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Description of key information

In a reliable combined repeated dose and reproductive/developmental toxicity screening test (OECD TG 422) with a test solution containing 25.4 % Sodium ethylenesulphonate by oral gavage in rats  no systemic toxicity occurred up to the high dose level of 2000 mg/kg bw/day. The NOAEL was determined to be greater than 2000 mg/kg bw/day for the test solution. Based on this study the derived repeated dose NOAEL for 100% Sodium ethylenesulphonate was greater than 500 mg/kg bw/day.
Repeated dose toxicity studies for the inhalation or dermal route are not required.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-02-23 to 2010-04-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reliable GLP compliant study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Procured from Charles River, USA and bred at IIBAT animal house facility.
- Age at study initiation: Young adult rats, between 12 and 14 weeks old Females were virgin.
- Weight at study initiation: males: 326-407g; females: 241-287g
- Housing: In standard polypropylene cages with stainless stell top grill; females were housed in groups of 5 animals during pre mating period. Males were housed individually during pre mating and post mating. One male and one female were kept together in a cage until the confirmation of mating. After confirmation of mating females were caged individually.
- Diet: ad libitum,
- Water: reverse osmosis water, ad libitum
- Acclimation period: Five days prior to experiment in the test room.

ENVIRONMENTAL CONDITIONS
- Temperature: between 19.6 and 22.0°C
- Humidity: between 50 and 59%
- Photoperiod: 12 light and 12 dark conditions
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION ON ORAL EXPOSURE:
The test substance was suspended in distilled water to achieve the nominal concentrations for each dose level. The dose formulations were prepared daily. The stability of sodium ethylenesulphonate in the vehicle was determined.

VEHICLE:
- Sodium ethylenesulphonate concentration in vehicle: 12.5, 25, 50 mg/mL
- Amount of vehicle: 10 mL/kg bw (dose volume)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Range Finding Study
- 7 days
Main Study
- at least 28 days
Dosing of both sexes began 2 weeks prior to mating, after acclimatization. Dosing was continued in both sexes during the mating period. Males were further dosed after the mating period until the minimum dosing period of 28 days was completed and then sacrificed. Dosing of mating confirmed females was continued throughout gestation until day 4 post partum.
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw/day of a 25% Sodium ethylenesulfonate solution
Basis:
actual ingested
No. of animals per sex per dose:
Range Finding Study
- 3 rats
Main Study
- 10 rats
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELECTION RATIONALE:
The dose levels for the main study were selected based in the results of a 7 day range-finding study which revealed no test substance-related finding (viability, clinical signs, macroscopical examination) up to and including the highest dose level of 2000 mg/kg bw, corresponding to 500 mg/kg bw/day in terms of pure Sodium etyhlenesulphonate.
MATING PROCEDURE
1:1 (one male to one female) mating was followed in this study. The females were housed with the same males until pregnancy occured.
Observations and examinations performed and frequency:
OBSERVATION PERIOD
Males were observed for 28 days and females were observed during premating (14 days), mating (1-6 days), and gestation (20-23 days), parturition and till day 4 post partum.

MORBIDITY/MORTALITY
All animals were observed twice daily for morbidity/mortality during the entire observation period.

DETAILED CLINICAL OBSERVATIONS
All animals were observed for toxicity signs once daily, preferably after dosing in the morning.


NEUROBEHAVIOURAL EXAMINATION
Functional observation battery (FOB) including auditory function, grip strength and locomotor activity assessment was conducted in selected 5 males and 5 females from each group. In males, FOB was conducted shortly before scheduled kill but before blood sampling for hematology or biochemistry. Females were tested for FOB during lactation, shortly before scheduled kill.


BODY WEIGHT
Body weight of individual male and female rats were recorded prior to the administration of the test substance (day 0) and weekly thereafter. During pregnancy, females were weighed on day 0, 7, 14 and 20 of pregnancy and within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum.

FOOD CONSUMPTION
Feed consumption was recorded daily during pre-mating (cage wise), pregnancy and lactation in females. The feed consumption was not recorded during mating period. In males, feed consumption was recorded daily only during pre-mating.

BLOOD COLLECTION
Blood was collected from orbital sinus in heparinised vials (for biochemistry) as well as in vials containing EDTA (for hematology) from 5 males and 5 females from each group. In males, it was done at the end of the pre-mating period and just prior to the procedure for killing the animals. In females, it was done at the end of the pre-mating period and just prior to the procedure for killing the animals.

HEMATOLOGY
The following parameters were determined:
Erythrocyte (RBC) count,
Hemoglobin (Hb) concentration,
Hematocrit (HCT),
Mean corpuscular volume (MCV),
Mean corpuscular hemoglobin (MCH),
Mean corpuscular hemoglobin concentration (MCHC),
Platelet count,
Total leucocyte (WBC) count,
Differential leucocyte count,
Clotting time.

