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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2008-08-14 till 2009-05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Sodium 2-hydroxyethanesulphonate
- Physical state: White crystalline solid
- Analytical purity: > 99%
- Lot/batch No.: EK 08/116
- Expiration date of the lot/batch: 2010-12-31
- Storage condition of test material: At room temperature (15 - 25 °C) in a closed container away from direct sunlight

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd., Füllinsdorf / Switzerland
- Age at study initiation: Ca. 7 weeks
- Weight at study initiation:
Males: 183 g to 202 g (mean 194 g)
Females: 138 g to 156 g (mean 146 g)
- Fasting period before study: for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 rat / mouse maintenance diet, ad libitum
- Water (e.g. ad libitum): Community tap-water from Itingen was available, ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%).
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.


VEHICLE
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Purity: bidistilled water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
After experimental start, samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration and stability. Samples of about 2 g of each concentration were taken during week 3, 8 and 13 after commencement of dosing to confirm homogeneity and concentration. The test item was used as analytical standard.
The test item concentrations were determined by IC coupled to a conductivity detector. The content was determined by external standard quantitation referring to the area under the test item peak.
Duration of treatment / exposure:
91/92 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Group 1: 15 males and 15 females (control group, where 10 animals were satellite A and the other 5 animals formed satellite B)
Group 2: 10 males and 10 females
Group 3: 10 males and 10 females
Group 4: 15 males and 15 females ( 10 animals were satellite A and the other 5 animals formed satellite B)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats (Harlan Laboratories Study B96974).
- Rationale for selecting satellite groups: the animals were allowed to a 28-day treatment-free recovery period
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once before commencement of administration as well as twice daily on days 1 to 3, once daily from on day 4 to the end of the treatment period, and once daily during days 1 to 28 (recovery period).
- Cage side observations checked: appearance (piloerection, salivation, hunched posture), motor (ataxia, tremor, prostration), behaviour (hyperactivity, somnolence), respiration (dyspnea, tachypnea, bradypnea)


BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatization, treatment and recovery periods and before necropsy


FOOD CONSUMPTION:
once during the acclimatization period and weekly thereafter


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once in all animals during acclimatization period; once in animals of the control and high dose groups, as well as in animals of the middle dose groups if test item-related changes were seen in the high dose (treatment period); once in the remaining animals of the control and high dose groups during the recovery period.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the dosing period, and after 17 weeks (satellite B animals)
- Anaesthetic used for blood collection: Yes (under light isoflurane anesthesia)
- Animals fasted: Yes (for approximately 18 hours before blood sampling but allowed access to water ad libitum)
- How many animals: all group animals at week 13, while 6 males and 6 females/group after 17 weeks, only groups 0 and 4
- Parameters checked in table No 1 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the dosing period, and after 17 weeks (satellite B animals)
- Animals fasted: Yes (for approximately 18 hours before blood sampling but allowed access to water ad libitum)
- How many animals: all group animals at week 13, while 6 males and 6 females/group after 17 weeks, only groups 0 and 4
- Parameters checked in table No. 2 were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: during the 18 hours fasting period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No. 3 were examined.


NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- functional observational battery (week 13)
- grip strength (week 13)
- locomotor activity (week 13)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No. 4)
HISTOPATHOLOGY: Yes (see table No. 4)

Statistics:
The following statistical methods were used to analyze body weight, locomotor activity, fore- and hindlimb grip strength, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
- The Dunnett-test
- The Steel-test
- Fisher's exact-test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
All rats survived until scheduled necropsy.
No test item-related clinical signs of toxicological relevance were noted during daily observations or weekly observations (weeks 1 - 12) at any dose level.

BODY WEIGHT AND WEIGHT GAIN
A trend to slightly lower mean absolute and relative body weights values were ascertained in males treated with 1000 mg/kg/day when compared with controls. This was considered to be a mild test item-related change. All other animals were unaffected.

FOOD CONSUMPTION
No differences of toxicological relevance were noted in the mean daily food consumption.


OPHTHALMOSCOPIC EXAMINATION
None of the ophthalmoscopic findings noted after 13 weeks’ treatment in rats treated with 1000 mg/kg/day were considered to be related to the test item.

