Sodium ethylenesulphonate

Endpoint summary

Description of key information

In a reliable combined repeated dose and reproductive/developmental toxicity screening test (OECD TG 422) with a similar multi-constituent substance solution containing 25.4% sodium ethylene sulphonate 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 ethylene sulphonate was greater than 500 mg/kg bw/day.

As this study was not dosed up to 1000 mg/kg bw/d active substance (sodium vinyl sulphonate SVS), ECHA requested a new OECD 422 study to be performed, dosed up to 1000 mg/kg bw/d active substance. This study was performed and is reported here too. Likewise, in this new study, dosed up to 1000 mg/kg bw/d no effects were noted for repeated dose toxicity or reproductive parameters and thus, the NOAEL for repeat dose toxicity, fertility and developmental toxicity in this study was set to the highest dose tested, i.e. 1000 mg/kg bw/d.

A sub-chronic oral toxicity study is to be performed and this endpoint will be re-assessed, once results are available.

Repeated dose toxicity studies for the inhalation or dermal route are not required.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name of the Test Item: Vinalyst 4330
Chemical Name (IUPAC): Sodium Vinyl sulfonate
Physical Appearance: Pale yellow liquid
Lot No.: SVS/R0821/12
Purity (as per Certificate of Analysis): 30.12% active (CAS No.: 3039-83-6) in aqueous solution
Storage Conditions: Ambient (21 to 29 °C)
Test Item code by Test facility: D1349-001
Date of Manufacture: 25 August 2021
Date of Retest: 25 August 2022

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source of Supply: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee, CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA
No. of Groups: 6 Groups; Main Groups: G1 - Vehicle control, G2 - Low dose, G3 - Mid dose, G4 - High dose; Recovery Groups: G1R - Vehicle control recovery group and G4R - High dose recovery group
No. of Animals / Group: Main Group: 24 (12 Males + 12 Females), Recovery Group: 10 (5 Males + 5 Females)
Total Number of Animals: 116 (58 Males + 58 Females)
A total of 68 females were received and evaluated pre-exposure for estrous cyclicity. The females that failed to exhibit typical 4 to 5-day cycles were excluded from the experiment and sacrificed after initiation of treatment on day 1. Finally, 58 females were selected for the study. Females used were nulliparous and non-pregnant.
Age at Receipt: 8 to 9 weeks
Age at initiation of treatment: 10 to 11 weeks
Body Weight at Receipt: Males: 237.60 to 286.08 g, Females: 200.11 to 225.20 g
Body Weight at initiation of treatment: Males: 335.91 to 415.73 g, Females: 235.49 to 281.14 g
During acclimatization period all the animals were identified within the cage by tail marking using a black permanent marker pen for males and red permanent marker pen for females. Additionally, a cage card was displayed which included study no., study code, cage no., sex, animal no., start date and end date of acclimatization period. During the treatment period and post randomization all the animals were identified by body marking using turmeric solution. Additionally, a cage card was displayed which included study no., study code, animal no., sex, dose, group no., cage no., dosing start date, dosing end date and date of necropsy.
Husbandry
Environmental Conditions: Animals were housed under standard laboratory conditions in an environmentally monitored, air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.1 to 24.2 °C and relative humidity 41 to 64%, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.
Housing: Animals were housed in a standard polysulphonate cage (size: L 43 x B 28 x H 21 cm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Processed corncob granules were provided as bedding material.
During acclimatization, two main group animals of same sex were housed as follows:
Pre-mating: Two animals of same sex and group per cage.
Cohabitation period (mating): Two animals (one male and one female) of same group per cage were housed.
Post-mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually.
Sterilized paper shreds were provided as a nesting material for main group females from gestation day 20 onwards.
For recovery group animals a maximum of two animals per cage per group of same sex were housed.
Feed: Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period. The contaminant analysis test report of the feed is included as an Annexure to the study report. A sample of feed from each batch used in the study was retained and was discarded on the day of finalization of study report.
Water: Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes. The contaminant analysis test reports for water and bedding material nearest to the experimental period are included as Annexures to the final report and maintained with the study raw data file.
Acclimatization: Healthy and young adult animals were acclimatized for five days to experimental room conditions and observed for clinical signs twice daily for mortality and morbidity. All the females were screened for normal estrous cycles (4 to 5 days) in a two-weeks pre-treatment period after initial 5 days of acclimatization period. All the animals (males and females) were observed for clinical signs twice daily for mortality and morbidity during this period. Veterinary examination of all the animals was performed on the day of receipt and on the day of randomization and grouping.
Grouping and Randomization: The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were randomly distributed to all the main and recovery groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ±20% (Males: -9.16 to 10.19%; Females: -8.69 to 7.31%) of the mean body weight of each sex. The grouping was done one day prior to initiation of treatment. Body weight of the animals were analyzed statistically for mean body weight to rule out statistically significant differences between groups within each sex.

Route of administration:

oral: gavage

Details on route of administration:

The vehicle and test item formulations were administered through oral (gavage) route as it is the probable route of exposure to human. Hence oral route was selected for dose administration.
Formulation Preparation: The prepared test item formulations were administered to the animals within established stability conditions. The required quantity of test item was weighed in a beaker. The test item was mixed with a small quantity of vehicle using glass rod and transferred into measuring cylinder. Again, a small quantity of vehicle was added to rinse the beaker and was transferred into the measuring cylinder. The rinsing procedure of beaker was repeated many times to ensure the transfer of entire contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 33.2, 99.6 and 332 mg/mL of test item [to achieve the concentrations of 10, 30 and 100 mg/mL of active content Sodium Vinyl Sulfonate (30.12 % v/v) in the provided test solution] for low, mid, and high dose groups respectively.
The test formulations were maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test item formulations during preparation and administration.

Vehicle:

water

Remarks:

The test item was clearly miscible with distilled water at the concentration of 332 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per in-house miscibility test results.

Details on oral exposure:

Administration of Test Item: The test item or vehicle was administered to the animals by oral (gavage) route once daily within the established stability conditions.
The main group males were treated for two weeks pre-mating, during mating period and up to the day before sacrifice (total of 29 days of administration). The main group females were administered as follows: The pregnant animals were treated for a two-week pre-mating period (14-days), during mating, pregnancy (gestation) and up to lactation day 13 (ranging from a total period of 49 to 58 days). Non-pregnant/dam with total implantation loss were treated for two weeks (14-days) during pre-mating, during cohabitation period until confirmation of mating and further 25 days from the day of confirmation of mating (group G1 - one female until experimental day 41, one female until experimental day 49; group G2 - one female until experimental day 43, one female until experimental day 45; G3 - one female until experimental day 42, one female until experimental day 46; G4 - one female until experimental day 41, one female until experimental day 41). The recovery group animals were treated until the first scheduled sacrifice of dams (total of 49 days of administration) and kept without treatment for further 14-days of observation period. The test item/vehicle was administered by oral (gavage) route using stainless steel intubation cannula attached to a disposable syringe. All the doses were administered in an equal volume of 10 mL/kg with the concentration of 33.2, 99.6 and 332 mg/mL of test item [to achieve the concentrations of 10, 30 and 100 mg/mL of active content Sodium Vinyl Sulfonate (30.12 % v/v) in the provided test solution] for low, mid, and high dose groups, respectively. Vehicle was administered to vehicle control group at an equal volume of 10 mL/kg body weight. The actual dose volume for each animal was calculated based on the most recent body weight. The test item formulations were administered as soon as possible after preparation. The prepared formulations were stirred using magnetic stirrer during administration to maintain homogeneity.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of Test Item Formulation: The stability of test item in dose formulations was established by the Analytical Department of Bioneeds India Private Limited with the concentration of 0.5 mg/mL (low dose) and 110 mg/mL (high dose) in distilled water under Bioneeds study no.: BIO-ANM 1855. The dose concentrations were stable up to 48 hours at room temperature. Prepared test item formulations were administered to the animals within established stability conditions.
Dose Formulation Analysis: Dose formulation analysis for dose concentration verification was done by Analytical Chemistry department of Bioneeds India Private Limited. The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 1855 and the results were presented in a separate report. Sampling and analysis of formulations was performed during day 1 (week 1) and day 27 (week 4) of the treatment. The samples were collected in duplicates (5 mL each) using a single layer from vehicle control, low, mid, and high dose concentrations. The prepared test item formulations were stirred using magnetic stirrer during sampling. The collected samples were transferred to the analytical chemistry department for dose formulation analysis. One set of aliquots of each formulation was analyzed. As during analysis of test item formulations on day 1, a laboratory incidence was reported, the sequence of process was stopped as the evaporator gas flow rate limit exceeded. The instrument and software were restarted. The sequence was started using the second set of aliquots of each formulation. During week 4, the second aliquot was stored as a backup purpose at established stability conditions. The second set of samples were discarded, as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, since the mean results were within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) was less than 10% during both the analysis time points.

Duration of treatment / exposure:

Main group males were treated for 29 days, daily. Pregnant animals were treated for a two-week pre-mating period (14-days), during mating, pregnancy (gestation) and up to lactation day 13 (ranging from a total period of 49 to 58 days). Non-pregnant females or dams with total implantation loss were treated for two weeks (14-days) during pre-mating, during cohabitation period until confirmation of mating and further 25 days from the day of confirmation of mating.

Frequency of treatment:

by gavage once daily

Dose / conc.:

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

Remarks:

Vehicle control G1 and vehicle control recovery group G1R

Dose / conc.:

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

Remarks:

Low dose group G2

Dose / conc.:

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

Remarks:

Mid dose group G3

Dose / conc.:

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

Remarks:

High dose G4 and high dose recovery group G4R

No. of animals per sex per dose:

12 males and 12 females per dose group were administered. Furthermore, 5 males and 5 females were used in the recovery groups (G1R and G4R).

Control animals:

yes, concurrent vehicle

Details on study design:

Dose Selection and Justification for Selection: A dose range finding study (Bioneeds Study No. BIO-DTX 084, see separate endpoint study record) was conducted with doses of 100, 300 and 1000 mg/kg body weight of active content Sodium Vinyl Sulfonate (30.12 % v/v in the provided test solution) for low, mid, and high dose groups, respectively based on the below mentioned references in consultation with the sponsor.
Based on the estimated LD50 being > 15000 mg/kg body weight for Sodium ethylene sulfonate 25% solution and the derived LD50 for 100% Sodium ethylene sulphonate greater than 3750 mg/kg body weight obtained from a study conducted as per OECD test guidelines 401 (Acute Oral Toxicity) in female rats and based on an estimated NOAEL of > 2000 mg of active ingredient SVS/kg body weight/day for Sodium ethylene sulfonate 25% solution and the derived repeated dose NOAEL for 100% Sodium ethylene sulphonate being greater than 500 mg/kg body weight /day obtained from a study conducted as per OECD test guidelines OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test a Dose range finding study was initiated and the results of this study revealed that oral administration of test item Vinalyst 4330 over a relatively limited period of time (for males with a total of 35 days and for females ranging from 49 to 66 days) did not reveal any indication of systemic, reproduction and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg body weight of active content Sodium Vinyl Sulfonate (30.12 % v/v in the provided test solution). Hence, the same doses i.e., 100, 300 and 1000 mg/kg body weight of active content Sodium Vinyl Sulfonate (30.12 % v/v in the provided test solution) were selected as low, mid, and high dose levels for the present definitive Combined Repeated and Reproduction/Developmental Toxicity Screening Test with Vinalyst 4330 in rats.
Mating Procedure and Grouping: The males and females were placed in 1:1 ratio. Every morning, the vaginal smear of each female was examined for presence of sperm in the vaginal smear. The female was placed with the same male until pregnancy occurred by evidence of sperm in the vaginal smear until two weeks had elapsed. The day with presence of sperm in the vaginal smear was considered as gestation day (GD) 0. All the females from each group were confirmed with mating within 14 days of cohabitation period with first male. Hence, no re-mating with proven males was considered in the study. The females confirmed with mating but not littered were sacrificed on day 26 after confirmation of mating.
The recovery group animals were not left for cohabitation. Analysis of findings and observations are described below.

Positive control:

none

Observations and examinations performed and frequency:

The following observations were done during experimental period.
Clinical Signs of Toxicity and Mortality/Morbidity: All the animals were observed once daily for general clinical signs of toxicity. These observations were made approximately at the same time on each day after dose administration. All the animals were observed twice daily for mortality and morbidity.
Detailed Clinical Examination: All animals were subjected to detailed clinical examinations on day 1 before treatment and weekly thereafter during treatment. These observations were made outside the home cage and approximately at the same time.
Body Weight recordings: The main group males were weighed at receipt, on the first day of dosing and weekly once thereafter and at termination. Main group females were weighed at receipt, on the first day of dosing, and weekly once thereafter during pre-mating and until confirmation of mating. Thereafter, all the pregnant animals were weighed on gestation days 0, 7, 14, and 20 and on lactation days 1, 4, 7, 13 and 14 (terminal body weight). The mated but not littered females were weighed on the day of mating and weekly once thereafter until termination including terminal body weight.
The recovery group animals (both sexes) were weighed at receipt, on the first day of dosing, at least weekly once thereafter, one day prior to termination and at termination.
Feed consumption (cage wise) was measured during following occasions:
Main Group Males: Cage wise feed consumption was measured for all males once a week during premating period coinciding with body weight recording. The feed consumption was also measured for males once in a week during post-mating period.
Main Group Females:
Cage wise feed consumption was measured for all females once in a week during premating period coinciding with body weight recording. Cage wise feed consumption was measured for all pregnant animals during gestation days 0 to 7, 7 to 14 and 14 to 20, during lactation days 1 to 4, 4 to 7 and 7 to 13. Cage wise feed consumption was measured weekly once for all mated, but not littered females. Cage wise feed consumption was measured for all non-pregnant/dam with total implantation loss animals once in a week coinciding with body weight recording.
Note: Feed consumption was not measured during following occasions: during cohabitation period for both sexes. after gestation day 20 and until parturition (pregnant animals). after day 20 from the day of confirmation of mating and until termination (mated but non-pregnant).
For recovery group animals (both sexes) cage wise feed consumption was measured once a week throughout the experimental period coinciding with body weight recording.
Average feed intake per animal (g/animal/day) was calculated using the amount of feed offered and left over in each cage and the number of rats per cage. Feed spillage was taken into consideration.
Ophthalmoscopic examination was carried out for all animals before initiation of treatment i.e., after grouping and randomization (day 1). Ophthalmological examination was also carried out at the end of dosing period for all males (shortly prior to scheduled sacrifice, i.e., day 29) and during lactation period for littered females (i.e., on lactation day 13, on the day of sacrifice, i.e., 25 days after confirmation of mating for mated but not littered/non-pregnant females) and during last week (i.e., day 65) for the recovery group animals.
Neurological/Functional examination was performed for five males and five females, randomly selected from each group, towards the end of the dosing period for males (shortly prior to scheduled sacrifice, i.e., on treatment day 28) and during the lactation period for females (shortly prior to scheduled sacrifice, i.e., on lactation day 13). Neurological/Functional examination was performed for all recovery group animals towards the end of the recovery period (shortly prior to scheduled sacrifice, i.e., on day 65). The following Neurological/Functional observations were performed:
- Home Cage Observations: Animals in the cage were observed for convulsions, tremors and palpebral closure and the scores were recorded.
- Handling Observations: During removal from the cage, the animals were observed for its ease to be removed out of the cage. During handling observation, the animals were observed for the resistance, lacrimation, red and crusty deposits around eyes, nose and mouth.
- Open Field Observations: Animals were placed in an open field arena and observed for their mobility, gait, arousal, rearing, urination, defecation, stereotypes and excessive grooming.
- Sensory Observations: Animals were observed for startle response, touch response, pupil response, response to nociceptive stimuli and righting reflexes.
- Neuromuscular Observations: As a part of the neuromuscular observations, the animals were dropped onto a recording paper sheet from a height of approximately 30 cm with painted paws to record the hind limb foot splay.
- Physiological Observation (Rectal temperature): Physiological temperature of the animals was recorded using a calibrated digital thermometer.
- Grip strength assessment: Assessment of hind limb and fore limb grip strength was carried out using a grip strength meter.
- Motor activity assessment: Assessment of motor activity of the animals was carried out using a Locomotor activity monitoring system.
Thyroid Hormone Estimations: Blood collection for Thyroxine (T4) Level estimation: Blood samples were collected from all the main group animals for measurement of serum T4 levels on the following schedule:
Two pups per litter on postnatal day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when the litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection for possible serum T4 assessments in case of availability of only one pup above the normal litter size.
All adult main group females at termination (lactation day 14).
- Two male pups per litter at termination (postnatal day 13).
- All adult main group males, at termination (after completion of at least 29 days of treatment).
Note: Pooled serum collected from PND 13 pups per litter was analyzed for T4 analysis. The assessment of serum T4 levels was performed for adult males and PND 13 pups. Further assessment of T4 levels in serum samples from the dams and PND 4 pups was not assessed as there were no treatment-related effects observed in the T4 levels of adult males or PND 13 pups.
Blood samples of all adult animals and PND 13 pups were collected using the retro-orbital plexus puncture method under Isoflurane anaesthesia with the help of a fine capillary tube. The blood from PND 4 pups was collected by jugular vein incision. The collected blood samples were centrifuged at 5000 rpm for 10 minutes; the serum was separated and stored at freezing conditions (-80 °C ±10 °C) till estimation. The serum samples were sent to bioanalytical department and estimated for thyroid hormonal levels (Thyroxine - T4) by the ELISA method using commercial assay kits. After estimation, the serum samples were stored at freezing conditions (-80 °C ±10 °C) till finalisation of study report.
Note: The assessment of serum T4 levels was not performed for recovery group animals.