CLINICAL CHEMISTRY
The following parameters were determined:
Glucose,
Urea,
Blood urea nitrogen (BUN),
Creatinine,
Total cholesterol,
Triglycerides,
Albumin,
Total protein,
Alanine aminotransferase (ALT),
Aspartate aminotransferase (AST),
Alkaline phosphatase (ALP),
Calcium,
Phosphorus,
Sodium,
Potassium

MATING CONFIRMATION/EVIDENCE OF COPULATION
Each morning the females were examined for the presence of sperm and/or vaginal plug. Day 0 of pregnancy is defined as the day on which vaginal plug or sperm is observed.

GESTATION LENGTH AND LITTER DATA
All dams were allowed to litter naturally and the size, weight of litter and sex of litter-mates were recorded at parturition (day 0) and at day 4 post partum. The duration of gestation length was recorded and was calculated from day 0 of pregnancy to the day of parturition. Each litter was examined as earliest after delivery to establish the numbers and sex of pups, still births, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed, litters were weighed within 24 hours of parturition (day 0 post partum) and day 4 post partum. Sex ratio (m/f) was calculated using formula as below:

Sex ration (m/f)= (No. of male pups/No. of female pups) x 100
Sacrifice and pathology:
SACRIFICE
Dams with offspring were sacrificed on day 4 post partum. Females which failed to deliver were sacrificed 26 days after the last day of mating period. Pups were sacrificed at day 4 post partum.

GROSS PATHOLOGY
Adult animals were examined macroscopically for any abnormalities and pathological changes. The number of implantation sites was recorded and the counting of corpora lutea was done. Special attention was paid to the organs of the reproduction system. The ovaries, testes, epididymis, accessory sex organs and all organs showing macroscopic lesions of all adult animals were preserved. Of the selected five males and five females, the following tissues were preserved in 10% neutral buffered formalin and intended for subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including Peyer's patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs, uterus, urinary bladder, mesenteric lymph nodes, mandibular lymph nodes, peripheral nerve (sciatic), and a section of bone marrow. Bone marrow aspirate was collected for bone marrow cytology.

ORGAN WEIGHT
Weights of following organs of all male adult animals were recorded.
1. Testes
2. Epididymis
In addition, weights of following organs for 5 adult males and females selected from each group was determined: liver, kidneys, adrenals, thymus, spleen, mesenteric lymph nodes, mandibular lymph nodes, brain and heart.

BONE MARROW CYTOLOGY
Bone marrow aspirate was conducted in selected 5 males and 5 females from each group for bone marrow cytology during necropsy.

HISTOPATHOLOGY
Detailed histological examination was performed on all the animals of control and high dose group with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure
1. Ovaries
2. Testes
3. Epididymis

In addition, full histopathological examination was carried out on the following tissues of the selected 5 males and 5 females of control and high dose group: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including Peyer's patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea, lungs, uterus, urinary bladder, mesenteric lymph nodes, mandibular lymph nodes, peripheral nerve (sciatic), and a section of bone marrow (sternum).



Statistics:
Body weight, food consumption, detailed signs of toxicity, FOB, hematology, biochemistry and organ weight, corpora lutea, implantations, litter data of rats belonging to the experimental groups assured for homogeneity. When the data was homogeneous then it was analysed using ANOVA. (Student's Newman - Keufs Test was employed for post - hoc comparison). When the data was not homogeneous it was analysed with Kruskal-Wallis One-Way ANOVA on Rank basis.
Details on results:
MORTALITY
No morbidity/mortality was observed in any of the animals during the entire observation period.

SIGNS OF TOXICITY
Test substance related signs of toxicity were not observed in any of the treated group throughout the observation period. No abnormal behavior was observed in offsprings.

BODY WEIGHT
No statistically significant changes were observed in body weights of males and females of treated groups when compared with the control group.

FEED CONSUMPTION
Test substance related statistically significant changes were not observed in feed consumption of males and females of treated groups when compared with the control group.

HEMATOLOGY
Statistically significant changes were not observed in hematology parameters of the treated groups when compared with the control group, except a slight decrease in mean corpusular volume (MCV) in the blood of the high dose males at day 28, which was well within normal limit and considered of no biological significance.

BIOCHEMISTRY
Statistically significant changes were not observed in biochemistry parameters of low, mid and high dose group of animals when compared with the control group animals.

FUNCTIONAL OBSERVATION BATTERY
No test substance related effects were observed in functional observational battery (FOB) parameters in treated groups of males (low, mid and high dose groups) and in the control group male animals. However, in females (500 mg/kg bw) auditory function (acoustic startle) was increased when compared to control group female animals.

MATING PERIOD/EVIDENCE OF COPULATION
No test substance related effect were observed on mating/mating period duration of females in treated groups and in the control group.

GESTATION LENGTH
There was no test substance related effect on gestation length of dams in any of the treated groups when compared to the control group.

IMPLANTATIONS
No test substance related effect was observed on the mean number of implantation sites in any of the treated groups when compared with the control group.

MEAN LITTER SIZE
No test substance related effect was observed on mean litter size in any of the treated groups when compared to the control group at day 0 and day 4 post partum.

MEAN LITTER SIZE
No test substance related effect was observed in mean litter weight on day 0 and day 4 post partum in any of the treated groups when compared with control group.