HAEMATOLOGY
A number of test item-related differences were noted in the hematology parameters of the rats treated with 1000 mg/kg/day. These changes were manifested by reduced mean corpuscular hemoglobin concentration values, elevated mean absolute and relative reticulocyte counts, and a ‘left-shift’ in the reticulocyte maturity indices indicative of increased reticulocyte turnover. Females treated with 1000 mg/kg/day also had reduced hemoglobin distribution width. All remained within the upper limits of the historical control data. Both sexes showed slightly elevated methemoglobin. In the absence of differences which exceeded the historical control values and/or Heinz body formation, these were considered to be of no toxicological relevance The remaining changes noted in animals at 1000 mg/kg/day (elevated mean absolute lymphocytes and monocytes in females) did not exceed the ranges of the historical control values or were not accompanied by concomitant changes in related parameters and were therefore considered to be incidental. All changes noted at 200 mg/kg/day or at 50 mg/kg/day were considered to be incidental. No test item-related findings remained nor were late effects in the animals previously treated with 1000 mg/kg/day noted after 4 weeks of recovery.

CLINICAL CHEMISTRY
Test item-related changes in the parameters of the clinical biochemistry were noted, and included reduced glucose levels in males and females at 1000 mg/kg/day. Although some outlying values were found, the overall mean lactate dehydrogenase activity was generally higher in males and females at 1000 mg/kg/day. The mean total bilirubin level was elevated in males and females at 1000 mg/kg/day when compared with the respective contr ls, possibly indicating some degree of cell friability. Elevated mean cholesterol levels and phospholipid levels were noted in males, whereas these
parameters were only marginally elevated in females at this dose level. In rats treated previously with 1000 mg/kg/day, the mean cholesterol level remained elevated after the recovery period in males when compared with the controls. Aspartate aminotransferase activity was elevated in females treated with 1000 mg/kg/day, whereas alanine aminotransferase activity was elevated in males of this dose level when compared with their respective controls. Differences in electrolytes included elevated sodium levels in males at 200 and 1000 mg/kg/day and in females at 50 mg/kg/day and 1000 mg/kg/day, reduced potassium levels in males at 1000 mg/kg/day and females at 200 mg/kg/day and 50 mg/kg/day, increased calcium levels at 1000 mg/kg/day, increased phosphorus in females at 1000 mg/kg/day, and increased chloride levels in males at 200 mg/kg/day were noted. The mean total protein levels were elevated in males and females; both values exceeded the upper limit of the historical control values.

URINALYSIS
Changes in the urine parameters of males treated with 1000 mg/kg/day included elevated mean urine volume and reduced mean relative density. Despite two marked outliers, the differences noted in both parameters of the remaining males at 1000 mg/kg/day also diverged from the values recorded in the control males and were considered to be test item related. The mean urinary pH values of the males treated with 1000 mg/kg/day and 200 mg/kg/day were lower than the control males and this was also considered to be a test item-related change. The protein level and ketones were reduced in males and females treated with 1000 mg/kg/day when compared with the controls. These changes were not considered to be associated with any toxicological relevance. The mean relative density and the mean protein level were also significantly lower in females at 200 mg/kg/day (p<0.05), but considered to be toxicologically irrelevant. All other parameters showed either minor differences which were unrelated to dose or were unaffected. No test item-related findings remained nor were late effects in the animals previously treated with 1000 mg/kg/day noted after 4 weeks of recovery.


ORGAN WEIGHTS
Reduced thymus weights were noted in test item treated males at all dose levels. A similar but less marked trend for lower thymus weights were noted in the test item-treated females. The mean spleen-to-body weight ratio of the females treated with 1000 mg/kg/day was elevated when compared with controls. The mean absolute spleen weight and the mean spleen-to-brain weight ratio were slightly elevated. The changes in spleen weights reflected the microscopical changes (increased hemopoiesis) seen in this organ.

GROSS PATHOLOGY
Macroscopically, males and females treated with 1000 mg/kg/day showed elevated incidence of liver foci were noted when compared with the respective controls. This finding was considered to be test item related. This finding was also noted in one female at 200 mg/kg/day after 13 weeks of treatment, but was not seen in any males at this dose level. It was not evident in either sex at 50 mg/kg/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopically, the livers of animals treated with 1000 mg/kg/day showed degeneration, necrosis (focal or of single hepatocytes), bile duct hyperplasia, focal hepatocytic hyperplasia (very likely regenerative), peribiliary fibrosis and an increased incidence and severity of mixed inflammatory cells infiltration in the parenchyma. Increased hemopoiesis was seen in the spleen of animals at 1000 mg/kg/day. After 28 days of recovery, there was the total reversibility of the findings: the livers and spleens of animals treated previously with 1000 mg/kg/day reverted to normal.


HISTORICAL CONTROL DATA (if applicable)
Used


OTHER FINDINGS:
Functional Observational Battery: No clinical signs were evident at any dose level during the functional observational battery performed at week 13 of treatment.