Sacrifice and pathology:

Necropsy and Gross pathology: The adult males were sacrificed after completion of 29 days of treatment. All pregnant females were sacrificed on lactation day 14 and the non-pregnant females were sacrificed after 25 days from the day of confirmation of mating. All the recovery group animals were sacrificed after completion of 14-days observation from the first scheduled sacrifice of dams. All the animals were fasted overnight, and water was made available ad libitum during fasting. The next day, body weight of all the fasted animals was recorded prior to necropsy. The animals were humanely sacrificed using CO2 asphyxiation and subjected to detailed gross necropsy, which included careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents as well as organs and tissues of each animal with special emphasis on reproductive organs. The vaginal smear of all main group females on the day of necropsy was performed and the stage of oestrus cycle was recorded to correlate with the histopathology of ovaries. The main group males and recovery group animals were necropsied in randomized manner. Dead pups and pups which were sacrificed on PND 4/13, examined for gross abnormalities with particular attention to the external reproductive genitals and the findings were recorded.
Tissue Collection, Organ Weights and Histopathology: The following organs, as applicable from all animals were collected, weighed, and preserved (see Table A and B in Any other information on materials and methods incl. tables). Adherent tissue/fat from the organs was trimmed and their wet weight was recorded for all animals. Paired organs were weighed together. The organ weight ratios as percentage of body weight were determined and presented in the report. All organs were preserved in 10% neutral buffered formalin (NBF) except testes, epididymides and eyes. Testes and epididymis were preserved in modified Davidson’s fixative for 48 hrs and then transferred to 10% NBF. Eyes with optic nerve were preserved in modified Davidson’s fixative for 48 hrs and then transferred to 50% isopropyl alcohol.
The thyroid along with the parathyroid was collected from one male and one female pup per litter on postnatal day 13. The thyroid along with the parathyroid from the adults and pups were preserved in 10% v/v Neutral Buffered Formalin. The thyroid along with the parathyroid from the adults were weighed post fixation.
Histopathology: Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure - stage aware histopathological evaluation). All organs and tissue samples were processed, embedded in paraffin, sectioned at 4 to 6 µm thickness, and stained with hematoxylin and eosin. The testes were sectioned at 3 to 5 µm thickness, stained with Periodic acid Schiff (PAS) stain and Haematoxylin and Eosin stain and subjected to histopathological examination. PAS stain aided spermatogenesis evaluation. The investigations were not extended to the lower dose groups and recovery groups as there were no tissue showing signs of treatment related effects at the high dose level (target organs) and no gross lesions were encountered. Bone marrow smear (from one femur) was prepared at the time of necropsy, fixed in methanol and stained with May-Grünwald-Giemsa stain. Bone marrow smears were subjected for qualitative evaluation.
Pup observations, Pup weight, Anogenital distance, and Nipples/Areolae Retention:
Pup Observations: The day of parturition was considered as postnatal day (PND) 1 for each pup. Individual pups from each litter was observed for external deformities or clinical signs once daily and for mortalities twice daily.
Pup Weight: Individual live pup weight from each litter was recorded on
PND 1 (within 24 hours of parturition), 4, 7, and 13. The mean pup weight per litter (sex-wise) was reported.
Anogenital Distance: The anogenital distance of each live pup from each litter was measured on PND 4. The mean pup anogenital distance and its ratio was reported per litter (sex-wise) as mentioned below: Anogenital distance (AGD) Ratio = Anogenital distance (mm) * 100/Cube root of PND 4 pup weight
Observation of Male Pups for Nipples/Areolae Retention: The number of nipples/areolae in male pups was counted on PND 13 and presented litter wise.

Clinical Pathology Investigations: The clinical pathology (haematological and clinical biochemistry) examinations were conducted in five males and five females randomly selected from each main group and from all recovery group animals. Blood samples of both males and females were collected on the day of scheduled terminal sacrifice (for main group males after completion of 29 days of dose administration, for main group females on lactation day 14 and for recovery group animals on the day of schedule sacrifice, i.e., day 68). One day before scheduled terminal sacrifice, the animals were fasted overnight. Water was provided ad libitum during the fasting period. Blood samples were collected from the animals separately into tubes containing K3-EDTA and sodium heparin for haematology and clinical chemistry analysis, respectively. 3.2% buffered tri-sodium citrate was used for Prothrombin time and activated partial thromboplastin time parameters. Blood samples were collected using the retro-orbital plexus puncture method under Isoflurane Anaesthesia with the help of a fine capillary tube.
The following hematology parameters were estimated using Advia 2120i hematology system (Siemens Limited): Haemoglobin concentration (HGB) [g/dL], Haematocrit (HCT) [%], Erythrocyte count (RBC) [106 cells/µL], Total leukocyte count (WBC) [103 cells/µL], Mean corpuscular volume (MCV) [fL], Mean corpuscular hemoglobin (MCH) [pg], Mean corpuscular haemoglobin concentration (MCHC) [g/dL], Platelet count (PLT) [103 cells/µL], Mean platelet volume (MPV) [ fL], Differential leucocytes count (DLC) [%], Reticulocyte Count [%], Absolute Reticulocyte Count [109 cells/L], Absolute differential leucocytes count (DLC) [103 cells/µL]. Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by Opticlot - 4 coagulation analyzer (Tulip Group).
The following clinical chemistry parameters were analyzed using Rx Imola clinical chemistry analyzer (Randox Laboratories): Alanine aminotransferase (ALT) [U/L], Aspartate aminotransferase (AST) [U/L], Alkaline phosphatase (ALP) [U/L], Total protein [g/dL], Albumin [g/dL], Glucose [mg/dL], Total cholesterol [mg/dL], Creatinine [mg/dL], Urea [mg/dL], Blood urea nitrogen (BUN) [mg/dL], Triglycerides [mg/dL], Phosphorous [mg/dL], Calcium [mg/dL], Globulin (instrument generated) [g/dL]. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Easylyte plus Na/K/Cl analyzer (Medica Corporation). The plasma was separated by centrifuging the blood samples at 5000 rpm for 10 minutes for determining the clinical chemistry and 1500 rpm for 15 minutes for determining the Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) parameters.

Other examinations:

Urinalysis: Urine was collected from five randomly selected males of each main group and for all recovery animals at termination. The selected animals were placed in urine collection cages overnight and not given access to feed, but water was provided ad libitum during their stay in the urine collection cages. The overnight urine volume (mL) collected from these animals was measured.
The volume of urine collected (mL), appearance and color were recorded by physical evaluation. The urine was analyzed using urine Dirui H 500 urine analyzer (Dirui Industries) for the following parameters: Blood [Ery/µL], Bilirubin [mg/dL], Urobilinogen [mg/dL], Ketones [mg/dL], Protein [mg/dL], Glucose [mg/dL], Microalbumin [mg/dL], Leucocytes [Leu/µL]. In addition, pH, nitrite and specific gravity was also analyzed. After analysis of the above parameters, the urine was centrifuged at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.
Note: Urinalysis was not conducted for main group females (dams).
Oestrus cyclicity was monitored for two weeks after five days of acclimatization to evaluate the normal oestrus cycle (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Oestrus cyclicity was monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudopregnancy. The status of oestrus cyclicity was determined on termination day.
Note: Recovery group females were not evaluated for oestrus cyclicity.
Delivery and Litter Observations: The day of littering was considered as lactation day (LD) 1 for the dam and postnatal day (PND) 1 for pups. The number of male/female pups born (live/dead/cannibalized) per litter were recorded and sex ratio (m/f) and live birth index was calculated per litter as mentioned below:
Sex ratio (m/f) = No. of male offspring/Number of female offspring
Live Birth Index (%) per litter = No. of pups born alive * 100/Total No. of pups born
The number of pups survived/dead/cannibalized per litter were recorded during lactation period and pup survival index (%) per litter between lactation day 1 to 4, 4 to 7 and 7 to 13 was calculated per litter as mentioned below:
Pup Survival index (%) on LD 4/7/13 = Total No. of live pups on LD 4/7/13 * 100/No. of pups born/4/7
The sex ratio (m/f) on LD 4, 7, and 13 was calculated per litter as mentioned below:
Sex ratio (m/f) on LD 4/7/13 = No. of male offspring on LD 4/7/13/No. of female offspring on LD 4/7/13
Reproductive Performance Evaluation: The following reproductive performance indices were calculated as mentioned below and presented in the study report.
Mating and Fertility Index: The male / female mating, and fertility indices were calculated as mentioned below:
Male Mating Index (%) = No. of males with confirmed mating * 100/Total No. of males cohabited
Female Mating Index (%) = No. of sperm-positive females * 100/ Total No. of females cohabited
Male Fertility Index (%) = No. of males impregnating a female * 100/Total No. of males mated
Female Fertility Index (%) = No. of pregnant females * 100/No. of sperm-positive females
Cohabitation Record and Copulatory Interval (Pre-coital Interval): The day of initiation of mating and day of confirmation of mating were recorded for each female, and the pre-coital interval was calculated as mentioned below:
Pre-coital Interval (Days) = Date of confirmation of mating – Date of initiation of cohabitation
Gestation Length: The day of confirmation of mating and day of parturition were recorded for each female and the gestation length per litter was calculated as mentioned below:
Gestation Length (days) = Date of parturition – Date of positive evidence of mating (GD 0)
Gestation Index: The gestation index (%) per group was calculated as mentioned below:
Gestation Index (%) = No. of females with live born * 100/No. of females with evidence of pregnancy
Parturition Index: The parturition index (%) per group was calculated as mentioned below:
Parturition Index (%) = No. of females littered * 100/No. of females with evidence of pregnancy
Female Fecundity or Pregnancy Index: The pregnancy index (%) per group was calculated as mentioned below:
Pregnancy Index (%) = No. of pregnant females * 100/No. of females with confirmed mating
Uteri Observations: The uterus of each female was observed for implantation sites and the count was recorded for comparison of number of implantation sites with the number of pups born for each litter to calculate the post-implantation loss. The postnatal loss per litter was calculated based on the number of pups born, and the number of pups survived till termination. The post-implantation loss and postnatal loss per litter was calculated as mentioned below:
Post-Implantation Loss (%): (No. of Implantations - No. of Viable pups) * 100/No. of Implantations
Post-implantation Loss (No.): No. of implantations - No. of live births
Total postnatal loss (No.): (Total no. of live pups delivered – Total no. of pups survived during postnatal period
Total postnatal loss (%): Total no. of pups dead or cannibalized during postnatal period * 100/Total no. of live pups delivered
The uterus of females with no visible implantation sites (non-pregnant) were immersed in 10% v/v ammonium sulphide stain under fume hood to visualize the implantation sites that had undergone very early resorptions. The uterus, along with the cervix and ovaries, were collected and preserved for all females.

Statistics:

The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of an appendix) was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version no. 27.
All analysis and comparisons were evaluated at 95% with the level of confidence of P<0.05 respectively, indicated by the aforementioned tests and were designated by the superscripts throughout the report as stated below: * Statistically significant (P<0.05) change than the vehicle control group.
Note: The data of females mated but not littered, non-pregnant animals and lactation data of females with total litter loss is presented in individual animal data and considered for mean calculations and statistical analysis for systemic toxicity end points.
The statistical analysis was followed to analyse the data, as follows: Body weight (weekly/gestation/lactation), Percent change in body weight (weekly/gestation/lactation), Feed consumption (weekly/gestation/lactation), Copulatory interval, Gestation length, Hematology, Clinical chemistry, Urinalysis (volume, specific gravity and UBG), Absolute/relative organ weights, Functional observation parameters, Mean pup weight per litter, Mean pup anogenital distance ratio per litter, and Serum T4 values (adults/pups) were analyzed using Parametric - One-way ANOVA with Dunnett’s post test. Implantations/litter, No. of pups/litter, Sex ratio/litter, Litter size, No. of early resorptions/litter, No. of late resorptions/litter, Prenatal loss/litter, and Postnatal loss/litter were investigated by Non-Parametric - Kruskal-Wallis statistics whereas Reproductive performances (frequency occurrences) was assessed using Cross Tabs - Chi-square test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity, and no mortality/morbidity was noted in any of the animals of both sexes from all the tested dose groups [G2 (100 mg of active ingredient SVS/kg body weight/day), G3 (300 mg of active ingredient SVS/kg body weight/day), G4/G4R (1000 mg of active ingredient SVS/kg body weight/day)] and G1/G1R [vehicle control group - 0 mg of active ingredient SVS/kg body weight/day] throughout the experimental period.
The detailed clinical examination conducted once in a week did not reveal any changes in any of the animals of both sexes from all the tested dose groups and vehicle control group. There was a wound observed in one female each from groups G1 and G4 from day 12 to day 14 and later it was recovered. This noted observation was considered as incidental and due to injury happened because of fight with the cage mate. Refer: Table 1

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in all the tested dose groups (main and recovery) of both sexes throughout the experimental period. The noted statistically significant increase in percent change in body weight on Day 7 with respect to Day 1 from group G3 males when compared with vehicle control group is considered as incidental and toxicologically insignificant. Refer: Table 2 & 5
Gestational Body Weight and Percent Change in Gestational Body Weight: There were no changes noted in mean gestational body weight and percent change in mean gestational body weight gain in all the tested dose groups when compared with the vehicle control group. Refer: Table 3 & 6
Lactation Body Weight and Percent Change in Lactation Body Weight: There were no changes noted in mean lactation body weight and percent change in mean lactation body weight gain in all the tested dose groups when compared with the vehicle control group. Refer: Table 4 & 7

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related changes noted in mean feed consumption in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control group. The noted statistically significant increase in mean feed consumption during week 5 from group G4R females when compared with vehicle control group is considered as incidental and toxicologically insignificant. Refer: Table 8
There were no changes noted in mean feed consumption measured during gestation period in all the tested dose groups when compared with the vehicle control group. Refer: Table 9
There were no changes noted in mean feed consumption during lactation period in all the tested dose groups when compared with the vehicle control group. Refer: Table 10

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in any of the animals from all the tested dose or vehicle control groups (main and recovery) of both sexes during ophthalmological examination conducted shortly before scheduled sacrifice. Refer: Table 11

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in obtained mean haematological values in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control group. However, the following statistically significant differences in mean haematological values were noted in the main and recovery dose groups when compared with vehicle control group:
- increase in activated prothrombin time (APTT) in group G2 males.
- decrease in hemoglobin, haematocrit (HCT) in group G3 females.
- increase in percent neutrophils in group G4R females.
- decrease in percent lymphocytes in group G4R females
These statistically significant changes are considered as incidental and unrelated to treatment, as the obtained mean values are alone occurrences in main group and similar changes were not noted in recovery group. Also, the obtained mean values are within in-house historical control range of same species and strain. Refer: Table 16

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in mean clinical chemistry values in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control groups. However, the following statistically significant differences in mean clinical chemistry values were noted in the tested dose groups when compared with vehicle control group.
- increase in glucose level in groups G4 males and group G4R females.
- increase cholesterol levels in groups G4 males and G4R females.
- increase albumin levels in group G3 males.
- decrease in alanine aminotransferase levels in group G3 males.
- increase in potassium levels in group G4R females
These statistically significant changes are considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain. Refer: Table 17

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in mean urinalysis values in all the tested dose groups (main group males and recovery group males and females) when compared with the vehicle control group.
The noted statistically significant increase in volume of urine from group G4R males when compared with vehicle control group is considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain. Refer: Table 18

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The neurological/functional observations such as, home cage, handling, open-field, sensory, physiological observations did not reveal any changes in any of the animals of both sexes from all the tested dose groups performed towards end of the dosing period for main groups (on day 28 for males and on lactation day 13 for dams) and performed towards end of recovery period for recovery groups (day 65). There were no test item related changes noted in mean fore/hind limb grip strengths, mean motor activity assessments and mean hind limb foot splay in all tested dose groups of both sexes when compared with vehicle control groups during conduct of neurological/functional examinations. The noted statistically significant decrease in average hind limb foot splay length for group G2 males when compared with vehicle control group is considered as incidental and unrelated to treatment, as the obtained mean value is alone occurrence and similar changes were not noted in mid or high dose groups. Also, the obtained mean value is within in-house historical control range of same species and strain. Refer: Table 12, 13, 14 & 15

Immunological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in mean absolute and relative organ weights in all the tested dose groups of both sexes (main and recovery) when compared with vehicle control group. However, the following statistically significant differences in absolute and relative organ weights were noted in the main and recovery dose groups when compared with vehicle control group:
- increase in absolute liver and lungs weight in group G3 males.
- increase in absolute liver and kidney weight in G4R males.
- increase in relative thymus weight in group G2 males.
- decrease in relative brain weight in group G3 males.
- increase in relative liver and kidney weight in group G4R males.
- decrease in relative spleen weight in group G2 and G4 females
These changes are considered as incidental and unrelated to treatment, as there were no gross pathological changes noted in any of these organs during necropsy and no microscopic changes noted in these organs during histopathological examination of high dose group animals. Moreover, all the obtained mean values of each organ are within in-house historical control range of same species and strain. Refer: Table 22 & 23

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in mean absolute and relative organ weights in all the tested dose groups of both sexes (main and recovery) when compared with vehicle control group. However, the following statistically significant differences in absolute and relative organ weights were noted in the main and recovery dose groups when compared with vehicle control group:
- increase in absolute liver and lungs weight in group G3 males.
- increase in absolute liver and kidney weight in G4R males.
- increase in relative thymus weight in group G2 males.
- decrease in relative brain weight in group G3 males.
- increase in relative liver and kidney weight in group G4R males.
- decrease in relative spleen weight in group G2 and G4 females
These changes are considered as incidental and unrelated to treatment, as there were no gross pathological changes noted in any of these organs during necropsy and no microscopic changes noted in these organs during histopathological examination of high dose group animals. Moreover, all the obtained mean values of each organ are within in-house historical control range of same species and strain. Refer: Table 22 & 23

