
distribution of pre coital intervals is comparable to controls although the proportion of animals mating on the first day is significantly lower (p<0.001) than controls. This is toxicologically not significant. At 12000 ppm and 5000 ppm there were no
significant differences in fertility compared to controls.

At 25000 ppm there was a slight increase (p<0.05) in the gestation lengths for the group compared to controls. All other gestation indices were comparable to controls.
At 12000 ppm and 5000 ppm there were no effects on gestation indices.

At 25000 ppm there was a statistically significant (p<0.001) reduction in group mean litter size at birth compared to controls. This difference in litter size was maintained throughout lactation, although offspring viability after birth was comparable to controls.
At 12000 ppm mean live birth index shows a lower (p<0.05) group mean value compared to controls for those females maintaining their litter until weaning. Offspring viability during lactation was comparable to controls.
At 5000 ppm live birth litter size and offspring viability during lactation were comparable to control values.

Details on results (P0)

During the pre-mating period food intake was recorded which resulted in mean food intake as follows (see tables attached):
Males:
Low dose: 376 mg piperazine dihydrochloride /kg bw/day corresponding to 204 mg piperazine base/kg bw/day
Mid dose: 925 mg piperazine dihydrochloride /kg bw/day corresponding to 501 mg piperazine base/kg bw/day
High dose: 2010 mg piperazine dihydrochloride /kg bw/day corresponding to 1089 mg piperazine base/kg bw/day
Females:
Low dose: 409 mg piperazine dihydrochloride /kg bw/day corresponding to 222 mg piperazine base/kg bw/day
Mid dose: 972 mg piperazine dihydrochloride /kg bw/day corresponding to 526 mg piperazine base/kg bw/day
High dose: 2056 mg piperazine dihydrochloride /kg bw/day corresponding to 1113 mg piperazine base/kg bw/day
Group mean test material intake was not calculated for females during gestation and lactation. However, individual food intake values were recorded. These were generally higher during gestation and 3-4 times higher during lactation, indicating that the mean intakes indicated above represent the lower band of test material intake.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
222 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: As piperazine base; based on reduction in BW gain, food intake, reduction in number of implantation sites and in live litter size at the next higher level tested
Dose descriptor:
NOAEL
Effect level:
204 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: As piperazine base; based on reduction in BW gain and food intake at the next higher level tested (see further at females)

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Similar clinical signs were observed as seen in the F0 generation for all treated females. There were no clinical signs in any animals on this study that could be attributable to a specific neurotoxic effect.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 25000 ppm, one female (number 454) was killed in extremis at/around the period of parturition. The female showed evidence of dystocia. At necropsy, the head of the dead foetus was visible at the vaginal entrance. A single large dead foetus was present within the vagina/cervix. Histopathology showed marked dilatation of one horn of the uterus. One female (number 471) was found dead during gestation. Previous clinical history included hunched posture and vocalisation for 14 days prior to death. At necropsy two foetuses and two resorptions present in utero with clotted blood adherent to placentae. These signs are consistent with findings of dystocia. In addition the glandular region of the stomach was thickened with areas of focal haemorrhage. Histopathology showed marked dilatation of 1 horn of the uterus with an area of haemorrhage.
At 12000 ppm one female (number 408) was killed in extremis due to dystocia. Hunched posture was seen on the day of termination. At necropsy the left horn of the uterus was surrounded by compacted fat with ovary adherent to this. The left horn of the uterus contained a single large foetus with head engaged at cervix.
At 5000 ppm one male (number 328) was killed in extremis due to reddening and scabbing around the right eye. At necropsy these findings were confirmed and additionally the eyelid was found swollen and thickened and the harderian gland
enlarged. One female (number 342) was killed in extremis after parturition. Clinical signs from the previous day included hunched posture, lethargy, pilo-erection, ptosis and chromodacryorrhoea. On the day of termination, pallor of the extremities was seen. Necropsy findings included displacement of upper incisors, minimal perinephric fat and pallor of the kidneys. Digested blood was found in the gastro-intestinal tract.
At 0 ppm there were no mortalities..
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Maturation:
At 25000 ppm there was a statistically significant difference in male and female bodyweights compared to controls (p<0.001) from the start of treatment until completion of the maturation phase for females and termination for males.
At 12000 ppm male bodyweight gain from week 2 was reduced compared to controls. This resulted in reduced bodyweights compared to controls and attained variable levels of statistical significance (from p<0.05 to p<0.001). Female bodyweight gain was also reduced from week 2 which resulted in statistically significant differences in group mean bodyweight compared to controls (p<0.01 - weeks 3 and 4, p<0.001 - weeks 5 to 12 of maturation).
At 5000 ppm male and female bodyweight gain was comparable to controls. On week (4) male group mean bodyweight was reduced compared to controls which attained statistical significance (p<0.05).

Gestation:
At 25000 ppm and 12000 ppm there was a significant reduction (p<0.001) in group mean bodyweight compared to controls throughout gestation.
At 5000 ppm there was a significant reduction (p<0.05) in group mean bodyweight compared to controls on Day 21 of gestation. During the rest of the gestation period, bodyweight was comparable to controls.