Grip Strength: At 1000 mg/kg/day, the mean hindlimb grip strength value of males was lower that that of the control males and considered to be related to the slight difference in mean body weight. The mean forelimb grip strength of these males compared favorably with those of the control males.
All other animals were unaffected.

Locomotor Activity: There were no test item-related effects on the mean locomotor activity of either gender.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: overall effects clinical signs; mortality; body weight; food consumption; ophthalmoscopic examination; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology
Dose descriptor:
NOAEL
Effect level:
426 mg/kg bw/day (actual dose received)
Based on:
other: Calculated as equimolar amount Coco Isethionate
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Recalculated as the equimolar amount of Coco Isethionate. Molweight sodium isethionate = 148 g/mol. Molweight Coco Isethionate = 315 g/mol. NOAEL = (200 mg/kg bw/day) *(315 g/mol)/ (148 g/mol) = 426 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Tabelle 5: Mean severity of Findings

Males

Females

Group

1

2

3

4

1

2

3

4

Dose (mg/g/day)

0

50

200

1000

0

50

200

1000

Number of animals examined

12

12

12

12

12

12

12

12

Liver

Mixed Cell Infiltration

7/1.0

8/1.0

9/1.0

11/1.8

5/1.2

5/1.0

7/1.0

12/1.5

Degeneration

-

-

-

3/1.3

-

-

-

4/2.0

Focal Necrosis

-

-

-

6/1.3

-

-

-

3/1.3

Single Cell Necrosis

-

-

-

7/1.1

-

-

-

4/1.5

Focal Hepatic Hyperplasia

-

-

-

3/1.7

-

-

-

3/1.7

Bile Duct Hyperplasia

-

-

-

½.0

-

-

-

6/1.5

Peribiliary fibrosis

-

-

-

½.0

-

-

-

5/1.4

Spleen

Incr. Hemopoiesis

-

-

½.0

4/1.8

2/2.0

-

-

8/2.3

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for the test item, Sodium 2-hydroxyethanesulphonate, was considered to be 200 mg/kg body weight/day.
Executive summary:

Oral administration of Sodium 2-hydroxyethanesulphonate to Wistar rats at doses of 50, 200 and 1000 mg/kg/day for 91/92 days resulted in no deaths, no clinical signs during daily or weekly observations, no clinical signs during the functional observational battery, no test item-related differences in the mean fore-or hindlimb grip strength or mean locomotor activity, no toxicologically relevant ophthalmoscopic changes, no differences in the mean food consumption, no changes in hematology parameters at 200 mg/kg/day or at 50 mg/kg/day, and no changes in urinalysis parameters at 50 mg/kg/day.

Although statistically significant differences were noted in the mean hindlimb grip strength values of males treated with 1000 mg/kg/day, these were considered to be secondary effects to the lower body weights.

Test item-related findings were generally restricted to slightly lower mean absolute and relative body weights in males treated with 1000 mg/kg/day, changes in the hematology parameters of the rats treated with 1000 mg/kg/day (reduced mean corpuscular hemoglobin concentration values, elevated mean absolute and relative reticulocyte counts, and a ‘left-shift’ in the reticulocyte maturity indices indicative of increased reticulocyte turnover, reduced hemoglobin distribution width in females only), the clinical biochemistry parameters at 1000 mg/kg/day (reduced glucose levels, elevated total bilirubin levels, elevated cholesterol and phospholipid levels, elevated aspartate or alanine aminotransferase activities at 1000 mg/kg/day) including electrolytes (elevated sodium levels at 50, 200 or 1000 mg/kg/day, reduced potassium levels at 50, 200 or 1000 mg/kg/day, increased calcium levels at 1000 mg/kg/day, increased phosphorus in females at 1000 mg/kg/day, increased chloride levels in males at 200 mg/kg/day), increased spleen weights in rats at 1000 mg/kg/day, macroscopical changes in the liver (a higher incidence of tan foci reported in the liver of males and females treated with 1000 mg/kg/day) after the treatment period only, microscopical changes in the liver (presence of degeneration, necrosis (focal or of single hepatocytes), bile ducts hyperplasia, focal hepatocytic hyperplasia, peribiliary fibrosis and an increased incidence and severity of mixed inflammatory cells infiltration in the parenchyma) and spleen (increased hemopoiesis) with complete post-recovery reversibility.

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for the test item, Sodium 2-hydroxyethanesulphonate, was considered to be 200 mg/kg body weight/day.