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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-08-19 till 2009-02-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform study under GLP without deviations
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MAFF, 12 Nohsan No. 8147 2000-11-04
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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: solid, white crystalline
- Analytical purity: => 99%
- Impurities (identity and concentrations): water (0.07%)
- Purity test date: 2008-04-30
- Lot/batch No.: EK 08/116
- Expiration date of the lot/batch: 2010-12-31
- Stability under test conditions: stable
- Storage condition of test material: At room temperature (15-25°C), tightly closed

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Rat, HanRcc: WIST(SPF) Harlan Laboratories Ltd., Laboratory Animal Services, 4414 Füllinsdorf / Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: 188 to 220 g
- Housing: females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. The females were removed and housed individually if: a) The daily vaginal smear was sperm positive, or b) A copulation plug was observed.
- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 rat/mouse maintenance diet was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Füllinsdorf , ad libitum
- Acclimation period: 7 days, under test conditions after health examination. Only animals without any visible signs of illness were used for the study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): relative humidity range: 30 - 70%)
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light):12-hour fluorescent light / 12-hour dark cycle with music during the light period.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: highly purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
The test item was weighed into a tared glass container on a suitable precision balance and the
vehicle was added to give the appropriate final concentration of the test item in the dose
formulation. The mixture was prepared using a magnetic stirrer or homogenizer as appropriate.
Homogeneity of the test item in the vehicle was maintained during the daily administration
period using a magnetic stirrer.

Storage of Dose Formulations
Dose formulations were stored at room temperature (20 ± 5 °C) protected from light in glass beakers.
Based upon the results of stability analyses performed within the RCC study no. B97031 (Validation of an analytical method for dose formulation analysis), dose formulations were stable for at least one week.


VEHICLE
- Amount of vehicle (if gavage): Dose Volume: 10 mL/kg body weight; daily adjustment to the actual body weight
- Purity: highly purified water
- Target does level: 0 mg/kg/day, 50 mg/kg/day, 200 mg/kg/day and 1000 mg/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples were analyzed by IC coupled to a conductivity detector following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard. The following acceptance criteria was applied to analytical results: sample contents should be within a range of ±20% of nominal. Formulations were considered homogenous if the maximum deviation from mean calculated from top, middle and bottom samples was no more than 15%.
The results obtained from storage stability samples should not deviate more than 10% from timezero reference (content or mean of homogeneity samples).
In conclusion, the results indicate the accurate use of the test item Sodium 2-hydroxyethanesulphonate and highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
Details on mating procedure:
- Impregnation procedure: After 7 days of acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
a) The daily vaginal smear was sperm positive, or
b) A copulation plug was observed.

-Male rats of the same source and strain were used only for mating. These male rats are in the
possession of Harlan Laboratories and were not considered part of the test system. The fertility
of these males had been proven and was continuously monitored.


Duration of treatment / exposure:
Exposure period: Day 0 post coitum (first treatment) to day 20 post coitum (last treatment)
Frequency of treatment:
Daily
Duration of test:
Duration of Treatment Period: Day 0 - 20 post coitum
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Group 1: 0 mg/kg/day (control group)
Basis:
nominal in water
Remarks:
Doses / Concentrations:
Group 2: 50 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
Group 3: 200 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
Group 4: 1000 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
22 mated females per group
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 Han Wistar rats, Harlan Laboratories Study B96996, using dose levels of 100, 300 and 1000 mg/kg/day.
- Rationale for animal assignment (if not random):Computer-generated random algorithm

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Viability/mortality was observed twice daily. Cage-side clinical signs (once daily, during acclimatization and up to day of necropsy) were noted daily.
- Cage side observations : yes


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations:Body weights recorded daily from day 0 until day 21 post coitum.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was recorded at 3-day intervals: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:


POST-MORTEM EXAMINATIONS: Yes
Maternal Data
- Sacrifice on gestation day # 21
Necropsy
The females were sacrificed by CO2 asphyxiation. Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea was performed and the data recorded. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain

OTHER: Fetus examinations: yes
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 resorptions: Yes
- Position of fetus: yes
Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea was performed and the data recorded. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain
Fetal examinations:
- Body weights of Live Fetuses: Yes: per dam
- External examinations: Yes: all per litter

Fetal Pathology
Fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
1. Microdissection technique (sectioning/dissection technique). At least one half of the fetuses from each litter was fixed in Bouin's fixative (one fetus per container). They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ethanol (one fetus per container). Descriptions of any abnormalities and variations were recorded.
2. The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol, glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations were recorded. The specimens were preserved individually in plastic vials.
If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher’s exact-test was applied if the variables could be dichotomized without loss of information.
Historical control data:
Historical control data of rats (WiIST Han/Brl (SPF Quality)); Dara from prenatal developmental toxicity studies, performed during 2004 and 2005; provided by: RCC Ltd., toxicology, 4414 Füllinsdorf / Switzerland

Reproduction data; spontaneous abnormal findings (external); abnormal findings from visceral examination of fetuses; abnormal findings and variations from skeletal and cartilage examinations of fetuses-summary; skeletal examination of fetuses (stage of development): fetus basis and litter basis; cartilage examination of fetuses (stage of development and variations): fetus basis and litter basis

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
All dams survived until the scheduled necropsy. No clinical symptoms or observations were noted during the study in any groups.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Clinical Signs or Observations - Females

 

 

Group 1
0 mg/kg

 

 

Group 2

50 mg/kg

 

Group 3

200 mg/kg

 

Group 4

1000 mg/kg

 

Number of females examined

22

 

22

22

22

No clinical signs or observations

22

 

22

22

22

Summary of Performance of Mated Females

Group

1

2

3

4

Dose (mg/kg)

(0)

(50)

(200)

(1000)

Female numbers

1 - 22

23 - 44

45 - 66

67 - 88

Number of mated females

22

22

22

22

Number of females with live fetuses at termination*

22

22

22

22

* Only dams with at least one live fetus at Caesarean section were used for the calculations of food consumption, body weight gain and corrected body weight gain data.

Maternal Data

Clinical Signs or Observations

All dams survived until the scheduled necropsy. No clinical symptoms or observations were noted during the study in any groups.

Food Consumption

In group 4, mean food consumption was marginally lower than control (-4.3% compared to the control group). Since this was not statistically significant and in absence of an effect on body weight was considered to be of no toxicological relevance. In groups 2 and 3, mean food consumption was similar to the control group (±0.0% and +0.4% compared to the control group, respectively).

Body Weights

Mean body weight and mean body weight gain were not considered to be affected by the treatment with the test item. Corrected body weight gain (corrected for the weight of the gravid uterus) was not considered to be affected by the treatment with the test item (15.8%, 14.1%, 14.9% and 14.0% in groups 1, 2, 3 and 4, respectively).

Reproduction Data

No test item-related effects on the reproduction data (pre-implantation loss, number of implantation sites, post-implantation loss and mean number of fetuses per dam) were noted at any dose level. In group 3, post-implantation loss was statistically significantly higher. Consequently, the number of fetuses was statistically significantly lower compared to the control group. This was considered to be incidental since there was no dose-dependency and within the range of the historical control data.

Macroscopical Findings

No abnormal findings were noted during the necropsy examination.

Fetal Data

External Examination

No test item-related abnormal findings were noted. In group 4, one fetus was noted to have the lower mandible markedly shortened and with only one central nostril. A shortened lower mandible was also noted in one fetus in the control group. These findings were noted as isolated cases in single fetuses and thus considered not to be related to the treatment with the test item.

Sex Ratios

No test item-related effects on the sex ratio of the fetuses were noted in any group. The percentage of male fetuses was 49.3%, 51.4%, 50.6% and 47.7% in groups 1, 2, 3 and 4, respectively.

Body Weights

Mean fetal weights were not affected by treatment with the test item at any dose level.

