Reaction mass of 2-Piperazin-1-ylethanol and Piperazine-1,4-diethanol and Piperazine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

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

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.

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.

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.

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

Reproductive effects observed:

not specified

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.

 

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Read across from one constituent of the mixture. Data taken from the EU risk assessment.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a two generation reproduction study in Sprague-Dawley CD rats performed according to OECD Test Guideline No. 416, groups of male and female animals were administered 0, 5000, 12000, or 25000 ppm (250, 600, or 1250 mg/kg bw/day) piperazine dihydrochloride (CAS 142-64-3) in the diet throughout maturation, mating, gestation and lactation phases for two successive generations (Wood and Brooks, 1994). Expressed as piperazine base, the doses represent 125, 300, and 625 mg/kg bw/day. The F0 males and females (32 per dose and sex) were dosed for 73 days for males and 17 days for females and paired within their respective dosage groups for up to 21 days. Subsequent exposure to diets continued throughout the breeding, gestation and lactation periods for both generations. At weaning of the offspring on day 21post 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 allowed to deliver their offspring that were observed for growth and development. The adult F0 animals as well as the F1 males and females were sacrificed and examined macroscopically post mortem. At the highest dose one F0 female was found dead on day 19post partum; no mortalities were seen at 300 or 125 mg/kg bw/day piperazine base. Also, 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. At 625 mg/kg bw/day piperazine base there was clear evidence of toxicity to the adult animals as judged by a statistically significant reduced body weight increase in both sexes for the F0 as well as F1 animals, an effect that was more pronounced in the second generation (F0 females, 3%; F0 males 9%; F1 females 17%, F1 males 20%). Further, there was a reduction in number of pregnancies, reaching statistical significance only in F1 (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), but no effects on live birth index, viability during lactation, or offspring physical development were noted when subjected to a set of reflexological tests. However, there was a delay in sexual maturation (appearance of vaginal opening for females and preputial separation for males) in both F1 males and females (not investigated in F2), but no significant differences in offspring sex ratios were noted at any dose level. However, it is likely that the delayed sexual observation could be related to the decreased body weights observed as from week 2 and onwards (roughly 25%, respectively). The reduced pregnancy index in combination with the decreased number of implantation sites and litter losses in F2-adults indicate pre- as well as post-implantation losses. At 300 mg/kg/day piperazine base, the effects on body weight gain were smaller, although statistically significant in F0 males (9%), but not in F0 females. In the F1 parental generation, bodyweights were significantly reduced in both males and females from week 2, and there was also a slight reduction in food consumption (F1 females, 9%; F1 males 9%). However, 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 (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 versus. 16.6 in controls). Further, there was a delay in sexual maturation (preputial separation) in F1 males (not investigated in F2), but no significant differences in offspring sex ratios. The group means 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/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/day piperazine base can be considered as a NOAEL, with 300 mg/kg/day as a LOAEL for this study, with effects mainly on fertility (i.e., reduced pregnancy index and decreased number of implantation sites, although litter losses in F2 may indicate post implantation losses as well).

Short description of key information:
No studies are available for hydroxyethylpiperazine mixture. Studies concerning toxicity to reproduction are available from piperazine dihydrochloride (CAS 142-64-3). This corresponds to 52.25% piperazine base which is one constituent of the hydroxyethylpiperazine mixture (piperazine CAS 110-85-0). Therefore these studies may be used as read across in order to assess the toxicity to reproduction of hydroxethylpiperazine mixture.
Two-generation study: NOAEL = 125 mg/kg bw/day (OECD 416, Wood and Brooks, 1994)

Justification for selection of Effect on fertility via oral route:
Only one study available

