Sodium 4-(2-hydroxyethyl)piperazin-1-ylethanesulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mar 2003 - Dec 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

27th July 1995

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

230-907-9

EC Name:

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

Cas Number:

7365-45-9

Molecular formula:

C8H18N2O4S

IUPAC Name:

2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen because it is a rodent species commonly accepted by regulatory authorities for this type of study and the Sprague-Dawley strain was selected since background data from previous studies are available at the laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, l’Arbresle, France
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: mean of 195 g (range: 178 g to 207 g) for the males and mean of 152 g (range: 138 g to 171 g) for the females
- Fasting period before study: no information
- Housing: suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm) and each cage contained two rats of the same sex and group
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

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

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route is selected as it is one anticipated route of secondary human exposure.

Vehicle:

water

Remarks:

purified

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle: the test item mixed with the required quantity of vehicle. Fresh test item dosage forms were prepared daily.

VEHICLE
- Amount of vehicle: 5 mL/kg bw/day (dosage-volume)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily (approx. at same time)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: preliminary study
- Fasting period before blood sampling for clinical biochemistry: yes

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day
- Cage side observations: Morbidity and mortality

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

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 27
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Please refer to table 1 under "Any other information on materials and methods incl. tables"

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 27
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Please refer to table 2 under "Any other information on materials and methods incl. tables"

URINALYSIS: Yes
- Time schedule for collection of urine:on day 27
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters examined: Please refer to table 3 under "Any other information on materials and methods incl. tables"

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Sacrifice
On completion of the treatment period, after at least 14 hours fasting, all animals were asphyxiated by carbon dioxide and killed by exsanguination.

Organ weights
The body weight of all animals killed at the end of the treatment period was recorded before sacrifice, and the organs specified below were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Macroscopic post-mortem examination
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

Preservation of tissues
For all study animals, the tissues specified below were preserved in 10% buffered formalin (except for the eyes and Harderian glands which were fixed in Davidson's fixative, and the testes and epididymides which were preserved in Bouin's fluid).

Microscopic examination
All tissues required for microscopic examination were embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS).
A microscopic examination was performed on:
- all tissues listed below for animals of the control and high-dose groups (groups 1 and 4) killed at the end of the treatment period,
- all macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3) killed on completion of the treatment period.

Tissues (preserved, examined microscopically (except *), organ weights (for ** only))
Organs
Macroscopic lesions
Adrenals
Aorta **
Brain (including medulla/pons cerebellar and cerebral cortex)
Cecum
Colon
Duodenum
Epididymides **
Esophagus
Eyes with Harderian glands
Femoral bone with articulation
Heart **
Ileum
Jejunum
Kidneys **
Liver **
Lungs with bronchi
Lymph nodes (mandibular and mesenteric)
Mammary gland area
Ovaries **
Pancreas
Pituitary gland
Prostate
Rectum
Salivary glands (sublingual and submandibular)
Sciatic nerve *
Seminal vesicles *
Skeletal muscle *
Skin *
Spinal cord (cervical, thoracic and lumbar)
Spleen **
Sternum with bone marrow *
Stomach with forestomach
Testes **
Thymus **
Thyroids with parathyroids **
Tongue *
Trachea
Urinary bladder
Uterus (horns and cervix)
Vagina *

Statistics:

Test for Normality of the distribution of untransformed and log-transformed data: Kolmogorov-Lilliefors test.
Assessment of homogeneity of variances between groups: Bartlett test and Fisher test.
Test for effects: Dunn test; t-test, Dunnett test, Mann-Whitney/Wilcoxon test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related observations were noted.