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes observed during conduct of necropsy in any of the adult animals of both sexes from main and recovery groups. The gross pathological examination conducted for cannibalised/dead pups, pups sacrificed on postnatal day 4 for blood collection and pups sacrificed at termination on postnatal day 13 did not reveal any gross pathological changes during conduct of necropsy in any of the tested dose and vehicle control groups. However, few pups at birth were found dead at birth, cannibalised and dead during post-natal period in both control and high dose group.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic findings in the study. Few microscopic findings observed in this study such as ultimobranchial cyst in thyroid glands, epithelial cyst in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Reproductive effect and pups observations
Oestrus Cyclicity: There were no test item-related irregularities observed in oestrus cyclicity of females from any of the tested dose groups during pre-mating and mating treatment periods. However, some of the animals from all the tested dose group were noted with occasional irregular oestrus cycles and majority of them during mating period. These noted irregularities in duration of oestrus cyclicity in all the tested dose groups are considered as incidental and unrelated to treatment as the same females were noted with normal, preceding (previous) and proceeding (following) oestrus cycles and there were no effects noted in reproductive performance of these females. A mean oestrus cycle length of 4.49, 4.79, 4.74 and 4.65 days was noted from groups G1, G2, G3 and G4, respectively. The mean length of oestrus cycle per female during treatment period was unaffected by the test item administration in all the tested dose groups. Refer: Table 19
Delivery and Litter observations: There were no test item related changes observed in birth parameters such as, total litter size, number of live pups born, sex ratio (m/f) and live birth index in all the tested dose groups when compared with the vehicle control group. Also, the pup survival index per litter during lactation period was unaffected by the test item in all the tested dose groups when compared with the vehicle control group. Refer: Table 20
Reproductive Performance
Male Mating Index: A total of 12 (out of 12) males were confirmed with mating during cohabitation period with a mating index of 100.0% from all the tested dose groups and vehicle control group. There were no statistically significant differences noted for male mating index in any of the tested dose groups when compared with the vehicle control group.
Male Fertility Index: A total of 10 (out of 12), 10 (out of 12), 10 (out of 12) and 12 (out of 12) mated males were confirmed as fertile by impregnating a female or siring a litter with a fertility index of 83.3%, 83.3%, 83.3% and 100.0% from the tested dose groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for male fertility index in any of the tested dose groups when compared with the vehicle control group.
Female Mating Index: A total of 12 (out of 12) females were confirmed as sperm positive during vaginal smear examination with a mating index of 100.0% from all the tested dose groups and vehicle control group.
Female Fertility Index: A total of 10 (out of 12), 10 (out of 12), 10 (out of 12) and 12 (out of 12) mated females were confirmed with presence of implantations / presence of live or dead pups / evidence of parturition with a fertility index of 83.3%, 83.3%, 83.3% and 100.0% from groups G1, G2, G3 and G4, respectively.
There were no statistically significant differences noted for female fertility index in any of the tested dose groups when compared with the vehicle control group.
Pre-coital Interval/Copulatory Interval/Mean time to Mating: A total of 12 pairs were left for cohabitation initially from each group. The mean pre-coital interval was 3.00, 3.42, 3.50 and 1.58 days for groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for mean pre-coital interval in any of the tested dose groups when compared with the vehicle control group.
Gestation Length/Duration of Pregnancy: The mean gestation length [day of confirmed as successfully mated to day of parturition] was 22.50, 22.60, 22.40 and 22.40 days for groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for mean gestation length in any of the tested dose groups when compared with the vehicle control group.
Implantation/ Fecundity/ or Pregnancy Index: A total of 10 (out of 12), 10 (out of 12), 10 (out of 12) and 12 (out of 12), mated females were confirmed as pregnant / with evidence of implantation sites with a fecundity implantation index of 83.3%, 83.3%, 83.3% and 100.0% from groups G1, G2, G3 and G4. In group G4, 10 females were littered and other 2 females were noted with early resorptions (evidence of implantation sites). There were no statistically significant differences noted for fecundity index in any of the tested dose groups when compared with the vehicle control group.
Pregnancy Index: A total of 10 (out of 12) mated females were confirmed with evidence of pregnancy with a pregnancy index of 83.3% from all the tested dose groups and vehicle control group.
Parturition Index: A total of 10 (out of 10), 10 (out of 10), 10 (out of 10) and 10 (out of 10), pregnant females were confirmed with parturition with a parturition index of 100.0% for all the tested dose groups and vehicle control group.
Gestation Index: A total of 10 (out of 10), 10 (out of 10), 10 (out of 10) and 10 (out of 10) pregnant females were confirmed with live born pups with a gestation index of 100.0% for all the tested dose groups and vehicle control group.
Implantation sites and Viable Pups: A mean number of 13.00, 13.20, 11.80 and 13.20 implantation sites and mean number of 12.30, 13.00, 11.50 and 11.90 viable pups were noted from groups G1, G2, G3 and G4, respectively. There were no statistically significant changes noted for both mean implantation sites and viable pups in any of the tested dose groups when compared with the vehicle control group.
Post-implantation Loss: A mean number of 0.70, 0.20, 0.30 and 1.30 post-implantation losses with a percentage of 5.21%, 1.33%, 3.20% and 10.34% were noted from groups G1, G2, G3 and G4 respectively. There were no statistically significant changes as for mean number and percentage of occurred post-implantation losses in any of the tested dose groups when compared with the vehicle control group.
Postnatal Loss: A mean number of 0.00, 0.40, 0.00 and 0.10 postnatal losses with a percentage of 0.00, 2.90, 0.00 and 10.00 were noted from groups G1, G2, G3 and G4, respectively. There were no statistically significant changes noted for mean number and percentage of occurred post-natal losses in any of the tested dose groups when compared with the vehicle control group.
In conclusion, there were no effects on male and female reproductive performance/indices noted from P generation in any of the tested dose groups when compared with vehicle control group. Refer: Table 21
There were no test item related external abnormalities or behavioural changes noted in any of the pups during daily observation from all the tested dose groups and vehicle control group during postnatal period. All the pups had normal behaviour during daily observations. Refer: Table 25
Mean Pup Weight per Litter during Postnatal Period
There were no changes noted in mean pup [both male and female] weight per litter in all the tested dose groups when compared with the vehicle control group, recorded on postnatal day (PND) 1, 4, 7 and 13. Refer: Table 26
Mean Pup Anogenital distance and Anogenital distance Ratio per Litter
There were no changes in mean pup [both male and female] anogenital distance measurement (mm) and its ratio per litter in all the tested dose groups when compared with the vehicle control group, recorded on postnatal day 4. Refer: Table 27
Nipple Retention in Male Pups per Litter
There were no occurrences or evidence of retention of nipples in any of the male pups examined on the postnatal day 13 from all the tested dose groups and vehicle control group litters. Refer: Table 28
Serum Thyroid Hormone (T4) Levels - Postnatal Pups
There were no changes in mean serum T4 hormone levels of pups at postnatal day 13 in any of the tested dose group litters when compared with vehicle control group litters. Refer: Table 29

Details on results:

Dose formulation analysis for dose concentration verification was performed during week 1 and 4 of the treatment. The results of dose concentration verifications for the prepared test item formulations and the mean results of analysis were within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) was less than 10%.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat. (dissolved fraction)

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

SUMMARY OF THE STUDY

Parameters ↓								Group & Dose (mg of active ingredient SVS/kg body weight/day)
								G1 & 0				G2 & 100				G3 & 300			G4 & 1000
Reproductive Indices
No. of Pairs started								12				12				12			12
Reproductive Performance / Indices (males)
Total No. of males cohabited								12				12				12			12
No. of males with confirmed mating								12				12				12			12
No. of males impregnating/siring a female								10				10				10			12
Male Mating Index (%)								100.0				100.0				100.0			100.0
Male Fertility Index (%)								83.3				83.3				83.3			100.0
Reproductive Performance and Indices (females)
Mean Oestrus cycle length (days)				Mean				4.49				4.79				4.74			4.65
				±SD				0.27				0.48				0.33			0.42
No. of females with regular oestrus cycle								9				7				7			8
Total No. of females cohabited								12				12				12			12
No. of sperm-positive females								12				12				12			12
No. of females with evidence of pregnancy								10				10				10			12
No. of females with live born pups								10				10				10			10
No. of females with conceiving days 1 to 5								9				10				8			12
No. of females with conceiving days >5								3				2				4			0
Pre-coital Interval (Days)				Mean				3.00				3.42				3.50			1.58
				±SD				3.25				2.84				3.06			1.00
Female Mating Index (%)								100.0				100.0				100.0			100.0
Female Fertility Index (%)								83.3				83.3				83.3			100.0
No. of females with Pregnancy≤21 days								0				1				2			2
No. of females with Pregnancy = 22 days								5				4				5			3
No. of females with Pregnancy≥23 days								5				5				3			5
Gestation Length (Days)				Mean				22.50				22.60				22.40			22.40
				±SD				0.53				0.97				1.26			0.97
Implantation/ Fecundity or Pregnancy Index (%)								83.3				83.3				83.3			100.0
Gestation Index (%)								100.0				100.0				100.0			100.0
Parturition index (%)								100.0				100.0				100.0			100.0
Delivery and Litter Observations
No. of Dams with live young born							10			10				10				10
No. of Dams with live young at PND 4							10			10				10				9
No. of Dams with live young at PND 7							10			10				10				9
No. of Dams with live young at PND 13							10			10				10				9
No. of Implants/dam		Mean					13.00			13.20				11.80				13.00
		±SD					4.08			3.01				3.65				3.81
No. of Live pups/dam at birth		Mean					12.30			13.00				11.50				11.90
		±SD					3.97			2.94				3.75				5.51
No. of Live pups/dam at PND 4		Mean					12.30			12.60				11.50				11.80
		±SD					3.97			2.95				3.75				5.73
No. of Live pups/dam at PND 7		Mean					11.00			11.60				10.50				11.67
		±SD					3.13			2.01				2.88				3.50
No. of Live pups/dam at PND 13		Mean					11.00			11.60				10.50				11.67
		±SD					3.13			2.01				2.88				3.50
Live Birth Index/dam (%)		Mean					97.98			100.00				98.47				90.32
		±SD					4.41			0.00				3.30				28.60
Pup Survival Index/dam on PND 4 (%)		Mean					100.00			97.10				100.00				100.00
		±SD					0.00			7.33				0.00				0.00
Pup Survival Index/dam on PND 7 (%)		Mean					100.00			100.00				100.00				100.00
		±SD					0.00			0.00				0.00				0.00
Pup Survival Index/dam on PND 13 (%)		Mean					100.00			100.00				100.00				100.00
		±SD					0.00			0.00				0.00				0.00
Sex ratio (m/f)/dam At Birth		Mean					1.51			1.60				1.12				1.87
		±SD					0.78			0.71				0.40				1.22
Sex ratio (m/f)/dam at PND 4		Mean					1.51			1.62				1.12				1.87
		±SD					0.78			0.74				0.40				1.22
Sex ratio (m/f)/dam at PND 7		Mean					2.63			2.24				1.40				3.16
		±SD					2.62			1.45				0.61				3.05
Sex ratio (m/f)/dam at PND 13		Mean					2.63			2.24				1.40				3.16
		±SD					2.62			1.45				0.61				3.05
Delivery and Litter Observations
Mean Male Pup weight (g)/dam	At birth				Mean				7.03				6.67				7.17			7.30
					±SD				0.79				0.82				0.79			0.77
	PND 4				Mean				9.78				9.71				10.53			10.62
					±SD				1.34				1.55				1.73			2.25
	PND 7				Mean				14.45				13.94				14.88			14.86
					±SD				2.55				2.19				1.94			2.88
	PND 13				Mean				24.07				23.85				24.62			23.41
					±SD				3.47				3.33				3.37			3.85
Mean Female Pup weight (g)/dam	At Birth				Mean				6.70				6.40				6.85			6.91
					±SD				0.78				0.89				0.70			0.81
	PND 4				Mean				9.41				9.40				10.09			10.02
					±SD				1.40				1.53				1.53			1.92
	PND 7				Mean				13.62				13.60				14.38			14.25
					±SD				2.64				2.05				2.20			2.59
	PND 13				Mean				22.36				22.81				23.80			22.46
					±SD				3.51				3.19				3.58			3.72
Mean Male Pup AGD Measurement/dam (mm) on PND 4					Mean				4.64				4.53				4.51			4.47
					±SD				0.39				0.34				0.21			0.24
Mean Male Pup AGD ratio/dam on PND 4					Mean				2.18				2.14				2.10			2.05
					±SD				0.21				0.20				0.19			0.14
Mean Female Pup AGD Measurement/dam (mm) on PND 4					Mean				2.53				2.54				2.48			2.55
					±SD				0.20				0.19				0.13			0.21
Mean Female Pup AGD ratio/dam on PND 4					Mean				1.21				1.21				1.13			1.19
					±SD				0.13				0.11				0.11			0.10
Male Pup Nipple Retention/dam on PND 13					Mean				0.00				0.00				0.00			0.00
					±SD				0.00				0.00				0.00			0.00
ABNORMAL PUPS DURING AT BIRTH
No. of Dams with live young born at birth						10					10				10				10
No. of Dams with 0 abnormal live pups						10					10				10				10
No. of Dams with ≥1 abnormal live pup						0					0				0				0
LOSS OF OFFSPRING
Pre-natal or post-implantation loss (implantations minus live births)
No. of Females with 0 post-implantation loss						7					9				7				7
No. of Females with 1 post-implantation loss						1					0				3				2
No. of Females with 2 post-implantation losses						1					1				0				0
No. of Females with 4 post-implantation losses						1					0				0				0
No. of Females with 11 post-implantation losses						0					0				0				2
No. of Females with 13 post-implantation losses						0					0				0				1
Post-implantation Loss (No.)			Mean			0.70					0.20				0.30				1.30
			±SD			1.34					0.63				0.48				3.43
Post-implantation Loss (%)			Mean			5.21					1.33				3.20				10.34
			±SD			9.28					4.22				5.74				28.68
Postnatal loss (live births minus alive at PND 13)
No. of Females with 0						10					8				10				11
No. of Females with 1						0					1				0				1
No. of Females with 3						0					1				0				0
Postnatal loss (No.)			Mean			0.00					0.40				0.00				0.10
			±SD			0.00					0.97				0.00				0.32
Postnatal Loss (%)			Mean			0.00					2.90				0.00				10.00
			±SD			0.00					7.33				0.00				31.62

±SD: Standard deviationNo.: Number; m/f: male/female; AGD: Anogenital distance; PND: Postnatal Day;

 

Table 1: Summary of clinical signs of toxicity, detailed clinical examination and mortality record

Group, Sex & Dose (mg of active ingredient SVS /kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1, M & 0	12	N (12)	NAD (12)	0 (12)
G2, M & 100	12	N (12)	NAD (12)	0 (12)
G3, M & 300	12	N (12)	NAD (12)	0 (12)
G4, M & 1000	12	N (12)	NAD (12)	0 (12)
G1, F & 0	12	N (12) 9 (1)@	NAD (12)	0 (12)
G2, F & 100	12	N (12)	NAD (12)	0 (12)
G3, F & 300	12	N (12)	NAD (12)	0 (12)
G4, F & 1000	12	N (12) 9 (1) @	NAD (12)	0 (12)
G1R, M & 0	5	N (5)	NAD (5)	0 (5)
G4R, M & 1000	5	N (5)	NAD (5)	0 (5)
G1R, F & 0	5	N (5)	NAD (5)	0 (5)
G4R, F & 1000	5	N (5)	NAD (5)	0 (5)

F: Female; N: Normal; NAD: No Abnormality Detected.

a: observed once daily; b: observed once weekly; c: observed twice daily.

@: There was a wound observed in one female each from groups G1 and G4 from day 12 to day 14 and later it was recovered. This noted observation was considered as incidental and due to injury happened because of fight with the cage mate. 

 

Table 2: Summary of body weight (g) records

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	29
G1, M & 0	Mean	378.74	394.33	417.01	427.50	441.19
	±SD	21.67	24.13	27.59	26.86	31.91
	n	12	12	12	12	12
G2, M & 100	Mean	378.12	398.17	419.64	432.26	446.79
	±SD	20.07	23.08	24.46	24.11	23.23
	n	12	12	12	12	12
G3, M & 300	Mean	378.30	406.49	427.67	441.41	464.48
	±SD	17.41	27.65	36.22	41.77	50.34
	n	12	12	12	12	12
G4, M & 1000	Mean	379.90	400.94	423.24	439.10	459.90
	±SD	16.68	23.96	25.11	30.55	38.58
	n	12	12	12	12	12
G1, F & 0	Mean	258.19	267.04	279.26	282.87
	±SD	12.21	12.96	11.22	15.60
	n	12	12	12	3
G2, F & 100	Mean	256.68	266.12	279.03	288.08
	±SD	11.35	10.38	13.14	10.79
	n	12	12	12	2
G3, F & 300	Mean	257.97	267.00	277.00	290.29
	±SD	12.27	17.34	19.54	16.72
	n	12	12	12	2
G4, F & 1000	Mean	256.84	267.72	278.30	-
	±SD	10.92	11.14	11.97	-
	n	12	12	12	-

F: Female; ±SD: Standard Deviation; n: Number of Animals.

#: The data obtained from females in cohabitation only considered for mean calculations.