Effects on developmental toxicity

Description of key information

No studies are available for hydroxyethylpiperazine mixture. Studies concerning developmental toxicity are available from piperazine phosphate (CAS 14538-56-5). Piperazine is one constituent of the hydroxyethylpiperazine mixture (piperazine CAS 110-85-0). Therefore these studies may be used as read across in order to assess the developmental toxicity of hydroxethylpiperazine mixture.
Teratology study (rat): NOAEL = 2100 mg/kg bw/day (Ridgway, 1987b)
Teratology study (rabbit): NOAEL = 42 mg/kg bw/day (Ridgway 1987a)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data taken from EU risk assessment of piperazine. Piperazine is a constituent of the test substance and therefore used as read across.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

no data

Duration of treatment / exposure:

The females were treated from days 6 to 18 of pregnancy

Frequency of treatment:

daily

Duration of test:

The dams were killed on day 28 of pregnancy and necropsy performed.

Remarks:

Doses / Concentrations:
42, 94, 210 mg/kg bw/day
Basis:
actual ingested
calculated as piperazine base

No. of animals per sex per dose:

16 animals per dose group

Control animals:

yes, concurrent vehicle

Maternal examinations:

Clinical signs, body weight and body weight gain, food consumption.

Fetal examinations:

The foetuses were subjected to detailed external, visceral and skeletal examination.

Details on maternal toxic effects:

Details on maternal toxic effects:
At 210 mg/kg bw/day clinical signs of neurotoxicity as demonstrated by excessive salivation and nervousness noted in all treated animals. Other adverse effects were anorexia, reduced or no faeces production, reduced food intake ( by 85%, day 6 to 14) coupled with body weight loss (high dose animals lost 9% of body weight whereas controls gained 6%). Two females were sacrificed and one female aborted.The sacrificed females were found to have intestinal abnormalities including erosion of the mucosa of the stomach or duodenum.
At 94 mg/kg bw/day piperazine base, there were no effects on body weight, although food consumption (-39%) and body weight gain were reduced during the 4 first days of dosing. One female aborted, and five females were observed with reduced feaces production for short periods. One female died, but this was ascribed to accidental dosing into the lungs. No effects were observed at 42 mg/kg bw/day piperazine base.

Dose descriptor:

NOAEL

Effect level:

42 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

94 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
At 210 mg/kg bw/day piperazine base was highly embryotoxic and also demonstrated teratogenicity. Post-implantation loss was high with 100% resorption in four litters. Foetal weights were reduced and there was a slight retardation in ossification. In addition 15 of 56 (23%) foetuses (total of 8 litters produced) exhibited major abnormalities (6 cases of cleft palate and 9 cases of umbilical hernia) as compared with two of 86 (1.7%) in controls. The frequencies of major abnormalities in the four groups, expressed per litter, were 2/14, 4/14, 0/14 and 5/8 (with one additional case in an aborted high dose litter) in the control, low, mid, and high dose respectively. There was also an increased incidence of poorly ossified hindlimbs (epiphyses; 86% versus 40% variants in controls, and astragalus; 5.7% versus 0% of minor cases in controls) probably related to the maternal toxicity.
At 94 as well as 42 mg/kg bw/day, post-implantation loss, foetal weights, extent of ossification, and foetal sex ratios were uneffected by the treatment. Also there was no significant increase in foetal abnormalities at the two lowest dose levels.

Dose descriptor:

NOAEL

Effect level:

94 mg/kg bw/day (actual dose received)

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Only at doses also causing maternal toxicity teratogenic effects can be seen in the rabbit.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

94 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Read across from one constituent of the mixture. Two teratology studies done with rats and rabbits. Data was taken from EU risk assessment.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There is a teratology study in rats (OECD 414) available with piperazine phosphate as test substance (Ridgway, 1987b). Groups of 24 female Charles River CD(SD)BR rats were administered 250, 1000, or 5000 mg/kg bw of piperazine phosphate (corresponding to 105, 420 or 2100 mg/kg piperazine base) by gavage during pregnancy days 6 to 15. Clinical signs, body weight and food consumption were recorded and the animals sacrificed at day 20 and the foetuses subjected to detailed external, visceral and skeletal examinations. Although there were no treatment-related deaths, signs of maternal toxicity were observed at the highest dose level, including excessive salivation, lethargy and a reduction in bodyweight gain (days 6-15), body weight (7 % at day 15), as well as food consumption (14 % during days 6-11 and 9 % days 11-15). At this dosage, a lower foetal weight was also recorded (7 %), but no evidence of teratogenicity was reported at any dose level. Pre- and post-implantation losses, litter size and sex rations were unaffected by piperazine treatment.

