Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data taken from EU risk assessment. Piperazine is one constituent (ca. 15%) of the test substance.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): piperazine dihydrochloride
- Piperazine content is 50%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on mating procedure:
no data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
throughout maturation, mating, gestation and lactation phase for two successive generations
Frequency of treatment:
daily
Details on study schedule:
no data
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250, 600, 1250 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
125, 300, 625 mg/kg bw/day
Basis:
nominal in diet
corresponds to the piperazine base
No. of animals per sex per dose:
32 males and 32 females in F0 generation, 28 males and 28 females in F1 generation
Control animals:
yes, plain diet
Details on study design:
The animals were administrated piperazine dihydrochloride at doses of 0, 250, 600 and 1250 mg/kg bw/day, corresponding to 0, 125, 300 and 625 mg/kg/day piperazine base, in the diet throughout maturation, mating, gestation and lactation phases for two successive generations.
The F0 animals were dosed for 73 days for males and 17 days for females and paired within their respective dosage groups for up to 21 days. Exposure to diets continued throughout breeding, gestation and lactating periods for both generations.
At weaning of the offspring on day 21 post partum, 28 males and 28 females per dose group were selected at random to form the parental F1 generation. The remaining generation was sacrificed and examined macroscopically.
F1 animals were given piperazine in the diet for 80 days, and all animals were observed for sexual development. Males and females were paired for up to 21 days and pregnant females were allowed to deliver their offspring that were observed for growth and development.

Examinations

Parental animals: Observations and examinations:
Parental animals were observed daily for clinical signs, and the body weights and food consumption recorded weekly during the maturation phase, which was continued for males after the mating phase. Mated females were weighted and food consumption recorded on specific days post coitum and post partum. The offspring were observed daily for clinical signs and the body weight recorded. During the lactation period the offspring were observed for intra-litter onset and duration of landmarks of physical development. On specific days of lactation, reflexological assessment of offspring was performed. These tests included investigation on surface-righting reflex (day 1 post partum), mid-air righting reflex (day 17 post partum), startle reflex (day 21 post partum) and pupil reflex (day 21 post partum).
Postmortem examinations (parental animals):
The adult parental F0 animals as well as the F1 males and females were sacrificed and examined macroscopically post mortem. Selected tissues and organs were weighed and/or retained in fixative. Selected tissues and organs from the highest dose and control animals from F0 as well as from F1 adults were subjected to histopathological examination. In addition, in all F1 females the implantation sites were counted. The macroscopic post mortem findings were recorded. The histological examination were limited to the sex organs and the pituitary.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

At the highest dose one F0 female was found dead on day 19 post partum; no mortalities were seen at 300 or 125 mg/kg bw/day piperazine base.
No significant treatment related internal or external macroscopic lesions were noted in any of the dose groups, and no significant histopathological abnormalities could be detected microscopically in tissue sections from the reproductive organs from either males or females.
After 11 weeks of treatment before mating there was clear a reduced body weight increase in both sexes at 625 mg/kg bw/day piperazine base for the F0 as well as F1 animals. This effect that was more pronounced in the second generation (F0 females, 3%; F0 males, 9%; F1 females, 17%; F1 males, 20%).
There was a reduction in number of pregnancies in F1 (pregnancy index 81.5% vs 100% in controls), and a reduced litter size at birth for both generations (59% and 32% of control values in F1 and F2, respectively). No effects on live birth index, viability during lactation, or offspring physical development were noted when subjected to a set of tests for reflexes. There was a delay in sexual maturation (appearance of vaginal opening for females and preputial separation for males) in both F1 males and females, but no significant differences in offspring sex ratios were noted at any dose level.
At 300 mg/kg bw/day piperazine base, the effects on body weight gain was statistically significant in F0 males (9%). In the F1 parental generation, bodyweights were significantly reduced in both males and females after week 2, and there was also a slight reduction in food consumption at day 11 before mating (only sign for females F1). The food conversion ratios were similar to control values. There was no effect on the number of pregnancies, but a statistically significant reduced litter size at birth was noted in both generations (pregnancy index 91% and 85% of control values in F0-offspring, and F1-offspring, respectively). There was a reduction of implantation sites in F1 females (group mean =13.2 vs 16.6 in controls). There was a delay in sexual maturation (preputial separation) in F1 males, but no significant differences in offspring sex ratios. The group mean day of completion of offspring sexual development was also increased in females, although the increase was not statistically significant. It is unclear whether the delayed sexual development could be related to the decreased growth rate (body weight at sexual maturation was decreased by roughly 9%).
At 125 mg/kg bw/day piperazine base, no effects that could be related to the administration of piperazine were noted. The only clinical signs observed in the study are bright yellow urine in the bedding of all exposed females (all groups), but not in control animals or exposed males. With respect to effects on reproduction, 125 mg/kg bw/day piperazine base can be considered as a NOAEL, 300 mg/kg bw/day as a LOAEL with effects mainly on fertility.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day
Based on:
other: piperazine base
Sex:
male/female
Basis for effect level:
other: Based on reduced pregnancy index, decreased number of implantation sites and decreased litter size
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day
Based on:
other: piperazine base
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
For reproduction a NOAEL of 125 mg/kg bw/day and a LOAEL of 300 mg/kg bw/day can be established, with decreased litter size as the main effect.
Executive summary:

In a two-generation study performed according to OECD Guideline 416, rats were fed dietary doses of piperazine-dihydrochloride at dose levels of 125, 300 and 625 mg/kg bw/day, calculated as piperazine base. Body-weight decreases were found in the mid and high doses of the parents. A NOAEL of 125 mg/kg bw/day was established, on the basis of reduced pregnancy index, decreased number of implantation sites and decreased litter size.