The data of day 21 body weight was not subjected to statistical analysis due to uneven number of variables.

 

Table 2 continued for recovery groups

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28	35	42	49	56	63	67
G1R, M & 0	Mean	371.76	389.45	408.75	421.95	439.19	452.19	467.48	482.52	496.36	512.59	519.35
	±SD	12.32	9.47	14.88	21.28	25.47	29.56	29.42	24.46	26.62	22.97	22.77
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	374.10	396.89	424.36	437.23	443.28	461.97	474.22	489.15	505.82	520.45	526.52
	±SD	13.06	14.51	23.50	23.50	19.84	9.65	15.98	13.51	18.58	17.71	16.90
	n	5	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	252.74	263.64	272.55	281.32	283.88	289.12	295.75	301.58	306.01	310.32	313.07
	±SD	6.40	9.24	8.17	8.58	13.69	16.13	15.98	18.82	17.34	16.52	15.69
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	252.52	262.98	276.31	278.96	290.38	297.03	308.76	314.33	321.83	324.89	328.16
	±SD	9.33	6.44	10.87	8.67	15.95	19.26	25.28	25.78	25.88	25.68	26.54
	n	5	5	5	5	5	5	5	5	5	5	5

F: female; M: Male; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

Table 3: Summary record of gestation body weight (g)

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Body Weight (g) on Gestation Day
		0	7	14	20
G1, F & 0	Mean	279.44	303.64	333.94	404.65
	±SD	9.70	17.39	27.84	52.42
	n	10	10	10	10
G2, F & 100	Mean	281.70	312.47	339.30	390.08
	±SD	17.11	15.08	15.92	39.41
	n	10	10	10	10
G3, F & 300	Mean	282.07	310.34	341.44	402.71
	±SD	22.81	28.87	26.98	45.25
	n	10	10	10	10
G4, F & 1000	Mean	279.73	313.86	353.56	424.84
	±SD	15.25	19.97	18.19	31.86
	n	10	10	10	10

F: Female; ±SD: Standard Deviation; n: Number of Dams.

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis but presented in individual animal data.

 

Table 4: Summary record of lactation body weight (g)

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Body Weight (g) on Lactation Day
		1	4	7	13
G1, F & 0	Mean	313.81	326.23	340.11	354.76
	±SD	28.01	32.95	30.18	28.27
	n	10	10	10	10
G2, F & 100	Mean	310.34	322.52	334.70	347.32
	±SD	12.12	11.85	14.01	13.30
	n	10	10	10	10
G3, F & 300	Mean	310.64	325.89	334.86	346.23
	±SD	21.07	29.35	28.88	24.83
	n	10	10	10	10
G4, F & 1000	Mean	330.16	341.25	349.83	359.32
	±SD	23.99	19.75	17.85	20.32
	n	10	10	10	10

F: Female; ±SD: Standard Deviation; n: Number of Dams.

 

Table 5: Summary of percent change in body weight gain (%) with respect to day 1 record

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14	1 to 21	1 to 29
G1, M & 0	Mean	4.11	10.10	12.90	16.52
	±SD	2.25	3.84	4.05	6.02
	n	12	12	12	12
G2, M & 100	Mean	5.28	10.98	14.35	18.24
	±SD	1.43	2.86	3.46	4.27
	n	12	12	12	12
G3, M & 300	Mean	7.36*	12.88	16.46	22.48
	±SD	2.96	4.89	6.20	8.09
	n	12	12	12	12
G4, M & 1000	Mean	5.49	11.38	15.52	20.98
	±SD	2.78	3.66	4.76	7.21
	n	12	12	12	12
G1, F & 0	Mean	3.43	8.26	14.13
	±SD	1.39	3.90	1.85
	n	12	12	3
G2, F & 100	Mean	3.72	8.71	11.79
	±SD	2.33	2.22	1.96
	n	12	12	2
G3, F & 300	Mean	3.44	7.28	8.57
	±SD	2.30	2.87	1.01
	n	12	12	2
G4, F & 1000	Mean	4.26	8.42	-
	±SD	2.42	3.98	-
	n	12	12	-

F: Female; M: Male; ±SD: Standard Deviation; n: Number of Animals.

#: The data obtained from females in cohabitation only considered for mean calculations. The data of day 1 to 21 body weight was not subjected to statistical analysis due to uneven number of variables.

* Statistically significant (P<0.05) change than the vehicle control group

 

Table 5 continued for recovery groups

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14	1 to 21	1 to 28	1 to 35	1 to 42	1 to 49	1 to 56	1 to 63	1 to 67
G1R, M & 0	Mean	4.80	10.01	13.60	18.27	21.73	25.90	29.97	33.71	38.09	39.91
	±SD	2.09	4.43	6.68	8.34	8.64	9.60	8.98	9.80	9.41	9.39
	n	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	6.10	13.39	16.86	18.47	23.55	26.79	30.81	35.24	39.15	40.78
	±SD	1.75	3.54	4.19	2.04	2.69	2.78	3.16	3.46	3.02	3.22
	n	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	4.30	7.85	11.30	12.30	14.37	16.99	19.28	21.06	22.77	23.87
	±SD	2.12	2.33	1.49	3.94	5.10	4.77	5.82	5.77	5.38	5.23
	n	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	4.20	9.47	10.54	14.97	17.56	22.16	24.36	27.31	28.52	29.81
	±SD	2.52	3.82	3.73	4.16	4.51	7.08	7.20	6.67	6.40	6.62
	n	5	5	5	5	5	5	5	5	5	5

F: Female; M: Male; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

 

Table 6: Summary record of percent change in body weight gain (%) during gestation period

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Percent Change in Body Weight Gain (%) during Gestation Day
		0 to 7	7 to 14	14 to 20
G1, F & 0	Mean	8.65	9.90	20.93
	±SD	4.80	4.96	9.15
	n	10	10	10
G2, F & 100	Mean	11.07	8.68	15.02
	±SD	4.33	4.53	11.06
	n	10	10	10
G3, F & 300	Mean	9.98	10.17	17.75
	±SD	3.76	2.68	6.68
	n	10	10	10
G4, F & 1000	Mean	12.20	12.77	20.16
	±SD	3.82	3.48	6.53
	n	10	10	10

F: Female; ±SD: Standard Deviation; n: Number of Dams.

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis but presented in individual animal data.

 

Table 7: Summary record of percent change in body weight gain (%) during lactation period

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Percent Change in Body Weight Gain (%) during Lactation Day
		1 to 4	4 to 7	7 to 13
G1, F & 0	Mean	3.90	4.40	4.41
	±SD	3.75	2.94	2.48
	n	10	10	10
G2, F & 100	Mean	3.95	3.78	3.79
	±SD	2.15	2.05	1.41
	n	10	10	10
G3, F & 300	Mean	4.78	2.79	3.52
	±SD	3.19	1.66	1.89
	n	10	10	10
G4, F & 1000	Mean	3.54	2.56	2.69
	±SD	4.34	1.89	1.24
	n	10	10	10

F: Female; ±SD: Standard Deviation; n: Number of Dams.

 

Table 8: Summary record of feed consumption (g/animal/day)

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Feed Consumption (g/animal/day) during Pre-mating Period
		Week 1	Week 2	Week 4
G1, M & 0	Mean	27.76	29.06	32.18
	±SD	1.22	0.63	1.20
	n	12 (6)	12 (6)	12 (6)
G2, M & 100	Mean	28.07	29.17	32.69
	±SD	2.44	1.85	1.81
	n	12 (6)	12 (6)	12 (6)
G3, M & 300	Mean	27.73	27.94	31.08
	±SD	3.94	3.83	4.02
	n	12 (6)	12 (6)	12 (6)
G4, M & 1000	Mean	27.55	28.58	31.71
	±SD	2.19	2.69	2.74
	n	12 (6)	12 (6)	12 (6)
G1, F & 0	Mean	25.59	20.67
	±SD	14.57	1.10
	n	12 (6)	12 (6)
G2, F & 100	Mean	20.57	21.17
	±SD	1.88	1.51
	n	12 (6)	12 (6)
G3, F & 300	Mean	19.51	21.19
	±SD	1.04	1.11
	n	12 (6)	12 (6)
G4, F & 1000	Mean	20.27	20.72
	±SD	2.03	1.16
	n	12 (6)	12 (6)

±SD: Standard Deviation; n: Number of Animals (Number of Cages).

Table 8 continued for recovery groups

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Feed Consumption (g/animal/day)
		Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Week 9	Week 10
G1R, M & 0	Mean	25.46	26.38	27.24	25.79	25.97	27.71	30.20	32.99	35.15	37.22
	±SD	0.92	0.74	2.78	0.84	1.20	1.73	1.93	2.24	2.83	2.29
	n	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)
G4R, M & 1000	Mean	28.06	28.27	28.35	26.63	27.05	29.14	31.32	34.15	36.69	38.68
	±SD	1.80	1.31	0.43	1.68	1.09	2.20	2.26	1.55	2.01	2.02
	n	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)
G1R, F & 0	Mean	20.15	20.92	20.91	21.00	20.97	24.08	27.03	30.70	31.26	33.03
	±SD	0.89	1.19	2.74	2.42	1.72	1.19	2.99	2.98	1.54	1.56
	n	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)
G4R, F & 1000	Mean	20.86	22.00	22.74	22.88	24.40*	24.73	26.61	29.17	31.41	33.76
	±SD	2.46	2.49	0.70	0.83	1.14	1.35	0.48	1.22	2.34	2.71
	n	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)

M: Male; F: Female; R: Recovery; ±SD: Standard Deviation; n: Number of Animals (Number of Cages).

 

Table 9: Summary record of feed consumption (g/animal/day) during gestation period

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Feed Consumption (g/animal/day) during Gestation Day (GD)
		0 to 7	7 to 14	14 to 20
G1, F & 0	Mean	22.87	25.64	28.82
	±SD	1.32	1.70	2.18
	n	10	10	10
G2, F & 100	Mean	22.74	24.93	28.26
	±SD	2.47	3.18	2.18
	n	10	10	10
G3, F & 300	Mean	21.83	24.31	26.91
	±SD	2.02	2.32	2.17
	n	10	10	10
G4, F & 1000	Mean	21.77	25.21	28.34
	±SD	2.53	2.21	1.88
	n	10	10	10

F: Female; ±SD: Standard Deviation; n: Number of Dams.

Note: Excluded the data of non-pregnant and total implantation loss females for mean calculations and statistical analysis but presented in individual animal data.

 

Table 10: Summary record of feed consumption (g/animal/day) during lactation period

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Feed Consumption (g/animal/day) during Lactation Day (LD)
		1 to 4	4 to 7	7 to 13
G1, F & 0	Mean	32.68	36.89	37.61
	±SD	3.98	2.09	0.92
	n	10	10	10
G2, F & 100	Mean	31.14	34.58	38.03
	±SD	2.05	2.41	1.02
	n	10	10	10
G3, F & 300	Mean	31.17	37.06	37.57
	±SD	3.56	3.87	0.84
	n	10	10	10
G4, F & 1000	Mean	32.41	35.86	38.67
	±SD	1.98	5.00	1.10
	n	10	10	10

F: Female; ±SD: Standard Deviation; n: Number of Dams.

 

Table 11: Summary tables of ophthalmological examination records

Day -1 (Grouping and randomization)
Group, Sex & Dose (mg of active ingredient SVS /kg body weight/day) →	G1, M & 0		G2, M & 100		G3, M & 300		G4, M & 1000
Number of Animals →	12		12		12		12
Observations↓
Region↓      Eye →	LE	RE	LE	RE	LE	RE	LE	RE
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N
Group, Sex & Dose (mg of active ingredient SVS /kg body weight/day) →	G1, F & 0		G2, F & 100		G3, F & 300		G4, F & 1000
Number of Animals →	12		12		12		12
Observations↓
Region↓      Eye →	LE	RE	LE	RE	LE	RE	LE	RE
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N
Day 29
Group, Sex & Dose (mg of active ingredient SVS /kg body weight/day) →	G1, M & 0		G2, M & 100		G3, M & 300		G4, M & 1000
Number of Animals →	12		12		12		12
Observations↓
Region↓      Eye →	LE	RE	LE	RE	LE	RE	LE	RE
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N
Littered females: Lactation day 13 & non-pregnant females: 26^th day from the day of confirmation of mating
Group, Sex & Dose (mg of active ingredient SVS /kg body weight/day) →	G1, F & 0		G2, F & 100		G3, F & 300		G4, F & 1000
Number of Animals →	12		12		12		12
Observations↓
Region↓      Eye →	LE	RE	LE	RE	LE	RE	LE	RE
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N

F: Female; M: Male; N: Normal/No Abnormality Detected; LE: Left Eye; RE: Right Eye.

 

Table 11 continued for recovery groups

Day -1 (Grouping and randomization)
Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day) →	G1R, M & 0		G4R, M & 1000
Number of Animals →	5		5
Observations↓
Region↓      Eye →	LE	RE	LE	RE
Eye Lids	N	N	N	N
Cornea	N	N	N	N
Iris	N	N	N	N
Aqueous Humour	N	N	N	N
Lens	N	N	N	N
Vitreous Humour	N	N	N	N
Retina/Optic disc	N	N	N	N
Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day) →	G1R, F & 0		G4R, F & 1000
Number of Animals →	5		5
Observations↓
Region↓      Eye →	LE	RE	LE	RE
Eye Lids	N	N	N	N
Cornea	N	N	N	N
Iris	N	N	N	N
Aqueous Humour	N	N	N	N
Lens	N	N	N	N
Vitreous Humour	N	N	N	N
Retina/Optic disc	N	N	N	N
Day 65
Group, Sex & Dose (mg of active ingredient SVS /kg body weight/day) →	G1R, M & 0		G4R, M & 1000
Number of Animals →	5		5
Observations↓
Region↓      Eye →	LE	RE	LE	RE
Eye Lids	N	N	N	N
Cornea	N	N	N	N
Iris	N	N	N	N
Aqueous Humour	N	N	N	N
Lens	N	N	N	N
Vitreous Humour	N	N	N	N
Retina/Optic disc	N	N	N	N
Group, Sex & Dose (mg of active ingredient SVS /kg body weight/day) →	G1R, F & 0		G4R, F & 1000
Number of Animals →	5		5
Observations↓
Region↓      Eye →	LE	RE	LE	RE
Eye Lids	N	N	N	N
Cornea	N	N	N	N
Iris	N	N	N	N
Aqueous Humour	N	N	N	N
Lens	N	N	N	N
Vitreous Humour	N	N	N	N
Retina/Optic disc	N	N	N	N

F: Female; M: Male; R: Recovery; N: Normal/No Abnormality Detected; LE: Left Eye; RE: Right Eye.

 

Table 12: Summary of neurological/functional observation battery (FOB) records for recovery groups

