4-(2-hydroxyethyl)piperazin-1-ylethanesulphonic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

The No Observed Effect Level (NOEL) for maternotoxicity and embryofetal development is 1000 mg/kg bw/day (reference 7.8.2 -1).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 2003 - Mar 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: mean body weight of 273 g (range: 230 g to 338 g).
- Fasting period before study: no
- Housing: housed individually in suspended wire-mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The test item was dissolved in the required quantity of vehicle in order to achieve the concentrations of 20, 60 and 200 mg/mL and then homogenized using a magnetic stirrer. The test item dosage forms were prepared daily and delivered to the study room at room temperature.

VEHICLE
- Amount of vehicle: 5 mL/kg/day

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Females were mated at the breeder's facilities (no detailed information available). The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum.

Duration of treatment / exposure:

14 days (from day 6 to day 19 post-coitum)

Frequency of treatment:

once per day (at approx. the same time)

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: results in preliminary study (CIT/Study No. 23826 RSR). In this study, no effect on maternotoxicity or on litter data parameters were observed at the dose-levels of 100, 300 and 1000 mg/kg/day; consequently the same dose-levels were chosen for the main study.
- Rationale for animal assignment: random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- at least twice a day during treatment period
- at least once a day on other days.
- Cage side observations were: Morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day

BODY WEIGHT: Yes
- Time schedule for examinations: on days 2, 6, 9, 12, 15, 18 and 20 post-coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: ovaries and uterus of females were examined to determine:
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of implantation sites (or uterine scars).
Also principal thoracic and abdominal organs were examined. A gross evaluation of placentas was also performed.

OTHER: Food consumption

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all
- Soft tissue examinations: Yes: approx. half per litter
- Skeletal examinations: Yes: approx. half per litter
In order to evaluate the relative importance of cartilage findings in fetuses from the high-dose group, a re-examination of the skeleton of the eight fetuses concerned has been conducted. This evaluation was also performed in a relevant number of fetuses from the control group.
- Head examinations: Yes: as part of soft tissue and skeletal examinations

In order to evaluate the relative importance/severity of the cartilage findings, the fetuses from the high-dose group have been re-examined. These findings concerned fetuses B28861-05, B28862-03, B28867-03, B28867-05, B28867-07, B28867-11, B28867-13 and B28867-17 which were considered to have "fused cartilage of cervical vertebra(e)" and/or "fused cartilage of thoracic vertebra(e)".

Statistics:

Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test. Data of non-pregnant females were not included in the group mean calculation.

Indices:

- Pre-implantation loss
- Post-implantation loss
- Fetal or litter incidence
- Mean proportion of affected fetuses

Historical control data:

Historical control data of the laboratory were considered in the evaluation of the study.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical sign was noted in the control and the 1000 mg/kg bw/day groups. At 100 mg/kg bw/day, soft feces was noted in one female on days 8 and 9 of pregnancy and regurgitation was noted in one female given 300 mg/kg bw/day on day 7 of pregnancy. In view of their low incidence and the absence of dose-relationship, these signs were not considered to be treatment-related.

Mortality:

no mortality observed

Description (incidence):

There was no death in any group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight, body weight changes and net body weight changes were unaffected by the treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption was unaffected by the treatment.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The only finding (serous content in uterine horns one non-pregnant female at 0 and 1000 mg/kg bw/day) was not related to the treatment with the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The post-implantation loss was not affected by the treatment at any dose-level.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There was no total resorption in any group.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was no total resorption in any group.

Dead fetuses:

no effects observed

Description (incidence and severity):

The mean number of live fetuses was similar in the control and the treated groups. There were no dead fetuses in any group.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal body weight was similar in the control and the treated groups.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

not examined

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No fetal variation was noted in any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No skeletal malformation was noted at 100 mg/kg bw/day. At 300 mg/kg/day, split sternebrae were noted in one fetus.
At 1000 mg/kg bw/day, the following findings were noted:
- absence of sacral vertebrae (2nd to 4th) and absence of all lumbar and caudal vertebrae in 1/125 fetuses,
- unossified metacarpal, supernumerary metatarsal and metacarpal, fused metatarsal, short fibula and tibia in 1/125 fetuses.

VARIATIONS
The significantly higher fetal incidence of incomplete ossification of hyoid, caudal vertebrae and pubis were not clearly dose-related and close or within CIT historical control data. Furthermore, the cartilage was generally present and normal at the level of these structures which indicated that these findings corresponded to a minimal delay in ossification while the significantly higher fetal incidence of thoracic vertebra was associated in some fetuses with fused thoracic cartilage.
Except for the above described findings, the fetal incidence of the recorded bone variations was generally similar in the control and the treated groups, not dose-related, not statistically significant and within the range of CIT historical control data.