DAM WITH LIVE PUPS
No test substance related effect was observed on the number of dams delivered with live pups in any of the treated groups as compared to control.

LOSS OF OFFSPRINGS
No test substance related effect was observed on loss of offspring (pre implantation, post implantation and post natal) in any of the treated groups when compared with the control group.

SEX RATIO OF PUPS
No test substance related effect was observed on sex ratio of the pups in any of the treated groups when compared with the control group.

EXTERNAL ABNORMALITIES IN PUPS
Gross external examination of live pups sacrificed on day 4 post-partum did not reveal any abnormality that could be attributed to the treatment.

ORGAN WEIGHT CHANGES
No statistically significant changes were observed in both absolute and relative organ weights in any of the treatment groups when compared with the control group.

BONE MARROW CYTOLOGY
No statistically significant changes were observed in bone marrow cytology in any of the treatment groups when compared with the control group.

GROSS PATHOLOGY
No test substance related gross pathological changes were observed in any of the treated and control group. All macroscopic findings were either related to agonal, spontaneous, or incidental lesion were of the type routinely observed in Wistar rats of this age.

HISTOPATHOLOGY
No test substance related adverse histopathological findings were observed in the high dose group. All microscopic findings were either related to agonal, spontaneous, or incidental lesions were of the type routinely observed in Wistar rats of this age.


Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No test substance related findings were noted up to and including the high dose level. Dose level given in terms of pure Sodium ethylenesulphonate (This dose level was reported as 2000 mg/kg bw/day of a 25 % solution).
Critical effects observed:
not specified
Conclusions:
Under the conditions of the combined repeated dose toxicity study in male and female rat with the test item, containing 25% Sodium ethylenesulphonate, the obtained NOAEL was greater than 2000 mg/kg bw/day for the tested solution. Based on this study the derived repeated dose NOAEL for 100% Sodium ethylenesulphonate was greater than 500 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: oral route

In a GLP compliant combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (IIBAT(d), 2010, OECD 422) a test solution containing 25.4 % Sodium ethylenesulphonate was administered daily to Wistar rats (4 groups of 10 male and 10 female animals each) via oral gavage at dose levels of 0, 500, 1000 or 2000 mg/kg bw/day (corresponding to approximately 0,125, 250 and 500 mg/kg bw/day of 100 % Sodium ethylenesulphonate, respectively). The treatment period covered a 2-week pre-mating and mating period in both sexes. Males were further dosed after the mating period until the minimum dosing period of 28 days was completed and then sacrificed. Dosing of mating confirmed females was continued throughout gestation until day 4 post partum. Mortality, clincal signs, body weight and food consumption were assessed at regular intervals. At the end of the study functional observation battery and motor activity parameters as well as haematology and clinical chemistry parameters were determined. At necropsy, selected organs were weighed and the animals were examined macroscopically and histopathologically. Relevant reproductive parameters and indices were determined.

No morbidity or mortality was observed in any of the animals during the entire observation period. Test substance related signs of toxicity were not observed in any of the treated group throughout the study period. No abnormal behavior was observed in offsprings. Body weights and feed consumption revealed no statistically significant changes. No statistically or biologically significant changes were observed at the evaluation of the haematology and clinical chemistry parameters. No test substance related effects were observed in functional observational battery (FOB) parameters in treated groups of males (low, mid and high dose groups) and in the control group male animals. However, in females (500 mg/kg bw) auditory function (acoustic startle) was increased when compared to control group female animals. Mating period, gestation length, mean number of implantation sites, mean litter size, number of dams delivered with live pups, loss of offsprings, sex ratio of pups and external abnormalities showed no test substance related abnormalities. Organ weight and bone marrow cytology revealed no statistically significant changes. No test substance related gross pathological changes and adverse histopathological findings were observed in any of the treated and control groups. All macroscopic and microscopic findings were either related to agonal, spontaneous, or incidental lesions were of the type routinely observed in Wistar rats of this age. In conclusion, under the conditions of the combined repeated dose toxicity study in male and female rats with the test item, containing 25% Sodium ethylenesulphonate, the obtained NOAEL was greater than 2000 mg/kg bw/day for the tested solution. The derived repeated dose NOAEL for 100% Sodium ethylenesulphonate was greater than 500 mg/kg bw/day. No specific target organ was identified.

Repeated dose toxicity: inhalation and dermal route of exposure

Repeated dose toxicity has been studied for the oral route of exposure. According to Annex IX of Regulation (EC) No 1907/2006 (REACH) only one route needs to be addressed for the evaluation of repeated dose toxicity. Thus, a repeated dose toxicity study for the inhalation or dermal route is not needed.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Most reliable study

Justification for classification or non-classification

Based on the results of the repeated dose toxicity testing, Sodium ethylenesulphonate does not need to be subjected to classification and labelling according to Directive 67/548/EEC (DSD) for danger of serious damage to health by prolonged exposure or for specific target organ toxicity after repeated exposure (STOT RE) according to Regulation (EC) No 1272/2008 (CLP).