Visceral Examination of Fetuses (Microdissection Technique)

During visceral examination of the fetuses, findings were noted in:

43% examined fetuses (in 100% litters) in group 1

51% examined fetuses (in 95% litters) in group 2

42% examined fetuses (in 95% litters) in group 3

38% examined fetuses (in 91% litters) in group 4

Abnormalities were noted in one fetus each in groups 1, 2 and 4 and were considered to be incidental. In group 1, one fetus was noted to have a small mouth and short tongue. In group 2, one fetus had thoracic situs inversus. In group 4, one fetus had small mouth, tongue absent, cleft and misshapen palate, fused nares, short nasal septum, interrupted and misshapen nasopharynx and small pituitary. These abnormalities occurred in single fetuses and did not give any indication of a test item-related effect.

The type and frequency of the noted variations were similar in all groups and did not indicate any dose-dependency. They were therefore considered not to be test item-related.

Skeletal Examinations of Fetuses (Abnormalities and Variations)

During skeletal examination of the fetuses, findings were noted in:

16% examined fetuses (in 59% litters) in group 1

8% examined fetuses (in 32% litters) in group 2

13% examined fetuses (in 45% litters) in group 3

13% examined fetuses (in 50% litters) in group 4

No test item-related findings were noted.

The type and frequencies of the noted less common variations were similar in nature for the group receiving the test item and the control group and did not indicate any dose-dependency, therefore they were not considered to be test item-related.

Bone and cartilage abnormalities were found in one fetus in group 3, which had right rib 1 short and left costal cartilage 1 short and right costal cartilage absent.

Bone Examination - Ossification Stage / Supernumerary Ribs

There were no test item-related effects on the ossification stage and the number of supernumerary ribs in any dose group.

The occasionally statistically significant differences did not show dose dependency and were caused by higher as well as lower stages of development, therefore were considered to be incidental.

Cartilage Examination - Additional Variations

During cartilage examination of the fetuses abnormal findings were noted in:

2% examined fetuses (in 14% litters) in group 1

4% examined fetuses (in 23% litters) in group 2

4% examined fetuses (in 18% litters) in group 3

4% examined fetuses (in 23% litters) in group 4

No test item-related findings were noted.

In group 4, statistically significantly higher incidence of branched xiphoid cartilage was noted when calculated on a litter basis. Since no statistically significance was noted when calculated on a fetus basis this finding was considered to be incidental.

Applicant's summary and conclusion

Conclusions:
Based on the above mentioned results and taking into consideration that the reduction of food consumption was not statistically significant, the NOAEL (No Observed Adverse Effect Level) for maternal and fetal organisms was considered to be 1000 mg/kg body weight/day.

Under the conditions described for this study, 2-Hydroxyethanesulphonate did not reveal teratogenic potential up to and including 1000 mg/kg body weight/day.
Executive summary:

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and fetus consequent to exposure of the female to the test item from day 0 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section).

Four groups of females were treated by gavage with Sodium 2-Hydroxyethanesulphonate once daily at dose levels of:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 50 mg/kg body weight/day

Group 3: 200 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

All females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section.

The following results were obtained:

1. Maternal Data

General Tolerability

All dams survived until the scheduled necropsy. No clinical symptoms related to treatment with the test item were noted during the study.

Food Consumption and Body Weights

Food consumption and body weight were not affected by the treatment with the test item. At 1000 mg/kg/day, mean food consumption was marginally lower than control, but as it was not statistically significant and in absence of an effect on body weight was considered to be of no toxicological relevance.

Reproduction Data

The relevant reproduction data (pre- and post-implantation loss and the mean number of fetuses per dam) were not affected by treatment with the test item at any dose level.

Macroscopical Findings

No macroscopical findings were noted during necropsy of the dams.

2. Fetal Data

External Examination

The abnormal findings observed during the external examinations were considered to be incidental since they occurred isolated in single fetuses.

Sex Ratios

No test item-related effects on fetal sex ratios were noted in any dose group.

Body Weights

No test item-related effects on fetal body weights were noted.

Visceral Examination

No test item-related abnormalities were noted during the visceral examination of fetuses.

Skeletal and Cartilage Examination

No abnormalities, which were considered to be test item-related, were noted during examination of fetal skeletons and cartilages.