Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 22.03.1996
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI
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, standard gamma irradiated pelleted food supplied by M/s. Tetragon Chemie Pvt. Ltd., Bangalore, India
- Water: reverse osmosis water, ad libitum
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature: between 19.6 and 22.0°C
- Humidity: between 50 and 59%
- Photoperiod: 12 light and 12 dark conditions

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on 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)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: 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.
- After successful mating each pregnant female was caged (how): single


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
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Range Finding Study: 3
Main Study: 10
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.
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for morbidity/mortality during the entire observation period.

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

BODY WEIGHT: Yes
- Time schedule for examinations: 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 AND COMPOUND INTAKE: Yes
- 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.

WATER CONSUMPTION AND COMPOUND INTAKE: No

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

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.

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
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.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
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 NECROPSY
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.

HISTOPATHOLOGY / ORGAN WEIGHTS
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.
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).

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

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
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.
Mortality:
no mortality observed
Description (incidence):
No morbidity/mortality was observed in any of the animals during the entire observation period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No statistically significant changes were observed in body weights of males and females of treated groups when compared with the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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 corpuscular 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.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No statistically significant changes were observed in bone marrow cytology in any of the treatment groups when compared with the control group.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
No test substance related effect was observed on mating/mating period duration of females in treated groups and in the control group. There was no test substance related effect on gestation length of dams in any of the treated groups when compared to the control group. 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. 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. 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.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day
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).
Key result
Dose descriptor:
NOAEL
Effect level:
> 2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Description (incidence and severity):
No test substance related effect was observed on the number of dams delivered with live pups in any of the treated groups of animals and in control group respectively. No test substance related effect was observed on mean litter size in any of the treated group of animals when compared to control group of animals at day 0 and day 4 post partum.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
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 of animals.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross external examination of live pups sacrificed on day 4 post-partum did not reveal any abnormality that could be attributed to the treatment.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 500 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
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 NOAEL for reproduction toxicity and fertility as well as the NOAEL for developmental toxicity in the F1 progeny for 100% Sodium ethylenesulphonate was greater than 500 mg/kg bw/day.
Executive summary:

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 25.09 % 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, clinical 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 25.09% Sodium ethylenesulphonate, the obtained NOAEL was greater than 2000 mg/kg bw/day for the tested solution. Based on this study the derived NOAEL for reproduction toxicity and fertility as well as the NOAEL for developmental toxicity in the F1 progeny for 100% Sodium ethylenesulphonate was greater than 500 mg/kg bw/day. No specific target organ was identified.