Day 28
Parameters↓	Group & Sex				G1 & M			G2 & M			G3 & M				G4 & M
	Dose (mg of active ingredient SVS /kg body weight/day)				0			100			300				1000
	Number of Animals (randomly selected)				5			5			5				5
Home Cage Observations
Home cage posture					1			1			1				1
Respiratory pattern					1			1			1				1
Clonic involuntary movements					1			1			1				1
Tonic involuntary movements					1			1			1				1
Vocalization					1			1			1				1
Palpebral closure					1			1			1				1
Handling Observations
Ease of removal from the cage					2(4), 3(1)			2(3), 3(2)			2(3), 3(2)				2(3), 3(2)
Ease of handling animal in hand					2(5)			2(4), 3(1)			2(4), 3(1)				2(4), 3(1)
Red or crusty deposits		Eyes			1			1			1				1
		Nose			1			1			1				1
		Mouth			1			1			1				1
Lacrimation					1			1			1				1
Salivation					1			1			1				1
Fur appearance					1			1			1				1
Piloerection					1			1			1				1
Eye prominence					1			1			1				1
Muscle tone					1			1			1				1
Open Field Observations
Mobility						1	1			1			1
Gait						1	1			1			1
Arousal						3	3			3			3
Number of rearing				Mean		4.2	4.4			4.4			4.4
				±SD		0.4	0.5			0.5			0.5
Numbers of urination (urine pools)				Mean		5.2	5.6			5.8			5.6
				±SD		0.8	0.5			0.4			0.5
Number of defecations				Mean		3.6	4.2			3.8			3.6
				±SD		0.5	0.8			0.4			0.5
Clonic involuntary movement						1	1			1			1
Tonic involuntary movement						1	1			1			1
Stereotype behaviour						1	1			1			1
No. of groomings				Mean		5.0	3.2			3.2			3.0
				±SD		2.7	0.4			0.4			0.0
Sensory Observations
Approach response						2	2			2			2
Auditory response						2	2			2			2
Touch response						2	2			2			2
Pupil reflex						2	2			2			2
Tail pinch response						2	2			2			2
Righting reflex						1	1			1			1
Physiological Observation
Body temperature (°F)				Mean		98.0	97.6			97.6			97.6
				±SD		0.9	1.0			1.1			0.9
Home Cage Observations: Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=Absent (Normal);  Palpebral closure - 1=Normal; Handling Observations: Ease of removal from the cage - 2=Normal; 3=Moderately difficult; Ease of handling animal in hand - 2=Normal; Red or crusty deposits - 1=Absent (Normal); Lacrimation - 1=None; Salivation- 1=Absent (Normal); Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal Open Field Observations:  Mobility - 1=Normal; Gait - 1=Normal; Arousal - 3=Normal; Clonic involuntary movement - 1=None; Tonic involuntary movement - 1=None; Stereotype behaviour - 1=Absent (Normal); Sensory Observations: Approach response - 2=Normal; Auditory response - 2=Normal; Touch response - 2=Normal; Pupil reflex - 2=Normal; Tail pinch response - 2=Normal; Righting reflex - 1=Present (Normal)
Lactation Day 13
Parameters↓	Group & Sex				G1 & F				G2 & F			G3 & F				G4 & F
	Dose (mg of active ingredient SVS/kg body weight/day)				0				100			300				1000
	Number of Animals (randomly selected)				5				5			5				5
Home Cage Observations
Home cage posture					1				1			1				1
Respiratory pattern					1				1			1				1
Clonic involuntary movements					1				1			1				1
Tonic involuntary movements					1				1			1				1
Vocalization					1				1			1				1
Palpebral closure					1				1			1				1
Handling Observations
Ease of removal from the cage					2(3), 3(2)				2(3), 3(2)			2(1), 3(4)				2(4), 3(1)
Ease of handling animal in hand					2(3), 3(2)				2(3), 3(2)			2(2), 3(3)				2(3), 3(2)
Red or crusty deposits			Eyes		1				1			1				1
			Nose		1				1			1				1
			Mouth		1				1			1				1
Lacrimation					1				1			1				1
Salivation					1				1			1				1
Fur appearance					1				1			1				1
Piloerection					1				1			1				1
Eye prominence					1				1			1				1
Muscle tone					1				1			1				1
Open Field Observations
Mobility						1	1			1				1
Gait						1	1			1				1
Arousal						3	3			3				3
Number of rearing				Mean		5.6	4.6			4.6				4.6
				±SD		1.1	0.9			1.1				0.9
Numbers of urination (urine pools)				Mean		5.0	4.8			5.0				5.0
				±SD		0.7	0.8			0.7				0.7
Number of defecations				Mean		3.2	3.4			3.2				3.4
				±SD		0.4	0.5			0.4				0.5
Clonic involuntary movement						1	1			1				1
Tonic involuntary movement						1	1			1				1
Stereotype behaviour						1	1			1				1
No. of groomings				Mean		3.8	4.6			4.2				4.2
				±SD		0.8	1.1			0.8				0.8
Sensory Observations
Approach response						2	2			2				2
Auditory response						2	2			2				2
Touch response						2	2			2				2
Pupil reflex						2	2			2				2
Tail pinch response						2	2			2				2
Righting reflex						1	1			1				1
Physiological Observation
Body temperature (°F)				Mean		97.9	98.1			97.9				98.0
				±SD		0.2	0.4			0.3				0.7
Home Cage Observations: Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=Absent (Normal);  Palpebral closure - 1=Normal; Handling Observations: Ease of removal from the cage - 2=Normal, 3=Moderately difficult; Ease of handling animal in hand - 2=Normal, 3=Moderate resistance; Red or crusty deposits - 1=Absent (Normal); Lacrimation - 1=None; Salivation- 1=Absent (Normal); Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal Open Field Observations:  Mobility - 1=Normal; Gait - 1=Normal; Arousal - 3=Normal; Clonic involuntary movement - 1=None; Tonic involuntary movement - 1=None; Stereotype behaviour - 1=Absent (Normal); Sensory Observations: Approach response - 2=Normal; Auditory response - 2=Normal; Touch response - 2=Normal; Pupil reflex - 2=Normal; Tail pinch response - 2=Normal; Righting reflex - 1=Present (Normal)

F: Female; M: Male; (): Number of animals; ±SD: Standard Deviation.

 

Table 12 continued for recovery groups

Day 65
Parameters↓	Group & Sex				G1R & M	G4R & M
	Dose (mg of active ingredient SVS /kg body weight/day)				0	1000
	Number of Animals				5	5
Home Cage Observations
Home cage posture					1	1
Respiratory pattern					1	1
Clonic involuntary movements					1	1
Tonic involuntary movements					1	1
Vocalization					1	1
Palpebral closure					1	1
Handling Observations
Ease of removal from the cage					2(3), 3(2)	2(2), 3(3)
Ease of handling animal in hand					2(3), 3(2)	2(3), 3(2)
Red or crusty deposits			Eyes		1	1
			Nose		1	1
			Mouth		1	1
Lacrimation					1	1
Salivation					1	1
Fur appearance					1	1
Piloerection					1	1
Eye prominence					1	1
Muscle tone					1	1
Open Field Observations
Mobility					1	1
Gait					1	1
Arousal					3	3
Number of rearing			Mean		5.6	5.6
			±SD		1.1	1.1
Numbers of urination (urine pools)			Mean		5.2	5.2
			±SD		0.8	0.8
Number of defecations			Mean		3.4	3.2
			±SD		0.5	0.8
Clonic involuntary movement					1	1
Tonic involuntary movement					1	1
Stereotype behaviour					1	1
No. of groomings			Mean		5.6	4.2
			±SD		0.5	1.3
Sensory Observations
Approach response					2	2
Auditory response					2	2
Touch response					2	2
Pupil reflex					2	2
Tail pinch response					2	2
Righting reflex					1	1
Physiological Observation
Body temperature (°F)			Mean		97.9	98.2
			±SD		0.2	0.4
Open Field Observations:  Mobility - 1=Normal; Gait - 1=Normal; Arousal - 3=Normal; Clonic involuntary movement - 1=None; Tonic involuntary movement - 1=None; Stereotype behaviour - 1=Absent (Normal); Sensory Observations: Approach response - 2=Normal; Auditory response - 2=Normal; Touch response - 2=Normal; Pupil reflex - 2=Normal; Tail pinch response - 2=Normal; Righting reflex - 1=Present (Normal) Home Cage Observations: Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=Absent (Normal);  Palpebral closure - 1=Normal; Handling Observations: Ease of removal from the cage - 2=Normal, 3=Moderately difficult;  Ease of handling animal in hand - 2=Normal; Red or crusty deposits - 1=Absent (Normal); Lacrimation - 1=None; Salivation- 1=Absent (Normal); Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal
Parameters↓	Group & Sex				G1R & F	G4R & F
	Dose (mg of active ingredient SVS /kg body weight/day)				0	1000
	Number of Animals				5	5
Home Cage Observations
Home cage posture					1	1
Respiratory pattern					1	1
Clonic involuntary movements					1	1
Tonic involuntary movements					1	1
Vocalization					1	1
Palpebral closure					1	1
Handling Observations
Ease of removal from the cage					2(3), 3(2)	2(4), 3(1)
Ease of handling animal in hand					2(2), 3(3)	2(2), 3(3)
Red or crusty deposits			Eyes		1	1
			Nose		1	1
			Mouth		1	1
Lacrimation					1	1
Salivation					1	1
Fur appearance					1	1
Piloerection					1	1
Eye prominence					1	1
Muscle tone					1	1
Open Field Observations
Mobility				1			1
Gait				1			1
Arousal				3			3
Number of rearing		Mean		5.0			5.0
		±SD		0.7			0.7
Numbers of urination (urine pools)		Mean		4.6			5.2
		±SD		0.5			0.8
Number of defecations		Mean		3.2			3.6
		±SD		0.4			0.9
Clonic involuntary movement				1			1
Tonic involuntary movement				1			1
Stereotype behaviour				1			1
No. of groomings		Mean		4.2			4.2
		±SD		1.3			1.3
Sensory Observations
Approach response				2			2
Auditory response				2			2
Touch response				2			2
Pupil reflex				2			2
Tail pinch response				2			2
Righting reflex				1			1
Physiological Observation
Body temperature (°F)		Mean		97.9			97.4
		±SD		0.4			0.7
Home Cage Observations: Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=Absent (Normal);  Palpebral closure - 1=Normal; Handling Observations: Ease of removal from the cage - 2=Normal; 3=Moderately difficult; Ease of handling animal in hand - 2=Normal; Red or crusty deposits - 1=Absent (Normal); Lacrimation - 1=None; Salivation- 1=Absent (Normal); Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal Open Field Observations:  Mobility - 1=Normal; Gait - 1=Normal; Arousal - 3=Normal; Clonic involuntary movement - 1=None; Tonic involuntary movement - 1=None; Stereotype behaviour - 1=Absent (Normal); Sensory Observations: Approach response - 2=Normal; Auditory response - 2=Normal; Touch response - 2=Normal; Pupil reflex - 2=Normal; Tail pinch response - 2=Normal; Righting reflex - 1=Present (Normal)

F: Female; M: Male; R: Recovery; (): Number of animals.

 

Table 13: Summary of locomotor activity / movement counts records

Day 28
Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Actimeter Readings
		Movement Counts (no.)
G1, M & 0	Mean	1995.0
	±SD	265.4
	n	5
G2, M & 100	Mean	1779.8
	±SD	139.1
	n	5
G3, M & 300	Mean	1843.6
	±SD	150.7
	n	5
G4, M & 1000	Mean	1985.6
	±SD	98.0
	n	5
Lactation Day 13
Group, Sex & Dose (mg of active ingredient SVS /kg body weight/day)		Movement Counts (no.)
G1, F & 0	Mean	1833.4
	±SD	201.2
	n	5
G2, F & 100	Mean	2064.4
	±SD	209.3
	n	5
G3, F & 300	Mean	1728.0
	±SD	84.3
	n	5
G4, F & 1000	Mean	1962.4
	±SD	114.8
	n	5
	n	5
Day 65
Group, Sex & Dose (mg of active ingredient SVS /kg body weight/day)		Movement Counts (no.)
G1R, M & 0	Mean	2006.2
	±SD	125.0
	n	5
G4R, M & 1000	Mean	2075.8
	±SD	78.6
	n	5
Day 65
Group, Sex & Dose (mg of active ingredient SVS /kg body weight/day)		Movement Counts (no.)
G1R, F & 0	Mean	2036.8
	±SD	146.6
	n	5
G4R, F & 1000	Mean	1978.4
	±SD	99.7
	n	5

M: Male; F: Female; ±SD: Standard Deviation; n: Number of Animals (randomly selected).

 

Table 15: Summary of average grip strength (kgf) measurement records

Day 28
Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)				Grip Strength (kgf)
				Fore limb	Hind limb
G1, M & 0	Mean			1.402	0.543
	±SD			0.090	0.046
	n			5	5
G2, M & 100	Mean			1.462	0.523
	±SD			0.100	0.046
	n			5	5
G3, M & 300	Mean			1.531	0.544
	±SD			0.071	0.049
	n			5	5
G4, M & 1000	Mean			1.540	0.554
	±SD			0.116	0.030
	n			5	5
Lactation Day 13
G1, F & 0	Mean			1.516	0.512
	±SD			0.039	0.041
	n			5	5
G2, F & 100	Mean			1.479	0.483
	±SD			0.024	0.033
	n			5	5
G3, F & 300	Mean			1.534	0.481
	±SD			0.019	0.014
	n			5	5
G4, F & 1000	Mean			1.525	0.505
	±SD			0.015	0.014
	n			5	5
Day 65
G1R, M & 0			Mean	1.590	0.569
			±SD	0.037	0.035
			n	5	5
G4R, M & 1000			Mean	1.580	0.604
			±SD	0.058	0.018
			n	5	5
G1R, F & 0		Mean		1.528	0.545
		±SD		0.039	0.049
		n		5	5
G4R, F & 1000		Mean		1.561	0.553
		±SD		0.045	0.037
		n		5	5

M: Male; F: Female; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

 

Table 15: Summary of average hindlimb foot splay records (cm)

Group, Sex & Dose (mg of active ingredient SVS/ kg body weight/day)		Hind Limb Foot Splay (cm)
Day 28
G1, M & 0	Mean	9.84
	±SD	0.29
	n	5
G2, M & 100	Mean	9.02*
	±SD	0.46
	n	5
G3, M & 300	Mean	9.13
	±SD	0.56
	n	5
G4, M & 1000	Mean	9.48
	±SD	0.53
	n	5
Lactation Day 13
G1, F & 0	Mean	7.49
	±SD	0.32
	n	5
G2, F & 100	Mean	7.33
	±SD	0.78
	n	5
G3, F & 300	Mean	7.26
	±SD	0.77
	n	5
G4, F & 1000	Mean	7.65
	±SD	0.42
	n	5
Day 65
G1R, M & 0	Mean	8.61
	±SD	1.17
	n	5
G4R, M & 1000	Mean	8.95
	±SD	0.61
	n	5
G1R, F & 0	Mean	7.38
	±SD	0.96
	n	5
G4R, F & 1000	Mean	7.60
	±SD	0.28
	n	5

F: Female; M: Male; ±SD: Standard Deviation; n: Number of Animals (randomly selected).

* Statistically significant (P<0.05) change than the vehicle control group

 

Table 16: Summary of haematological records

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Hemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Hemoglobin	Mean Corpuscular Hemoglobin Concentration	Platelet Count
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)
		(10^3 cells/µL)	(10^6 cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3 cells/µL)
G1, M & 0	Mean	9.90	7.84	15.54	45.90	61.30	20.18	33.60	1161.80
	±SD	1.75	1.77	2.45	2.87	14.98	2.10	3.65	104.20
	n	5	5	5	5	5	5	5	5
G2, M & 100	Mean	9.24	8.90	16.96	48.80	54.86	19.04	34.76	992.80
	±SD	2.49	0.48	0.87	2.60	1.60	0.76	0.59	319.22
	n	5	5	5	5	5	5	5	5
G3, M & 300	Mean	7.21	8.81	16.32	47.00	53.38	18.54	34.74	1297.00
	±SD	1.45	0.58	0.76	3.09	0.38	0.51	0.83	161.06
	n	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	9.17	8.73	16.46	47.42	54.34	18.82	34.68	1189.80
	±SD	2.26	0.36	0.61	2.21	1.53	0.38	0.49	188.01
	n	5	5	5	5	5	5	5	5

M: Male; ±SD: Standard Deviation; n: Number of Animals (randomly selected).

 

Table 16: Summary of haematological records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Mean Platelet Volume	Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(MPV)	(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(fL)	(%)	(%)	(%)	(%)	(%)	(%)
G1, M & 0	Mean	7.22	9.90	30.18	65.12	2.24	1.06	0.32
	±SD	0.68	16.21	7.38	8.53	0.68	0.55	0.13
	n	5	5	5	5	5	5	5
G2, M & 100	Mean	7.20	2.11	31.40	63.68	2.12	1.34	0.40
	±SD	0.47	0.46	9.47	9.13	0.66	0.39	0.12
	n	5	5	5	5	5	5	5
G3, M & 300	Mean	6.94	2.72	27.84	67.16	2.20	1.72	0.20
	±SD	0.21	0.24	4.30	4.86	0.77	0.38	0.07
	n	5	5	5	5	5	5	5
G4, M & 1000	Mean	6.88	2.44	29.88	63.88	3.14	1.40	0.28
	±SD	0.18	0.82	10.50	10.88	1.33	0.32	0.08
	n	5	5	5	5	5	5	5

M: Male; ±SD: Standard Deviation; n: Number of Animals (randomly selected).

 

Table 16: Summary of haematological records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^9 cells/L)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(Seconds)	(Seconds)
G1, M & 0	Mean	546.54	3.00	6.43	0.22	0.11	0.03	21.38	17.20
	±SD	711.42	1.08	1.30	0.10	0.06	0.01	2.38	1.13
	n	5	5	5	5	5	5	5	5
G2, M & 100	Mean	188.70	2.82	5.94	0.20	0.12	0.04	21.72	24.36*
	±SD	48.05	0.96	2.22	0.11	0.03	0.02	2.42	6.08
	n	5	5	5	5	5	5	5	5
G3, M & 300	Mean	238.48	1.97	4.88	0.15	0.13	0.02	20.16	23.06
	±SD	15.37	0.24	1.26	0.03	0.04	0.01	1.23	4.42
	n	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	212.66	2.76	5.81	0.30	0.13	0.03	21.08	19.36
	±SD	73.25	1.27	1.65	0.17	0.04	0.01	0.68	0.97
	n	5	5	5	5	5	5	5	5

M: Male; ±SD: Standard Deviation; n: Number of Animals (randomly selected).

* Statistically significant (P<0.05) change than the vehicle control group

 

Table 16: Summary of haematological records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Hemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Hemoglobin	Mean Corpuscular Hemoglobin Concentration	Platelet Count
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)
		(10^3 cells/µL)	(10^6 cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3 cells/µL)
G1, F & 0	Mean	10.52	8.04	16.26	46.76	58.26	20.24	34.74	990.20
	±SD	2.91	0.46	0.81	1.87	1.68	0.44	0.57	196.51
	n	5	5	5	5	5	5	5	5
G2, F & 100	Mean	7.73	7.66	15.76	45.28	59.36	20.66	34.82	880.40
	±SD	1.94	0.58	0.42	1.72	4.46	1.39	0.99	264.39
	n	5	5	5	5	5	5	5	5
G3, F & 300	Mean	11.06	7.50	15.12*	43.12*	57.50	20.18	35.08	803.00
	±SD	1.42	0.30	0.65	1.87	1.87	0.59	1.25	437.90
	n	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	10.04	7.94	15.84	45.28	57.04	19.94	34.98	1160.40
	±SD	3.43	0.29	0.55	1.51	1.86	0.82	0.29	244.59
	n	5	5	5	5	5	5	5	5

F: Female; ±SD: Standard Deviation; n: Number of Animals (randomly selected).