Mortality:

no mortality observed

Description (incidence):

No deaths occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight changes were unaffected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was similar in all groups.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

In different treated groups, a few variations were noted but the values for these parameters were very low and inconsistent between sexes (e.g. decreased Activated Partial Thromboplastin Time in males of group 4). These changes were consequently considered not to be related to test item administration but rather to reflect biological variability.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

At 1000 mg/kg bw/day, glucose concentration decreased slightly in males (-9 %, p<0.05) and calcium concentration increased slightly in females (+3 %, p<0.05); all individual values were within laboratory historical control data, not consistent with male or female values and without a clear dose-relationship. In males of the three treated groups, inorganic phosphorus was decreased (p<0.05 and p<0.01) compared to control; as the differences were not dose-related and concerned one sex only, there were not considered to be consecutive to test item administration.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No marked effects were noted in any urine parameters. The decrease in pH values measured in treated animals at 1000 mg/kg bw/day was not ascribed to treatment with the test substance as it was very slight.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Some differences from the controls in organ weights were observed. However, they were minor, not dose-related and/or without dose-relationship. They were thus considered to be of no toxicological importance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The few macroscopic findings were those which are commonly encountered in the laboratory rat of this strain and age and were thus considered to be of no toxicological importance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Spleen
Marginally higher severity of extramedullary hematopoiesis was observed in the animals given 1000 mg/kg bw/day as summarized in table 4 under "Any other information on results incl. tables". The marginally higher severity of extramedullary hematopoiesis, especially in the females, was the contribution of one female with marked (grade 4) extramedullary hematopoiesis. As this finding was recorded with such severity from time to time in control female rats from this strain and age, the marginally higher severity of extramedullary hematopoiesis was considered to be fortuitous and to bear no relationship to the treatment with the test item.

Other organs
Medio-lobular minimal vacuolated hepatocytes with occasional macro-vacuoles were observed in 4/10 males receiving 1000 mg/kg bw/day vs. 1/10 control males. However peri/medio-lobular vacuolated hepatocytes were also observed in the treated and control females with similar incidence and severity (minimal). This was thus considered to be of no toxicological significance.
Slight osseous metaplasia was observed unilaterally in the adrenal of 1/10 females given 1000 mg/kg bw/day, at the junction of the glomerulosa and the fasciculata. Slight diffuse hyperplasia was noted in the pars distalis of the pituitary gland of 1/10 females given 1000 mg/kg bw/day.
Considering the low incidence and severity of the above-mentioned findings in the treated females and their absence from the top-dose group in the males, they were considered to be of spontaneous nature and to bear no relationship to the treatment with the test item.
All the other microscopic findings were those which are commonly recorded spontaneously in the untreated laboratory rat of this strain and age and they were thus considered to be without relationship to the test item treatment.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 4 Incidence and severity of extramedullary hematopoiesis in the spleen

Dose-level (mg/kg bw/ day)	0		1000
Sex	Male	Female	Male	Female
Incidence	10/10	10/10	10/10	10/10
Severity	1.9	2.3	2.1	2.7

 

Applicant's summary and conclusion

Conclusions:

In GLP-study according to OECD TG 407, the test item, when administered orally by gavage to Sprague-Dawley rats for 4 weeks, resulted in a No Observed Effect Level (NOEL) of 1000 mg/kg bw/day.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 4 weeks (GLP-study according to OECD TG 407). Three treated groups of ten male and ten female Sprague-Dawley rats received the test item by gavage at the dose-levels of 100, 300 or 1000 mg/kg bw/day for 4 weeks. An additional group of ten males and ten females received the vehicle alone (purified water) under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used. The animals were checked daily for mortality and clinical signs. Body weight was recorded before allocation of animals into the study and once a week thereafter. Food consumption was recorded once a week. Haematology, blood biochemical investigations and urinalysis were performed at the end of the treatment period. On completion of the treatment period, the animals were killed and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on designated tissues from animals of the control and high-dose groups.

No death occurred during the study. No treatment-related clinical signs were noted and no relevant differences from controls were observed in the body weight of the treated animals. The food consumption was unaffected and no overt signs of toxicity were observed in haematological, blood biochemical or urinalysis parameters. Organ weights were unaffected by treatment and no macroscopic or histopathological findings revealed a treatment-related effect.

Under the experimental conditions of the study, the No Observed Effect Level (NOEL) was established at 1000 mg/kg bw/day in Sprague-Dawley rats.