A further teratology study was available with piperazine phosphate with rabbits (OECD 414, Ridgway, 1987b). Groups of 16 animals were dosed by oral intubation of 0, 100, 225, and 500 mg piperazine phosphate per kg bw and day suspended in 1% w/v methyl cellulose. The doses correspond to 0, 42, 94, or 210 mg/kg piperazine base). The females were treated from days 6 to 18 of pregnancy, while registering clinical signs, bodyweights and food consumption. The dams were killed on day 28 of pregnancy and necropsy performed. The foetuses were subjected to detailed external, visceral and skeletal examination. At 210 mg/kg/day piperazine base overt signs of toxicity were observed in the treated dams including signs of neurotoxicity as demonstrated by excessive salivation and nervousness noted in all treated animals. Other symptoms of adverse effects were anorexia, reduced or no faeces production, reduced food intake (e.g., by 85% days 6-14) coupled with body weight loss (high dose animals lost 9% of body weight whereas controls gained 6%). Two females were killed in extremis and one female aborted. The sacrificed females were found to have intestinal abnormalities including erosion of the mucosa of the stomach or duodenum. At 94 mg/kg/day piperazine base, there were no effects on body weight, although food consumption (-39 %) and body weight gain were transiently reduced during the 4 first days of dosing. One female aborted, and five females were observedwith reduced faeces production for short periods. One female died, but this was ascribed to accidental dosing into the lungs. No effects were observed at 42 mg/kg/day piperazine base. Although borderline, 94 mg/kg/day piperazine base may be considered to constitute the maternal LOAEL in this study.At 210 mg/kg bw, piperazine base was highly embryotoxic and also demonstrated teratogenicity. Post-implantation loss was high with 100% resorptions in four litters. Foetal weights were reduced and there was a slight retardation of ossification. In addition, 15 of 56 (23%) foetuses (in a total of 8 litters produced) exhibited major abnormalities (6 cases of cleft palate and 9 cases of umbilical hernia) as compared with two of 86 (1.7%) in controls. The frequencies of major abnormalities in the four groups, expressed per litter, were 2/14, 4/14, 0/14, and 5/8 (with one additional case in an aborted high dose litter) in the control, low, mid, and high dose, respectively. It is known that specific and rare abnormalities have also been observed in food-deprivation studies in rabbits. Thus, they can be considered to be secondary to the maternal toxicity. There was also an increased incidence of poorly ossified hindlimbs (epiphyses; 86% versus 40% variants in controls, and astragalus; 5.7% versus 0% of minor cases in controls) probably related to the maternal toxicity. At 94 as well as at 42 mg/kg bw piperazine base post-implantation loss, foetal weights, extent of ossification, and foetal sex ratios were unaffected by the treatment. Also, there was no significant increase in foetal abnormalities at the two lowest dose levels. Overall, the effects observed at 210 mg/kg bw/day piperazine base are considered to be secondary to maternal toxicity.

In summary, piperazine does not to appear to be teratogenic in the rat. In rabbits, such effects may be elicited at a dose level that is also toxic to the mother animal. The maternal LOAEL is 94 mg/kg bw/day, and the NOAEL 42 mg/kg/day piperazine base. The NOAEL for teratogenicity is 94 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:
Rabbits showed developmental effects at maternal toxicity. In rats no developmental toxicity was observed.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is considered to be classified for reproductive toxicity (R62/63) under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is considered to be classified for reproductive toxicity (Category 2) under Regulation (EC) No. 1272/2008.