* Statistically significant (P<0.05) change than the vehicle control group

 

Table 16: Summary of haematological records, continued

Group, Sex & Dose  (mg of active ingredient SVS/kg body weight/day)		Mean Platelet Volume	Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(MPV)	(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(fL)	(%)	(%)	(%)	(%)	(%)	(%)
G1, F & 0	Mean	7.36	2.91	30.18	63.70	3.36	1.50	0.26
	±SD	0.34	1.02	14.50	14.48	1.31	0.84	0.15
	n	5	5	5	5	5	5	5
G2, F & 100	Mean	7.22	2.76	33.08	59.76	3.90	1.56	0.16
	±SD	0.54	0.37	8.84	10.20	2.43	1.13	0.09
	n	5	5	5	5	5	5	5
G3, F & 300	Mean	7.84	3.48	40.78	53.28	3.36	1.18	0.14
	±SD	1.23	0.84	3.26	3.60	1.30	0.64	0.05
	n	5	5	5	5	5	5	5
G4, F & 1000	Mean	7.18	2.49	31.54	61.70	3.82	1.38	0.20
	±SD	0.15	0.61	5.75	6.11	2.01	0.41	0.00
	n	5	5	5	5	5	5	5

F: Female; ±SD: Standard Deviation; n: Number of Animals (randomly selected).

 

Table 16: Summary of haematological records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^9 cells/L)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(Seconds)	(Seconds)
G1, F & 0	Mean	236.46	3.17	6.68	0.37	0.16	0.03	20.72	24.68
	±SD	88.66	1.68	2.45	0.21	0.10	0.02	2.66	7.84
	n	5	5	5	5	5	5	5	5
G2, F & 100	Mean	211.58	2.48	4.73	0.27	0.13	0.01	21.74	20.96
	±SD	29.90	0.69	1.89	0.09	0.10	0.00	2.17	6.74
	n	5	5	5	5	5	5	5	5
G3, F & 300	Mean	259.48	4.51	5.88	0.39	0.12	0.02	21.52	18.22
	±SD	53.66	0.67	0.71	0.20	0.06	0.01	1.68	3.18
	n	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	197.76	3.27	6.06	0.41	0.13	0.02	22.20	21.78
	±SD	50.43	1.46	1.67	0.33	0.04	0.01	3.43	4.73
	n	5	5	5	5	5	5	5	5

F: Female; ±SD: Standard Deviation; n: Number of Animals (randomly selected).

 

Table 16: Summary of haematological records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Hemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Hemoglobin	Mean Corpuscular Hemoglobin Concentration	Platelet Count
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)
		(10^3 cells/µL)	(10^6 cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3 cells/µL)
G1R, M & 0	Mean	12.68	8.74	16.32	47.46	54.32	18.70	34.44	972.60
	±SD	9.66	0.47	0.91	2.61	0.75	0.37	0.93	99.66
	n	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	11.62	8.18	16.00	47.92	59.06	19.62	33.34	887.80
	±SD	2.94	0.79	1.31	1.55	7.03	1.97	2.36	108.35
	n	5	5	5	5	5	5	5	5

M: Male; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

 

Table 16: Summary of haematological records, continued

Group, Sex & Dose  (mg of active ingredient SVS/kg body weight/day)		Mean Platelet Volume	Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(MPV)	(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(fL)	(%)	(%)	(%)	(%)	(%)	(%)
G1R, M & 0	Mean	7.56	2.18	28.52	66.26	3.04	1.18	0.28
	±SD	0.36	0.81	8.85	9.78	0.98	0.78	0.16
	n	5	5	5	5	5	5	5
G4R, M & 1000	Mean	8.46	2.74	26.50	67.42	3.68	0.72	0.42
	±SD	1.75	0.64	7.13	8.63	1.92	0.08	0.08
	n	5	5	5	5	5	5	5

M: Male; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

 

Table 16: Summary of haematological records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^9 cells/L)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3cells/µL)	(Seconds)	(Seconds)
G1R, M & 0	Mean	189.26	4.23	7.73	0.44	0.13	0.05	20.16	21.84
	±SD	63.70	4.88	4.24	0.48	0.07	0.06	1.83	4.43
	n	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	220.34	3.24	7.65	0.44	0.08	0.05	20.04	20.06
	±SD	31.52	1.82	0.89	0.27	0.03	0.02	2.61	5.25
	n	5	5	5	5	5	5	5	5

M: Male; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

 

Table 16: Summary of haematological records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Hemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Hemoglobin	Mean Corpuscular Hemoglobin Concentration	Platelet Count
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)
		(10^3 cells/µL)	(10^6 cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3 cells/µL)
G1R, F & 0	Mean	6.50	8.27	15.96	45.38	55.10	19.42	35.18	1062.60
	±SD	1.06	0.68	0.76	2.27	2.76	1.49	0.95	89.02
	n	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	5.99	7.90	15.38	44.28	56.04	19.46	34.70	1019.40
	±SD	1.48	0.33	0.81	2.07	1.50	0.49	0.47	127.59
	n	5	5	5	5	5	5	5	5

F: Female; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

 

Table 16: Summary of haematological records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Mean Platelet Volume	Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(MPV)	(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(fL)	(%)	(%)	(%)	(%)	(%)	(%)
G1R, F & 0	Mean	7.54	2.03	15.58	79.64	2.04	1.48	0.18
	±SD	0.30	1.27	5.57	5.95	0.87	0.81	0.08
	n	5	5	5	5	5	5	5
G4R, F & 1000	Mean	7.42	2.10	22.60*	72.10*	2.52	1.64	0.22
	±SD	0.18	0.49	3.25	3.29	0.36	0.67	0.04
	n	5	5	5	5	5	5	5

F: Female; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

* Statistically significant (P<0.05) change than the vehicle control group

 

Table 16: Summary of haematological records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^9 cells/L)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(Seconds)	(Seconds)
G1R, F & 0	Mean	161.44	0.99	5.20	0.13	0.09	0.01	20.32	20.42
	±SD	84.20	0.31	1.13	0.06	0.06	0.01	2.47	6.51
	n	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	167.26	1.36	4.30	0.15	0.10	0.01	18.64	22.38
	±SD	46.10	0.41	1.03	0.05	0.06	0.00	1.54	4.28
	n	5	5	5	5	5	5	5	5

F: Female; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

 

Table 17: Summary of clinical chemistry records

Group, Sex & Dose (mg of active ingredient SVS /kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)
G1, M & 0	Mean	100.20	29.24	0.53	64.60	40.00	6.56
	±SD	5.67	3.54	0.01	5.81	7.45	0.34
	n	5	5	5	5	5	5
G2, M & 100	Mean	105.80	28.80	0.51	73.00	38.00	6.62
	±SD	7.79	1.81	0.04	6.93	9.19	0.18
	n	5	5	5	5	5	5
G3, M & 300	Mean	111.80	27.20	0.51	65.40	40.20	6.50
	±SD	5.26	4.26	0.03	7.27	10.33	0.32
	n	5	5	5	5	5	5
G4, M & 1000	Mean	118.00*	28.78	0.50	81.60*	39.20	6.40
	±SD	10.46	3.86	0.01	12.38	9.83	0.27
	n	5	5	5	5	5	5

M: Male; ±SD: Standard Deviation; n: Number of Animals (randomly selected).
* Statistically significant (P<0.05) change than the vehicle control group

 

Table 17: Summary of clinical chemistry records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Calcium	Phosphorous
		(ALB)	(ALT)	(AST)	(ALP)	(CAL)	(PHO)
		(g/dL)	(U/L)	(U/L)	(U/L)	(mg/dL)	(mg/dL)
G1, M & 0	Mean	3.20	44.60	95.40	129.60	10.00	5.64
	±SD	0.11	2.97	11.19	38.04	0.21	0.35
	n	5	5	5	5	5	5
G2, M & 100	Mean	3.30	42.60	83.60	115.40	10.08	5.52
	±SD	0.11	5.18	12.10	13.81	0.30	0.31
	n	5	5	5	5	5	5
G3, M & 300	Mean	3.42*	36.00*	80.60	145.80	10.14	5.76
	±SD	0.10	3.16	7.80	31.51	0.11	0.35
	n	5	5	5	5	5	5
G4, M & 1000	Mean	3.26	39.20	84.80	157.40	10.08	5.87
	±SD	0.14	2.05	11.12	27.00	0.18	0.40
	n	5	5	5	5	5	5

M: Male; ±SD: Standard Deviation; n: Number of Animals (randomly selected).

* Statistically significant (P<0.05) change than the vehicle control group

 

Table 17: Summary of clinical chemistry records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(GLO)	(BUN)	(Na)	(K)	(CLO)
		(g/dL)	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, M & 0	Mean	3.36	13.66	140.86	3.54	104.94
	±SD	0.43	1.65	1.60	0.25	2.21
	n	5	5	5	5	5
G2, M & 100	Mean	3.32	13.46	141.76	3.85	106.98
	±SD	0.21	0.84	1.15	0.13	1.51
	n	5	5	5	5	5
G3, M & 300	Mean	3.08	12.71	141.56	3.48	105.90
	±SD	0.24	1.99	0.78	0.23	0.90
	n	5	5	5	5	5
G4, M & 1000	Mean	3.14	13.45	140.18	3.74	104.84
	±SD	0.25	1.80	0.48	0.35	2.35
	n	5	5	5	5	5

M: Male; ±SD: Standard Deviation; n: Number of Animals (randomly selected).

 

Table 17: Summary of clinical chemistry records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)
G1, F & 0	Mean	116.40	47.62	0.66	76.40	115.60	7.20
	±SD	20.70	13.18	0.03	24.36	63.77	0.34
	n	5	5	5	5	5	5
G2, F & 100	Mean	107.20	49.90	0.63	90.00	144.00	6.78
	±SD	14.82	12.45	0.05	15.17	97.59	0.22
	n	5	5	5	5	5	5
G3, F & 300	Mean	106.20	58.66	0.68	82.40	150.60	6.90
	±SD	10.43	6.20	0.03	12.97	71.65	0.19
	n	5	5	5	5	5	5
G4, F & 1000	Mean	110.60	60.06	0.68	88.40	132.80	6.96
	±SD	4.56	13.89	0.02	16.04	45.84	0.42
	n	5	5	5	5	5	5

F: Female; ±SD: Standard Deviation; n: Number of Animals (randomly selected).

 

Table 17: Summary of clinical chemistry records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Calcium	Phosphorous
		(ALB)	(ALT)	(AST)	(ALP)	(CAL)	(PHO)
		(g/dL)	(U/L)	(U/L)	(U/L)	(mg/dL)	(mg/dL)
G1, F & 0	Mean	3.59	79.40	142.00	141.80	10.40	6.99
	±SD	0.15	32.38	66.79	85.72	0.41	2.41
	n	5	5	5	5	5	5
G2, F & 100	Mean	3.48	87.80	163.20	128.20	10.30	6.98
	±SD	0.21	21.74	51.16	31.05	0.25	1.39
	n	5	5	5	5	5	5
G3, F & 300	Mean	3.49	111.00	160.00	227.40	10.52	8.55
	±SD	0.08	13.06	55.44	106.23	0.54	1.47
	n	5	5	5	5	5	5
G4, F & 1000	Mean	3.60	89.40	120.40	163.20	10.42	7.79
	±SD	0.16	20.97	24.28	56.05	0.15	1.72
	n	5	5	5	5	5	5

F: Female; ±SD: Standard Deviation; n: Number of Animals (randomly selected).

 

Table 17: Summary of clinical chemistry records, continued

Group, Sex & Dose  (mg of active ingredient SVS/kg body weight/day)		Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(GLO)	(BUN)	(Na)	(K)	(CLO)
		(g/dL)	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, F & 0	Mean	3.61	22.25	138.58	3.90	103.86
	±SD	0.29	6.16	2.18	0.56	3.04
	n	5	5	5	5	5
G2, F & 100	Mean	3.30	23.32	139.78	3.73	104.70
	±SD	0.17	5.82	1.76	0.40	2.04
	n	5	5	5	5	5
G3, F & 300	Mean	3.41	27.41	136.60	3.95	103.20
	±SD	0.15	2.90	1.83	0.29	4.11
	n	5	5	5	5	5
G4, F & 1000	Mean	3.36	28.07	138.36	4.01	103.36
	±SD	0.28	6.49	3.18	0.69	2.12
	n	5	5	5	5	5

F: Female; ±SD: Standard Deviation; n: Number of Animals (randomly selected).

 

Table 17: Summary of clinical chemistry records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)
G1R, M & 0	Mean	118.00	36.28	0.53	40.40	27.20	6.60
	±SD	12.41	3.22	0.04	4.88	9.68	0.14
	n	5	5	5	5	5	5
G4R, M & 1000	Mean	125.20	38.22	0.54	48.80	38.80	6.68
	±SD	14.13	3.99	0.04	12.21	14.77	0.11
	n	5	5	5	5	5	5

M: Male; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

 

Table 17: Summary of clinical chemistry records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Calcium	Phosphorous
		(ALB)	(ALT)	(AST)	(ALP)	(CAL)	(PHO)
		(g/dL)	(U/L)	(U/L)	(U/L)	(mg/dL)	(mg/dL)
G1R, M & 0	Mean	3.11	60.20	110.00	149.60	9.68	5.34
	±SD	0.09	9.65	20.21	38.53	0.29	1.00
	n	5	5	5	5	5	5
G4R, M & 1000	Mean	3.23	56.00	118.20	99.20	9.94	5.15
	±SD	0.09	9.67	45.86	39.80	0.24	0.45
	n	5	5	5	5	5	5

M: Male; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

 

Table 17: Summary of clinical chemistry records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(GLO)	(BUN)	(Na)	(K)	(CLO)
		(g/dL)	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1R, M & 0	Mean	3.49	16.95	141.28	3.58	104.80
	±SD	0.09	1.51	0.86	0.51	1.66
	n	5	5	5	5	5
G4R, M & 1000	Mean	3.45	17.86	142.36	3.62	103.62
	±SD	0.14	1.86	1.31	0.60	1.46
	n	5	5	5	5	5

M: Male; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

 

Table 17: Summary of clinical chemistry records, continued

Group, Sex & Dose (mg of active ingredient SVS /kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)
G1R, F & 0	Mean	115.40	35.10	0.59	71.60	41.40	7.54
	±SD	16.47	4.48	0.02	4.16	13.33	0.11
	n	5	5	5	5	5	5
G4R, F & 1000	Mean	146.20*	32.76	0.58	97.20*	48.60	7.98
	±SD	5.63	3.87	0.04	15.82	36.04	0.39
	n	5	5	5	5	5	5

F: Female; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.
* Statistically significant (P<0.05) change than the vehicle control group

 

Table 17: Summary of clinical chemistry records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Calcium	Phosphorous
		(ALB)	(ALT)	(AST)	(ALP)	(CAL)	(PHO)
		(g/dL)	(U/L)	(U/L)	(U/L)	(mg/dL)	(mg/dL)
G1R, F & 0	Mean	4.05	40.60	85.20	33.20	10.44	4.04
	±SD	0.37	5.73	13.22	10.76	0.27	0.42
	n	5	5	5	5	5	5
G4R, F & 1000	Mean	4.11	44.20	122.40	43.00	10.76	3.53
	±SD	0.66	28.03	92.74	10.72	0.48	0.51
	n	5	5	5	5	5	5

F: Female; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

 

Table 17: Summary of clinical chemistry records, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(GLO)	(BUN)	(Na)	(K)	(CLO)
		(g/dL)	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1R, F & 0	Mean	3.49	16.40	142.38	3.03	105.92
	±SD	0.28	2.10	0.98	0.17	2.68
	n	5	5	5	5	5
G4R, F & 1000	Mean	3.87	15.31	142.76	3.39*	105.30
	±SD	0.36	1.81	1.32	0.17	1.97
	n	5	5	5	5	5

F: Female; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.
* Statistically significant (P<0.05) change than the vehicle control group

 

Table 18: Summary of urinalysis records

Examination	Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		G1, M & 0	G2, M & 100	G3, M & 300	G4, M & 1000
	Number of Animals		5	5	5	5
Physical Examination	Color	Pale Yellow	5	4	5	5
		Yellow	-	1	-	-
	Appearance	Clear	5	4	5	5
		Turbid	-	1	-	-
	Volume (mL)	Mean	4.7	4.2	4.4	4.6
		±SD	0.9	0.8	0.7	0.8
Chemical Examination	pH	Mean	7.0	7.4	7.5	7.1
		±SD	0.4	0.4	0.4	0.4
	Specific gravity (SG)	Mean	1.017	1.017	1.016	1.017
		±SD	0.003	0.003	0.005	0.003
	Urobilinogen (UBG) (mg/dL)	Mean	0.2	0.2	0.2	0.2
		±SD	0.0	0.0	0.0	0.0
	Bilirubin (BIL) (mg/dL)	Neg	5	5	5	5
	Ketones (KET) (mg/dL)	Neg	1	1	1	-
		5	4	4	3	5
		15	-	-	1	-
	Blood (BLD) (Ery/µL)	Neg	2	5	5	5
		Ca10	1	-	-	-
		Ca25	1	-	-	-
		Ca80	1	-	-	-
	Protein (PRO) (mg/dL)	Neg	-	-	-	1
		Trace	4	2	3	3
		30	1	2	2	1
		100	-	1	-	-
	Nitrite (NIT)	Neg	3	5	5	4
		Pos	2	-	-	1
	Glucose (GLU)  (mg/dL)	Neg	5	5	5	5
	Micro Albumin (MALB) (mg/dL)	>15	5	5	5	4
		15	-	-	-	1
Microscopic Examination	Epi Cells	0	4	4	4	5
		0-1	1	1	1	-
	Casts	Absent	5	5	5	5
	Crystals	Present	5	5	5	5

M: Male; ±SD: Standard Deviation; Pos: Positive; Neg: Negative; Ca: Calculated.