Lactation:
At 25000 and 12000 ppm there was a statistically significant reduction in group mean bodyweight compared to controls (p<0.001) throughout lactation except 12000 ppm - Day 1 of lactation (p<0.01).
At 5000 ppm there were no significant effects on bodyweight throughout lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Maturation:
At 25000 ppm there was a reduction in male and female food consumption compared to controls which varied in the level of statistical significance (p<0.005 to p<0.001) achieved throughout the maturation period.
At 12000 ppm there was a reduction in male and female food consumption compared to controls throughout the maturation period. Occasionally the reduction achieved statistical significance (p<0.05 to p<0.01).
At 5000 ppm there were minimal differences in male and female food consumption compared to controls. A statistically significant (p<0.05) difference in male food consumption occurred on one occasion.

Gestation:
At 25000 ppm there was a statistically significant (p<0.001) reduction in group mean food consumption throughout gestation compared to controls.
At 12000 ppm there was a statistically significant (p<0.01) reduction in group mean food consumption throughout gestation compared to controls.
At 5000 ppm there was a statistically significant (p < 0.05) reduction in group mean food consumption from Day 7 to Day 21 of gestation compared to controls.

Lactation:
At 25000 ppm there was a statistically significant reduction (p<0.001) in group mean food consumption during Days 1 to 14 postpartum.
At 12000 ppm there was a statistically significant reduction (p< 0.01) in group mean food consumption compared to controls during days 7 to 14 postpartum.
At 5000 ppm there were no significant differences in food consumption compared to controls.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Maturation:
At 25000 ppm an overall reduction in food conversion ratio was seen compared to controls.
At 12000 ppm and 5000 ppm the food conversion ratios for F0 and F1 generations were similar to control values.

Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment related findings for adults at post mortem macroscopic examination.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no significant abnormalities seen at histopathology.
All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Reproductive function / performance (P1)

Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 25000 ppm there was a significant (p<0.05) reduction in pregnancy rate. A number of females were found to have uterine resorptions only, when the uteri were examined post mortem.
At 12000 ppm and 5000 ppm there were no significant differences in fertility parameters examined.

At 25000 ppm there was a slight increase (p<0.05) in the gestation lengths for the group compared to controls. All other gestation indices were comparable to controls.
At 12000 ppm and 5000 ppm there were no effects on gestation indices.

At 25000 ppm there was a significant reduction in live birth litter size (p<0.001). Offspring viability postpartum was comparable to controls if the number of viable pups was taken into consideration. Evaluation of the uteri of females at necropsy
showed that a significant number of females had embryo implantations but all resorbed prior to term. The number of implantations per female was still reduced compared to control implantation number.
At 12000 ppm there was a significant reduction (p<0.01) in live birth litter size and a significant reduction in uterine implantation number (p<0.001). Offspring viability during lactation was comparable to controls.
At 5000 ppm live birth litter size, implantation number and offspring viability were comparable to controls.

Details on results (P1)

During the pre-mating period food intake was recorded which resulted in mean food intake as follows (see tables attached):
Males:
Low dose: 536 mg piperazine dihydrochloride /kg bw/day corresponding to 290 mg piperazine base/kg bw/day
Mid dose: 1183 mg piperazine dihydrochloride /kg bw/day corresponding to 641 mg piperazine base/kg bw/day
High dose: 2635 mg piperazine dihydrochloride /kg bw/day corresponding to 1427 mg piperazine base/kg bw/day
Females:
Low dose: 511 mg piperazine dihydrochloride /kg bw/day corresponding to 277 mg piperazine base/kg bw/day
Mid dose: 1220 mg piperazine dihydrochloride /kg bw/day corresponding to 661 mg piperazine base/kg bw/day
High dose: 2725 mg piperazine dihydrochloride /kg bw/day corresponding to 1476 mg piperazine base/kg bw/day
Group mean test material intake was not calculated for females during gestation and lactation. However, individual food intake values were recorded. These were generally higher during gestation and 2-4 times higher during lactation, indicating that the mean intakes indicated above represent the lower band of test material intake.

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Effect level:
277 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: As piperazine base; based on reduction in BW gain, food intake, reduction in number of implantation sites and in live litter size at the next higher level tested
Dose descriptor:
NOAEL
Effect level:
290 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: As piperazine base; based on reduction in BW gain and food intake at the next higher level tested (see further at females)

Target system / organ toxicity (P1)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
The proportion and range of clinical signs observed for offspring were comparable.for all groups.
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 25000 ppm there was a significant reduction in group mean litter weights (p<0.001) compared to controls. This is reflective of smaller litter sizes. Individual offspring values were comparable to controls.
At 12000 ppm there was a significant difference in litter weights compared to controls (p<0.05) at Day 21.
At 5000 ppm there were no significant differences.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
At 25000 ppm the differences in completion of development landmarks were considered incidental.
At 12000 and 5000 ppm there were no significant differences.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment related macroscopic post mortem findings among interim deaths or terminal kills.
Histopathological findings:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Offspring Reflexological Assessment: there were no significant differences.