Visceral malformations:

no effects observed

Description (incidence and severity):

No malformation was observed in the control and in the 100 and 1000 mg/kg bw/day groups. At 300 mg/kg bw/day, malpositioned anus and absence of kidneys and ureters were observed in one fetus which presented an absence of tail and micromelia at external examination.

VARIATIONS
Dilated ureter was noted in 1/117 fetuses of the control group, 2/143 fetuses at 100 mg/kg bw/day, 5/125 fetuses at 300 mg/kg bw/day and 3/114 fetuses at 1000 mg/kg bw/day. The few following other variations were observed at 300 mg/kg bw/day: one enlarged ovary and dilated uterine horn in one fetus and dilated renal pelvis in two other fetuses.

Other effects:

no effects observed

Description (incidence and severity):

The cartilage was not affected at 100 and 300 mg/kg bw/day. At 1000 mg/kg bw/day, significantly higher incidence of fused cartilage of cervical and thoracic vertebrae were recorded. No other relevant findings were noted at this dose-level. The significantly higher number of live fetuses with both fused cartilage of thoracic and cervical vertebrae, concerned six fetuses from three different litters and was recorded at the highest dose-level only.

Re-examination:
After re-examination and when compared with fetuses from the control group and, after careful evaluation of all skeletons of the eight fetuses concerned, no cartilage abnormalities were detected.
After analyzing the probable causes of this misdiagnosis the following explanations were retained:
- double staining can result in excessive staining with Alcian blue (when the staining is too pronounced, delicate structures may be difficult to observe and may appear abnormally shaped). After more than 10 years in glycerin, fetuses were remarkably clarified and all ossified structures and cartilages were very well identifiable,
- a too intense staining may be associated with artefacts such as apparent fused cartilages. In the case of this study, the apparent fusion may correspond to intense staining of the lamina ventralis covering the 7th cervical vertebra in direction of the 1st thoracic vertebra and/or an excessive staining of the proximal cartilage portion of the 1st rib.

Details on embryotoxic / teratogenic effects:

No malformation (external, visceral or skeletal) were noted at 100 mg/kg bw/day. At 300 and 1000 mg/kg bw/day, two fetuses from two different litters presented with multiple malformations: At 300 mg/kg bw/day, one fetus presented with unilateral micromelia, absence of tail, malpositioned anus and absence of kidneys and ureters and one other fetus split sternebra. At 1000 mg/kg bw/day, one fetus presented short bilateral hindlimb, unossified metacarpal, supernumerary metatarsal and metacarpal bones and short fibula and tibia and one other fetus presented absence of sacral and all lumbar and caudal vertebra. Because these findings were low in incidence and not dose-related, they were considered to be fortuitous in origin.
At 1000 mg/kg/day, although the higher incidence of fused cervical and thoracic cartilage concerned three litters, it was considered to be related to the treatment. Furthermore, the significantly higher incidence of incomplete ossification of thoracic vertebra associated with that of fused corresponding cartilage was considered to be of toxicological significance.

Re-examination:
Originally, for eight fetuses of the high dose group fused cartilage was reported. Upon request, the relative importance/severity of these findings were evaluated by re-examining the respective fetuses and comparing them with fetuses of the control group. Regarding the originally stated observations of "fused cartilage of cervical vertebra(e)" and "fused cartilage of thoracic vertebra(e)" it was clarified that these two observations correspond to a single observation, namely the fusion of a part the cartilage of the 7th cervical vertebra with part of the cartilage of the first thoracic vertebra.
After re-examination and when compared with fetuses from the control group, no cartilage abnormalities were detected.
A probable reason of the first diagnosis is that too intense staining with Alcian blue, which had cleared after 10 years in glycerin, may be associated with artefacts such as apparent fused cartilages.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Examination of fetal skeletal cartilage after re-examination

Dose   Parameter		0 mg/kg bw/d	100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
Litter evaluated	N	19	23	21	19
Fetuses evaluated	N	128	155	137	125
Cartilage of Thoracic vertebra(e): present
Fetal incidence	N	6	14	9	17*
	%	4.7	9.0	6.6	13.6
Litter incidence	N	5	11	5	7
	%	26.3	47.8	23.8	36.8
Affected Fetuses/Litter	Mean %	5.1	9.9	6.3	12.0
	SD	9.6	14.0	14.8	22.4
Total fetal skeletal cartilage
Fetal incidence	N	108	145	112	106
	%	84.4	93.5	81.8	84.8
Litter incidence	N	19	23	21	18
	%	100.0	100.0	100.0	94.7
Affected Fetuses/Litter	Mean %	85.3	92.8	80.6	82.0
	SD	19.1	11.6	21.1	27.5