 

Table 18: Summary of urinalysis records, continued

Examination	Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		G1R, M & 0	G4R, M & 1000
	Number of Animals		5	5
Physical Examination	Color	Pale Yellow	4	4
		Yellow	1	1
	Appearance	Clear	4	4
		Turbid	1	1
	Volume (mL)	Mean	5.3	8.3*
		±SD	1.8	1.8
Chemical Examination	pH	Mean	6.5	7.0
		±SD	0.8	0.7
	Specific gravity (SG)	Mean	1.019	1.014
		±SD	0.007	0.004
	Urobilinogen (UBG) (mg/dL)	Mean	0.2	0.2
		±SD	0.0	0.0
	Bilirubin (BIL) (mg/dL)	Neg	4	4
		1	1	1
	Ketones (KET) (mg/dL)	Neg	-	2
		5	5	3
	Blood (BLD) (Ery/µL)	Neg	2	4
		Ca25	2	1
		>=Ca200	1	-
	Protein (PRO) (mg/dL)	Neg	4	3
		Trace	-	2
		30	1	-
	Nitrite (NIT)	Neg	2	3
		Pos	3	2
	Glucose (GLU) (mg/dL)	Neg	5	5
	Micro Albumin (MALB) (mg/dL)	>15	1	2
		15	4	3
Microscopic Examination	Epi Cells	0	5	4
		0-1	-	1
	Casts	Absent	5	5
	Crystals	Absent	1	1
		Present	4	4

M: Male; R: Recovery; ±SD: Standard Deviation; Pos: Positive; Neg: Negative; Ca: Calculated.
* Statistically significant (P<0.05) change than the vehicle control group

 

Table 18: Summary of urinalysis records, continued

Examination	Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		G1R, F & 0	G4R, F & 1000
	Number of Animals		5	5
Physical Examination	Color	Pale Yellow	4	5
		Yellow	1	-
	Appearance	Clear	5	5
		Turbid	-	-
	Volume (mL)	Mean	6.2	8.0
		±SD	0.8	3.0
Chemical Examination	pH	Mean	7.2	6.8
		±SD	1.1	0.7
	Specific gravity  (SG)	Mean	1.014	1.013
		±SD	0.007	0.004
	Urobilinogen (UBG) (mg/dL)	Mean	0.6	0.2
		±SD	0.8	0.0
	Bilirubin (BIL) (mg/dL)	Neg	4	5
		1	1	-
	Ketones (KET) (mg/dL)	Neg	3	5
		5	2	-
		15	-	-
	Blood (BLD) (Ery/µL)	Neg	3	2
		Ca10	-	1
		Ca25	-	2
		Ca80	2	-
	Protein (PRO)  (mg/dL)	Neg	4	4
		Trace	-	1
		>=300	1	-
	Nitrite (NIT)	Neg	2	4
		Pos	3	1
	Glucose (GLU) (mg/dL)	Neg	5	5
	Micro Albumin (MALB) (mg/dL)	Neg	-	1
		>15	1	1
		15	4	3
Microscopic Examination	Epi Cells	0	4	4
		0-1	1	-
		1-2	-	1
	Casts	Absent	5	5
	Crystals	Present	5	5

F: Female; R: Recovery; ±SD: Standard Deviation; Pos: Positive; Neg: Negative; Ca: Calculated.

 

Table 19: Summary of vaginal smear examination records for determination of oestrous cyclicity

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)	Total No. of Females Evaluated		No. of Females with Complete Regular Cycles	No. of Females with at least one Irregular Oestrus Cycle		Mean Oestrus Cycle Length (days)
G1, F & 0	12	n	9	3	Mean	4.51
		%	75.0	25.0	±SD	0.30
					n	12
G2, F & 100	12	n	7	5	Mean	4.79
		%	58.3	41.7	±SD	0.48
					n	12
G3, F & 300	12	n	7	5	Mean	4.74
		%	58.3	41.7	±SD	0.33
					n	12
G4, F & 1000	12	n	8	4	Mean	4.65
		%	66.7	33.3	±SD	0.42
					n	12

F: Female; ±SD: Standard Deviation; n: Number of Animals; -: Not Applicable.

 

Table 20: Summary of delivery and litter observations

Group & Dose (mg of active ingredient SVS/kg body weight/day)		Delivery Record At birth
		Litter Size (No.)	Live Pups (No.)			Dead Pups (No.)			Cannibalized Pups (No.)				Sex ratio (m/f)	Live birth index (%)
			Male	Female	Total	Male	Female	Total	Undetermined	Male	Female	Total
G1 & 0	Mean	12.60	7.00	5.30	12.30	0.00	0.20	0.20	0.10	0.00	0.00	0.10	1.51	97.98
	±SD	4.12	2.62	2.50	3.97	0.00	0.63	0.63	0.32	0.00	0.00	0.32	0.78	4.41
	n	10	10	10	10	10	10	10	10	10	10	10	10	10
G2 & 100	Mean	13.00	7.70	5.30	13.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1.60	100.00
	±SD	2.94	2.45	1.64	2.94	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.71	0.00
	n	10	10	10	10	10	10	10	10	10	10	10	10	10
G3 & 300	Mean	11.70	6.10	5.40	11.50	0.10	0.10	0.20	0.00	0.00	0.00	0.00	1.12	98.47
	±SD	3.83	2.60	1.51	3.75	0.32	0.32	0.42	0.00	0.00	0.00	0.00	0.40	3.30
	n	10	10	10	10	10	10	10	10	10	10	10	10	10
G4 & 1000	Mean	13.00	7.00	4.90	11.90	0.30	0.50	0.80	0.30	0.00	0.00	0.30	1.87	90.32
	±SD	4.11	3.68	2.60	5.51	0.95	1.27	2.20	0.95	0.00	0.00	0.95	1.22	28.60
	n	10	10	10	10	10	10	10	10	10	10	10	9	10
		LD 1 to LD 4
G1 & 0	Mean		7.00	5.30	12.30	0.00	0.00	0.00		0.00	0.00	0.00	1.51	100.00
	±SD		2.62	2.50	3.97	0.00	0.00	0.00		0.00	0.00	0.00	0.78	0.00
	n		10	10	10	10	10	10		10	10	10	10	10
G2 & 100	Mean		7.50	5.10	12.60	0.20	0.20	0.40		0.00	0.00	0.00	1.62	97.10
	±SD		2.59	1.60	2.95	0.63	0.42	0.97		0.00	0.00	0.00	0.74	7.33
	n		10	10	10	10	10	10		10	10	10	10	10
G3 & 300	Mean		6.10	5.40	11.50	0.00	0.00	0.00		0.00	0.00	0.00	1.12	100.00
	±SD		2.60	1.51	3.75	0.00	0.00	0.00		0.00	0.00	0.00	0.40	0.00
	n		10	10	10	10	10	10		10	10	10	10	10
G4 & 1000	Mean		7.00	4.80	11.80	0.00	0.10	0.10		0.00	0.00	0.00	1.87	90.00
	±SD		3.68	2.78	5.73	0.00	0.32	0.32		0.00	0.00	0.00	1.22	31.62
	n		10	10	10	10	10	10		10	10	10	9#	10
		LD 5 to LD 7
G1 & 0	Mean		7.00	4.00	11.00	0.00	0.00	0.00		0.00	0.00	0.00	2.63	100.00
	±SD		2.62	2.16	3.13	0.00	0.00	0.00		0.00	0.00	0.00	2.62	0.00
	n		10	10	10	10	10	10		10	10	10	10	10
G2 & 100	Mean		7.50	4.10	11.60	0.00	0.00	0.00		0.00	0.00	0.00	2.24	100.00
	±SD		2.59	1.45	2.01	0.00	0.00	0.00		0.00	0.00	0.00	1.45	0.00
	n		10	10	10	10	10	10		10	10	10	10	10
G3 & 300	Mean		6.10	4.40	10.50	0.00	0.00	0.00		0.00	0.00	0.00	1.40	100.00
	±SD		2.60	0.84	2.88	0.00	0.00	0.00		0.00	0.00	0.00	0.61	0.00
	n		10	10	10	10	10	10		10	10	10	10	10
G4 & 1000	Mean		7.78	3.89	11.67	0.00	0.00	0.00		0.00	0.00	0.00	3.16	100.00
	±SD		2.91	2.03	3.50	0.00	0.00	0.00		0.00	0.00	0.00	3.05	0.00
	n		9	9	9	9	9	9		9	9	9	9	9
		LD 8 to LD 13
G1 & 0	Mean		7.00	4.00	11.00	0.00	0.00	0.00		0.00	0.00	0.00	2.63	100.00
	±SD		2.62	2.16	3.13	0.00	0.00	0.00		0.00	0.00	0.00	2.62	0.00
	n		10	10	10	10	10	10		10	10	10	10	10
G2 & 100	Mean		7.50	4.10	11.60	0.00	0.00	0.00		0.00	0.00	0.00	2.24	100.00
	±SD		2.59	1.45	2.01	0.00	0.00	0.00		0.00	0.00	0.00	1.45	0.00
	n		10	10	10	10	10	10		10	10	10	10	10
G3 & 300	Mean		6.10	4.40	10.50	0.00	0.00	0.00		0.00	0.00	0.00	1.40	100.00
	±SD		2.60	0.84	2.88	0.00	0.00	0.00		0.00	0.00	0.00	0.61	0.00
	n		10	10	10	10	10	10		10	10	10	10	10
G4 & 1000	Mean		7.78	3.89	11.67	0.00	0.00	0.00		0.00	0.00	0.00	3.16	100.00
	±SD		2.91	2.03	3.50	0.00	0.00	0.00		0.00	0.00	0.00	3.05	0.00
	n		9	9	9	9	9	9		9	9	9	9	9

F: Female; ±SD: Standard Deviation; n: Number of Dams; m/f: Male/Female; %: Percentage.

#: In group G4, one dam (Rg6836) delivered with 1 live pup and 7 dead pups. However, the one live pup died on postnatal day 3.  Hence, for sex ratio the data of this animal has been excluded.

 

Table 21: Summary records of reproductive performance

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)	No. of Males with Evidence of Mating (No. of Males used for Mating)	Male Mating Index (%)	No. of Males Capable of Impregnating a Female (No. of males used for Mating)	Male Fertility Index (%)
G1, M & 0	12 (12)	100.0	10 (12)	83.3
G2, M & 100	12 (12)	100.0	10 (12)	83.3
G3, M & 300	12 (12)	100.0	10 (12)	83.3
G4, M & 1000	12 (12)	100.0	12 (12)	100.0

M: Male.

 

Table 21: Summary records of reproductive performance, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Pre-Coital Interval / Copulatory Interval		Conceiving Days (1 to 5)	Conceiving Days (5 to More)	Gestation Length (Days)
G1, F & 0	Mean	3.00	N	9	3	22.50
	±SD	3.25	%	75.00	25.00	0.53
	n	12				10
G2, F & 100	Mean	3.42	N	10	2	22.60
	±SD	2.84	%	83.33	16.67	0.97
	n	12				10
G3, F & 300	Mean	3.50	N	8	4	22.40
	±SD	3.06	%	66.67	33.33	1.26
	n	12				10
G4, F & 1000	Mean	1.58	N	12	0	22.40
	±SD	1.00	%	100.00	0.00	0.97
	n	12				10

F: Female; ±SD: Standard Deviation; N: Pre-coital interval: Number of females with evidence of mating; n: Gestation length: Number of pregnant females.

 

Table 21: Summary records of reproductive performance, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)	Female Mating Index (%)		Female Fertility Index (%)
	No. of Females with Evidence of Mating (No. of Females used for Mating)	Female Mating Index (%)	No. of Females Confirmed as Fertile (No. of Females used for Mating)	Female Fertility Index (%)
G1, F & 0	12 (12)	100.0	10 (12)	83.3
G2, F & 100	12 (12)	100.0	10 (12)	83.3
G3, F & 300	12 (12)	100.0	10 (12)	83.3
G4, F & 1000	12 (12)	100.0	12 (12)	100.0

F: Female.

 

Table 21: Summary records of reproductive performance, continued

Group, Sex & Dose                                      (mg of active ingredient SVS /kg body weight/day)	Implantation Index (%)		Pregnancy Index (%)		Gestation Index (%)		Parturition index (%)
	No. of females evidence with implantation sites (confirmed with mating)	Implantation Index (%)	No. of females confirmed with pregnancy (confirmed with mating)	Pregnancy Index (%)	Females with Live Born Pups at Parturition (with evidence of pregnancy)	Pregnancy Gestation Index (%)	No. of Females Littered (with evidence of pregnancy)	Parturition index (%)
G1, F & 0	10 (12)	83.3	10 (12)	83.3	10 (10)	100.0	10 (10)	100.0
G2, F & 100	10 (12)	83.3	10 (12)	83.3	10 (10)	100.0	10 (10)	100.0
G3, F & 300	10 (12)	83.3	10 (12)	83.3	10 (10)	100.0	10 (10)	100.0
G4, F & 1000	12 (12)	100.0	10 (12)	83.3	10 (10)	100.0	10 (10)	100.0

F: Female.

 

Table 21: Summary records of reproductive performance, continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Post-implantation Loss (%)				Postnatal Loss (%)
		No. of Implantations	No. of Viable (Live) Pups	Post-implantation Loss (No.)	Post-implantation Loss (%)	Total No. of Pups Found Dead/ Cannibalized during lactation period	Post-natal Loss (%)
G1, F & 0	Mean	13.00	12.30	0.70	5.21	0.00	0.00
	±SD	4.08	3.97	1.34	9.28	0.00	0.00
	n	10	10	10	10	10	10
G2, F & 100	Mean	13.20	13.00	0.20	1.33	0.40	2.90
	±SD	3.01	2.94	0.63	4.22	0.97	7.33
	n	10	10	10	10	10	10
G3, F & 300	Mean	11.80	11.50	0.30	3.20	0.00	0.00
	±SD	3.65	3.75	0.48	5.74	0.00	0.00
	n	10	10	10	10	10	10
G4, F & 1000	Mean	13.20	11.90	1.30	10.34	0.10	10.00
	±SD	4.16	5.51	3.43	28.68	0.32	31.62
	n	10	10	10	10	10	10

F: Female; ±SD: Standard Deviation; n: Number of Dams.

 

Table 23: Summary of absolute organ weight records (g)

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys
G1, M & 0	Mean	0.0651	0.3273	1.0530	3.5328	1.4708	1.4896	2.7988
	±SD	0.0120	0.0602	0.6163	0.1626	0.1321	0.1034	0.2223
	n	5	5	5	12	12	5	5
G2, M & 100	Mean	0.0721	0.4516	0.8074	3.4968	1.4422	1.4115	2.8952
	±SD	0.0103	0.0855	0.1016	0.1876	0.1312	0.1963	0.3391
	n	5	5	5	12	12	5	5
G3, M & 300	Mean	0.0663	0.3779	0.8940	3.5797	1.5112	1.5892	3.2414
	±SD	0.0104	0.1223	0.0811	0.2480	0.1280	0.1442	0.4926
	n	5	5	5	12	12	5	5
G4, M & 1000	Mean	0.0762	0.3878	0.8623	3.4445	1.5021	1.6555	3.1296
	±SD	0.0175	0.0439	0.1176	0.2832	0.1850	0.1716	0.4514
	n	5	5	5	12	12	5	5

M: Male; ±SD: Standard Deviation; n: Number of Animals.

 

Table 23: Summary of absolute organ weight records (g), continued

Group, Sex & Dose (mg of active ingredient SVS/kg body weight/day)		Brain	Liver	Prostate (P)	Seminal vesicles with coagulating glands (SC)	Lungs	Thyroid along with parathyroid^#
G1, M & 0	Mean	2.1398	10.7633	1.4139	1.5412	2.2493	0.0247
	±SD	0.0861	0.7687	0.3515	0.3529	0.3424	0.0050
	n	5	5	12	12	5	12
G2, M & 100	Mean	2.0706	11.3407	1.3461	1.6064	2.4237	0.0235
	±SD	0.1785	1.0457	0.2084	0.4380	0.3994	0.0036
	n	5	5	12	12	5	12
G3, M & 300	Mean	2.1309	13.4131*	1.3368	1.7543	2.8609*	0.0234
	±SD	0.0826	2.1061	0.2485	0.5815	0.1063	0.0045
	n	5	5	12	12	5	12
G4, M & 1000	Mean	2.1460	12.8157	1.5199	1.4856	2.5022	0.0242
	±SD	0.1203	1.4829	0.1923	0.4319	0.2149	0.0043
	n	5	5	12	12	5	12

M: Male; ±SD: Standard Deviation; n: Number of Animals.

* Statistically significant (P<0.05) change than the vehicle control group

 

Table 23: Summary of absolute organ weight records (g), continued

Group, Sex & Dose (mg of active ingredient SVS/ kg body weight/day)		Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix	Heart
G1, F & 0	Mean	0.0779	0.2427	0.7202	0.1426	0.5589	1.1989
	±SD	0.0044	0.0578	0.0495	0.0189	0.1031	0.0421
	n	5	5	5	12	12	5
G2, F & 100	Mean	0.0872	0.3061	0.6318	0.1375	0.4686	1.3014
	±SD	0.0088	0.1073	0.0444	0.0477	0.1791	0.1198
	n	5	5	5	12	12	5
G3, F & 300	Mean	0.0850	0.1783	0.7140	0.2026	0.4437	1.1214
	±SD	0.0070	0.0398	0.0740	0.2969	0.1245	0.0831
	n	5	5	5	12	12	5
G4, F & 1000	Mean	0.0784	0.2278	0.6184	0.1510	0.5698	1.1930
	±SD	0.0056	0.0531	0.0895	0.0188	0.1877	0.1138
	n	5	5	5	12	12	5

F: Female; ±SD: Standard Deviation; n: Number of Animals.