Details on results (F1)

No effects on sex ratio.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 089 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: as piperazine base; no treatment-related effects; lowest dose chosen (male parents 1089 /mg/kg bw; female parents 1113 mg/kg bw)

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
The proportion and range of clinical signs observed for offspring were comparable for all groups.
Description (incidence and severity):
At 25000 ppm there was a reduction in group mean litter weights (p < 0.001) which was principally a result of reduced litter size. Initial individual offspring bodyweights on Days 1 to 4 were significantly higher than controls due to reduced
litter size.
At 12000 ppm there was a reduction in group mean litter weights of variable statistical significance. This is also reflective of litter size as individual bodyweights are comparable to controls.
At 5000 ppm there were no significant differences compared to controls.
Description (incidence and severity):
At 25000 ppm, the time of completion of sexual development of selected F1 offspring was increased compared to controls which was statistically significant (p<0.001) for males and females.
At 12000 ppm, the time of completion of sexual development was increased compared to controls which was statistically significant (p<0.01 for males and p<0.001 for females).
At 5000 ppm there were no significant differences compared to controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment related macroscopic post mortem findings among interim deaths or terminal kills.
Description (incidence and severity):
.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Offspring Reflexological Assessment: there were no significant differences.

Details on results (F2)

No effects on sex ratio.

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
277 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: as piperazine base; delay in sexual development at next two higher levels tested. Lowest dose chosen (male parents 290 mg/kg bw; female parents 277 mg/kg bw)

Target system / organ toxicity (F2)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Treatment through the reproductive cycle of the rat for two successive generations with Piperazine Dihydrochloride at dose levels of 5000 ppm and above resulted in dose-related effects on the adults. Dose levels of 12000 ppm and above resulted in a dose-related reduction in litter size. The effects seen on adults and offspring were enhanced in the second generation. The no effect level for effects on the reproduction parameters evaluated was 5000 ppm.
The dietary level of 5000 ppm resulted in a mean intake of piperazine base of 204 mg/kg bw/day for P0 males and 290 mg/kg bw/day for P1 males. For P0 females the mean intake of piperazine base was at least 222 mg/kg bw/day, and for P1 females 277 mg/kg bw/day, as individual food intake values were higher during gestation and 2-4 times higher during lactation.
Executive summary:

The test material was administered orally, by dietary inclusion, to groups of male and female rats throughout the maturation, mating, gestation and lactation phases of two successive generations. The dose levels were 5000, 12000 and 25000 ppm of the test material in the diet with a similar size control group receiving untreated diet.

Following 10 weeks of treatment, groups of 32 male and 32 female F0 generation rats were paired to produce F1 litters. At weaning of the offspring, animals were selected at random to form the parental F1 generation. Parental animals were observed daily for clinical signs. Bodyweights and food consumption were recorded weekly during the maturation phase which was continued for males after the mating phase. Mated females were weighed and food consumption recorded on specific days post coitum and post partum. The offspring were observed daily for clinical signs. The litter signs and individual pup bodyweights were recorded on specific days post partum. During the lactation period the offspring were observed for intra—Iitter onset and duration of landmarks of physical development. On specific days of lactation, reflexological assessment of offspring was performed. Post weaning sexual development was assessed for selected F1 males and females. Post mortem macroscopic examinations were performed on all adults and offspring, including decedents. Reproductive organs from all parental animals were preserved in fixative. Histopathology was carried out on specific organs from selected parental F0 and F1 animals.

At 25000 ppm there was evidence of adult toxicity including reduced bodyweight gain and food consumption thoughout the dosing period. The effects were more marked in the F1 generation. Evaluation of reproductive performance showed a reduction in the number of pregnancies and a reduced live litter size at birth for both generations. Evaluation of the uterine implantation sites of the F1 generation indicated a number of females with no live offspring at birth had a small number of resorbed embryos in utero. There were no effects on offspring viability during lactation. There were no effects on the reproductive organs.

At 12000 ppm, similar, but less marked effects on bodyweight gain and food consumption were noted. There was no effect on the number of pregnancies but there was a reduced live litter size in both generations. Evaluation of the uterine implantation data showed a reduction in implantation sites for F1 females.

At 25000 and 12000 ppm there was a delay in sexual development of F1 males and females.

At 5000 ppm there were slight and inconsistent effects on adult bodyweight gain and food consumption for both generations. There were no effects on any of the reproductive parameters evaluated.

Treatment through the reproductive cycle of two successive generations of the rat with piperazine dihydrochloride at dose levels of 5000 ppm and above resulted in dose related effects on the adults. Dose levels of 12000 ppm and above resulted in a dosage related reduction in live litter size. The effects on adults and offspring were enhanced in the second generation. The no effect level for effects on reproductive parameters evaluated was 5000 ppm.