 

f=Fishers exact test * = p<0.05

Conclusions:

In GLP-study according to OECD TG 414, the test item, when administered orally by gavage to pregnant female rats from day 6 to day 19 post-coitum was well tolerated at all dose-levels. At all doses (100, 300 and 1000 mg/kg bw/day), no effect on litter data parameters and fetal development were observed. In conclusion, 1000 mg/kg bw/day is the No Observed Effect Level (NOEL) for maternotoxicity and embryofetal development.

Executive summary:

The objective of this prenatal developmental toxicity GLP-study according to OECD TG 414 was to evaluate the potential toxic effects of the test item on the pregnant and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (day 6 to day 19 post-coitum inclusive).

Four groups of 24 mated female Sprague-Dawley rats, received the test item by oral administration (gavage) at 100, 300 or 1000 mg/kg bw/day from day 6 to day 19 post-coitum. A group of 24 mated females was given the vehicle alone (purified water) under the same experimental conditions and acted as the control group. Clinical signs and mortality were checked daily. Body weight and food consumption were recorded at designated intervals. On day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The gravid uterus was weighed to allow calculation of the net body weight gain of females. The fetuses were removed by hysterectomy. The litter parameters were recorded, namely: number of corpora lutea, implantation sites, early and late resorptions, dead and live fetuses. The fetuses were weighed, sexed and submitted to external examination of the soft tissue and the other half to a detailed examination of the skeleton (bone and cartilage).

Results

Mortality: There was no mortality in any group.

Clinical signs: There were no treatment-related clinical signs in any group.

Food consumption and body weight: The food consumption and body weight were unaffected by the treatment with the test item.

Macroscopic post-mortem examination: No macroscopic findings that were related to the treatment were noted at any dose-level.

Litter data: No treatment-related effect was noted on litter data parameters, namely: corpora lutea, implantation sites, post-implantation loss, number of live fetuses and fetal body weight.

Fetal examination: No fetal external, soft tissue or skeletal variations or malformations were observed that were considered to be attributable to the treatment with the test item.

In conclusion, the test item when administered to pregnant female rats from day 6 to day 19 post-coitum was well tolerated at all dose-levels. No effect on litter data parameters and fetal development were observed.

In conclusion, 1000 mg/kg bw/day is the No Observed Effect Level (NOEL) for maternotoxicity and embryofetal development.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study according to OECD TG, RL1.

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

Developmental toxicity study

The objective of this prenatal developmental toxicity GLP-study according to OECD TG 414 was to evaluate the potential toxic effects of the test item on the pregnant and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (day 6 to day 19 post-coitum inclusive) (reference 7.8.2 -1).

Four groups of 24 mated female Sprague-Dawley rats, received the test item by oral administration (gavage) at 100, 300 or 1000 mg/kg bw/day from day 6 to day 19 post-coitum. A group of 24 mated females was given the vehicle alone (purified water) under the same experimental conditions and acted as the control group. Clinical signs and mortality were checked daily. Body weight and food consumption were recorded at designated intervals. On day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The gravid uterus was weighed to allow calculation of the net body weight gain of females. The fetuses were removed by hysterectomy. The litter parameters were recorded, namely: number of corpora lutea, implantation sites, early and late resorptions, dead and live fetuses. The fetuses were weighed, sexed and submitted to external examination of the soft tissue and the other half to a detailed examination of the skeleton (bone and cartilage).

Results

Mortality: There was no mortality in any group.

Clinical signs: There were no treatment-related clinical signs in any group.

Food consumption and body weight: The food consumption and body weight were unaffected by the treatment with the test item.

Macroscopic post-mortem examination: No macroscopic findings that were related to the treatment were noted at any dose-level.

Litter data: No treatment-related effect was noted on litter data parameters, namely: corpora lutea, implantation sites, post-implantation loss, number of live fetuses and fetal body weight.

Fetal examination: No fetal external, soft tissue or skeletal variations or malformations were observed that were considered to be attributable to the treatment with the test item.

In conclusion, the test item, when administered to pregnant female rats from day 6 to day 19 post-coitum was well tolerated at all dose-levels. No effect on litter data parameters and fetal development were observed.

In conclusion, 1000 mg/kg bw/day is the No Observed Effect Level (NOEL) for maternotoxicity and embryofetal development.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on developmental toxicity, the test item does not need to be classified and labelled according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.