 

Table 23: Summary of absolute organ weight records (g), continued

Group, Sex & Dose (mg of active ingredient SVS/ kg body weight/day)		Kidneys	Brain	Liver	Lungs	Thyroid along with parathyroid
G1, F & 0	Mean	2.0007	1.9936	10.9616	1.7627	0.0225
	±SD	0.0273	0.1581	0.6743	0.1447	0.0032
	n	5	5	5	5	12
G2, F & 100	Mean	2.0546	2.0258	12.2315	2.1365	0.0236
	±SD	0.2419	0.0922	1.2946	0.3023	0.0070
	n	5	5	5	5	12
G3, F & 300	Mean	1.9456	2.0325	11.7661	1.9613	0.0212
	±SD	0.0586	0.0371	0.9510	0.2521	0.0038
	n	5	5	5	5	12
G4, F & 1000	Mean	2.0504	2.0743	11.5623	2.0986	0.0228
	±SD	0.1529	0.1127	0.7940	0.4238	0.0045
	n	5	5	5	5	12

F: Female; ±SD: Standard Deviation; n: Number of Animals.

 

Table 23: Summary of absolute organ weight records (g), continued

Group, Sex & Dose (mg of active ingredient SVS/ kg body weight/day)		Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys
G1R, M & 0	Mean	0.0614	0.2762	1.0458	3.4416	1.4459	1.2219	2.2849
	±SD	0.0159	0.0445	0.2906	0.0905	0.0616	0.2236	0.3113
	n	5	5	5	5	5	5	5
G4R, M & 1000	Mean	0.0712	0.3076	1.3710	3.6244	1.4259	1.4645	2.8096*
	±SD	0.0098	0.0792	0.6894	0.1692	0.1743	0.2401	0.3089
	n	5	5	5	5	5	5	5

M: Male; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

* Statistically significant (P<0.05) change than the vehicle control group

 

Table 23: Summary of absolute organ weight records (g), continued

Group, Sex & Dose (mg of active ingredient SVS/ kg body weight/day)		Brain	Liver	Prostate (P)	Seminal vesicles with coagulating glands (SC)	Lungs	Thyroid along with parathyroid
G1R, M & 0	Mean	2.0333	9.9369	1.2398	1.5664	2.1478	0.0230
	±SD	0.1516	1.3292	0.3848	0.4113	0.3358	0.0021
	n	5	5	5	5	5	5
G4R, M & 1000	Mean	2.1282	12.0208*	1.3221	1.8001	2.4389	0.0239
	±SD	0.2057	1.4916	0.3765	0.4789	0.4957	0.0038
	n	5	5	5	5	5	5

M: Male; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

* Statistically significant (P<0.05) change than the vehicle control group

 

Table 23: Summary of absolute organ weight records (g), continued

Group, Sex & Dose (mg of active ingredient SVS/ kg body weight/day)		Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix	Heart
G1R, F & 0	Mean	0.0714	0.2981	0.5873	0.1544	0.7927	1.1026
	±SD	0.0144	0.0344	0.0612	0.0321	0.1554	0.0971
	n	5	5	5	5	5	5
G4R, F & 1000	Mean	0.0752	0.3382	0.6032	0.1345	0.7865	1.1358
	±SD	0.0115	0.0472	0.0304	0.0170	0.1660	0.1844
	n	5	5	5	5	5	5

F: Female; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

 

Table 23: Summary of absolute organ weight records (g), continued

Group, Sex & Dose (mg of active ingredient SVS/ kg body weight/day)		Kidneys	Brain	Liver	Lungs	Thyroid along with parathyroid
G1R, F & 0	Mean	1.8781	2.0946	8.6061	1.9087	0.0208
	±SD	0.1244	0.0428	0.6344	0.2390	0.0037
	n	5	5	5	5	5
G4R, F & 1000	Mean	1.8695	1.9591	9.6881	2.0766	0.0204
	±SD	0.1466	0.1421	1.7576	0.2280	0.0012
	n	5	5	5	5	5

F: Female; R: Recovery; ±SD: Standard Deviation; n: Number of Animals.

 

Table 24: Summary of serum T4 hormone levels (ng/mL) for adults

Group, Sex & Dose (mg of active ingredient SVS/ kg body weight/day)		Serum T4 Levels (ng/mL)
G1, M & 0	Mean	92.058
	±SD	12.208
	n	12
G2, M & 100	Mean	77.190*
	±SD	12.805
	n	12
G3, M & 300	Mean	88.138
	±SD	13.776
	n	12
G4, M & 1000	Mean	88.694
	±SD	15.259
	n	12

M: Male; ±SD: Standard Deviation; n: Number of Animals.

* Statistically significant (P<0.05) change than the vehicle control group

 

Table 25: Summary records of pup observations during postnatal period

Group, Sex & Dose (mg of active ingredient SVS/ kg body weight/day)		At Birth (PND 1)	PND 1 to 4	Pups Sacrificed for Blood Collection on PND 4 (No.) *	PND 5 to 7^$	PND 8 to 13^$
G1, F & 0	No. of Dams^#	10	10	6	10	10
	No. of Live Pups	123	123	13	110	110
	Pup Observation/ No. of Pups observed	N/123	N/123	-	N/110	N/110
G2, F & 100	No. of Dams^#	10	10	5	10	10
	No. of Live Pups	130	126	10	116	110
	Pup Observation/ No. of Pups observed	N/130	N/126, D/4	-	N/116	N/110
G3, F & 300	No. of Dams^#	10	10	5	10	10
	No. of Live Pups	115	115	10	105	105
	Pup Observation/ No. of Pups observed	N/115	N/115	-	N/105	N/105
G4, F & 1000	No. of Dams^#	10	10	7	10	10
	No. of Live Pups	119	118	13	105	105
	Pup Observation/ No. of Pups observed	N/119	N/118, D/1	-	N/105	N/105

F: Female; N: Normal; PND: Postnatal Day; #: Dams confirmed with littering.

*: Pups selected for blood collection from dams with litter size of more than 10; $: Pups sacrificed on PND 4 were excluded.

 

Table 26: Summary records of mean pup weight (g) per litter

Group & Dose (mg of active ingredient SVS/ kg body weight/day)		PND 1		PND 4		PND 7		PND 13
		Mean Male Pup Weight (g)	Mean Female Pup Weight (g)	Mean Male Pup Weight (g)	Mean Female Pup Weight (g)	Mean Male Pup Weight (g)	Mean Female Pup Weight (g)	Mean Male Pup Weight (g)	Mean Female Pup Weight (g)
G1 & 0	Mean	7.03	6.70	9.78	9.41	14.45	13.62	24.07	22.36
	±SD	0.79	0.78	1.34	1.40	2.55	2.64	3.47	3.51
	n	10	10	10	10	10	10	10	10
G2 & 100	Mean	6.67	6.40	9.71	9.40	13.94	13.60	23.85	22.81
	±SD	0.82	0.89	1.55	1.53	2.19	2.05	3.33	3.19
	n	10	10	10	10	10	10	10	10
G3 & 300	Mean	7.17	6.85	10.53	10.09	14.88	14.38	24.62	23.80
	±SD	0.79	0.70	1.73	1.53	1.94	2.20	3.37	3.58
	n	10	10	10	10	10	10	10	10
G4 & 1000	Mean	7.30	6.91	10.62	10.02	14.86	14.25	23.41	22.46
	±SD	0.77	0.81	2.25	1.92	2.88	2.59	3.85	3.72
	n	9	10	9	9	9	9	9	9

±SD: Standard Deviation; n: Number of Litters (number of pups); PND: Postnatal Day.

 

Table 27: Summary record of mean pup anogenital distance (AGD) measurement (mm) and anogenital distance (AGD) ratio per litter on postnatal day 4

Group & Dose (mg of active ingredient SVS/kg body weight/day)		AGD Measurement (mm)		AGD Ratio
		Male	Female	Male	Female
G1 & 0	Mean	4.63	2.53	2.18	1.21
	±SD	0.39	0.20	0.21	0.13
	n	10	10	10	10
G2 & 100	Mean	4.53	2.54	2.14	1.21
	±SD	0.34	0.19	0.20	0.11
	n	10	10	10	10
G3 & 300	Mean	4.51	2.48	2.10	1.13
	±SD	0.21	0.13	0.19	0.11
	n	10	10	10	10
G4 & 1000	Mean	4.47	2.55	2.05	1.19
	±SD	0.24	0.21	0.14	0.10
	n	9	9	9	9

±SD: Standard Deviation; n: Number of Litters (number of pups); AGD: Anogenital Distance.

 

Table 28: Summary records of observation of male pups for retention of nipple/areolae (No.)

Group & Dose (mg of active ingredient SVS/kg body weight/day)		Mean No. of Pups with Retention of Nipples/ Areolae on Postnatal Day 13
G1 & 0	Mean	0.00
	±SD	0.00
	n	10 (70)
G2 & 100	Mean	0.00
	±SD	0.00
	n	10 (75)
G3 & 300	Mean	0.00
	±SD	0.00
	n	11 (61)
G4 & 1000	Mean	0.00
	±SD	0.00
	n	9 (70)

±SD: Standard Deviation; n: Number of Litters (number of male pups).

 

Table 29: Summary records of pup serum T4 hormone levels (ng/mL) on postnatal day 13 (PND 13)  

Group & Dose (mg of active ingredient SVS/kg body weight/day)		Serum T4 Levels (ng/mL)
G1, F & 0	Mean	85.454
	±SD	7.831
	n	10
G2, F & 100	Mean	85.604
	±SD	7.473
	n	10
G3, F & 300	Mean	83.803
	±SD	3.817
	n	10
G4, F & 1000	Mean	78.111
	±SD	8.099
	n	9

±SD: Standard Deviation; n: Number of Litters (number of pups); PND: Postnatal Day

Conclusions:

In conclusion, the oral administration of test item Vinalyst 4330 over the time of the study, carried out according to the OECD guideline 422 (for main group males with a total of 29 days, for main group females ranging from 48 to 57days and for recovery group animals with a total period of 49 days) did not produce any indication of systemic, reproduction and developmental toxicity at the dose levels of 100, 300 and 1000 mg of active ingredient SVS/kg body weight/day under experimental conditions employed. Therefore, the no-observed-adverse-effect-level (NOAEL) of test item Vinalyst 4330 is considered as 1000 mg/kg body weight for systemic, reproduction and developmental toxicity end points.

Executive summary:

The objective of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test of Vinalyst 4330 by Oral Gavage in Sprague Dawley Rats was to determine the possible health hazards likely to arise from repeated exposure to the test item over a relatively limited period of time. This study was also conducted to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition and to estimate the No Observed Adverse Effect Level (NOAEL).

A total of 116 (58 males + 58 females) Sprague Dawley rats were selected for the study and distributed to four main (G1, G2, G3 and G4) and two recovery groups (G1R and G4R). Each main group consisted of 12 males and 12 females and each recovery group consisted of 5 males and 5 females. The animals in group G1/G1R were administered with vehicle [distilled water], animals in groups G2, G3 and G4/G4R were administered with test item at the dose levels of 100, 300 and 1000 mg of active ingredient SVS/kg body weight/day for low, mid and high/high dose recovery, respectively. Vehicle and test item formulations were administered orally by gavage with the dose volume of 10 mL/kg body weight/day once daily at similar times on each day for 7 days a week.

All the main group males were administered with test item during pre-mating (14-days), during mating and during post-mating period (total of 29 days). The pregnant females from main groups were administered with test item during pre-mating (14-days), during mating, pregnancy (gestation) and up to lactation day 13. The non-pregnant females were administered with test item during pre-mating (14-days), during mating until confirmed as mated and further 25 days from the day of confirmation of mating. All the recovery group animals were administered with test item for 49 days followed by a 14-day observation period without treatment.

The test item formulations were stable up to 48 hours at room temperature in distilled water with the concentrations of 0.5 mg/mL and 110 mg/mL. The dose concentration analysis for prepared test item formulations during treatment period was performed during week 1 and 4. The obtained mean results were within the range of 90 to 110% of nominal concentration and the relative standard deviation (% RSD) was less than 10%.

For systemic toxicity assessment, all the animals (main and recovery groups) of both sexes were observed once daily for clinical signs, twice daily for mortality and morbidity and once in a week for detailed clinical examination. Body weight of all the animals (main and recovery groups) of both sexes were recorded once in a week until termination. Further, the body weight of all pregnant females was recorded on gestation day (GD) 0, 7, 14 and 20 during pregnancy and on lactation day (LD) 1, 4, 7, 13 and 14 during lactation. Body weight of all non-pregnant females was recorded once in a week until termination. Feed consumption for all the animals (main and recovery groups) of both sexes were recorded once in a week during pre-mating. Further, feed consumption of all pregnant females was recorded during GD 0 to 7, 7 to 14 and 14 to 20 and during LD 1 to 4, 4 to 7 and 7 to 13. Ophthalmological examination was carried out once before treatment and at the end of the dosing period for all males and females (main and recovery groups). Neurological/functional observations were performed for five randomly selected males and five randomly selected females from each main group towards end of the dosing period, for all animals from recovery groups towards end of the recovery period. The investigations of haematology and clinical chemistry for five randomly selected males and five randomly selected females from main group and all animals from recovery groups was conducted at termination. The investigations of urinalysis for five randomly selected males from each main group and for all recovery groups animals was conducted at termination. Serum thyroxine hormone (T4) levels were estimated for all main group males by ELISA method. The organs were collected and weighed on the day of termination for all animals (main and recovery groups) of both sexes and organ weight relative to terminal body weight was calculated. All the animals (main and recovery groups) of both sexes were observed for both external and internal gross pathological changes during conduct of necropsy. Histopathological examination was conducted on all the tissues collected from the groups G1 and G4 animals with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

For reproductive toxicity assessment, all the males (main groups) were evaluated for reproductive performances such as, mating and fertility indices. All the females (main groups) were evaluated for reproductive performances such as, mating, fertility, gestation and parturition indices. The females were also evaluated for pre-coital interval and gestation length. All females were evaluated for oestrus cyclicity during pre-mating period and the vaginal smear examination was continued until evidence of mating. At birth (delivery) parameters such as, number of live/dead pups born, litter size, sex ratio (m/f) and live birth index per litter were observed / calculated. The litter observations during lactation period such as, number of survived or dead pups, sex ratio (m/f) and pup survival index per litter were observed / calculated. The total number of implantation sites was recorded for each litter during necropsy and the post-implantation and postnatal losses per litter was calculated.

For developmental toxicity assessment, all survived pups from each litter were observed once daily for external behavioural changes and twice daily for mortality until termination [postnatal day (PND) 13]. All pups from each litter were weighed individually on PND 1, 4, 7 and 13 and measured for anogenital distance on PND 4. All male pups were observed for retention of nipples on PND 13. All the pups were subjected for both external and internal gross pathological changes during conduct of necropsy. The collected serum from PND 13 pups was subjected to estimation of thyroxine hormone (T4) levels using ELISA method.

There were no clinical signs of toxicity and no mortality/morbidity noted in any of the animals from all the tested dose groups. Detailed clinical examination did not reveal any changes in any of the tested dose group animals. No changes were noted in mean body weight, percent change in mean body weight gain and mean feed consumption in all the tested dose groups of both sexes. The ophthalmological examination and neurological/functional examinations did not reveal any test item-related changes in any of the animals from all the tested dose groups of both sexes. The obtained mean haematology, clinical chemistry and urinalysis values did not reveal any test item-related changes in all the tested dose groups. The estimated serum T4 levels did not reveal any test item related changes. The absolute and relative organ weights of both sexes did not reveal any test item related changes from all the tested dose groups. No gross pathological changes noted in any of the animals from all the tested dose groups during necropsy. No treatment related microscopic findings noted in any of the tissues/organs subjected for histopathological evaluation from high dose group animals of both sexes.

There were no effects noted on reproductive performance of both sexes in all the tested dose groups. No test item-related irregularities observed in oestrus cyclicity of the females from all the tested dose groups during treatment period. No changes were noted in ‘at birth’ (delivery) and or ‘litter observations’ in all the tested dose groups. No effects were noted for live birth index and pup survival index in all the tested dose groups. No test item-related post-implantation losses were noted in all the tested dose groups.

There were no test item-related developmental or external behavioural changes and no test item-related mortalities noted during postnatal period in any of pups from all the tested dose group litters. The mean pup weight, mean anogenital distance and its ratio in either sex of pups per litter were unaffected by the test item in all the tested dose groups. There were no incidences of retention of nipples in male pups examined on PND 13 from all the tested dose and control group litters. The estimated serum T4 levels of PND 13 (from all litters) pups did not reveal any changes in all the tested dose group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all the tested dose and control group litters.

In conclusion, the oral administration of test item Vinalyst 4330 over the time of the study, carried out according to the OECD guideline 422 (for main group males with a total of 29 days, for main group females ranging from 48 to 57 days and for recovery group animals with a total period of 49 days) did not produce any indication of systemic, reproduction and developmental toxicity at the dose levels of 100, 300 and 1000 mg of active ingredient SVS /kg body weight/day under experimental conditions employed. Therefore, the no-observed-adverse-effect-level (NOAEL) of test item Vinalyst 4330 is considered as 1000 mg/kg body weight for systemic, reproduction and developmental toxicity end points.

Reference 2

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

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 or test system 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 - 407 g; females: 241 - 287 g
- Housing: In standard polypropylene cages with stainless steel 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 ethylene sulphonate in the vehicle was determined.

VEHICLE:
- Sodium ethylene sulphonate 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 ethylene sulfonate 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 etyhlene sulphonate.
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 occurred.

Observations and examinations performed and frequency:

OBSERVATION PERIOD
Males were observed for 28 days and females were observed during pre-mating (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 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.

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 ethylene sulphonate (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 ethylene sulphonate, 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 ethylene sulphonate was greater than 500 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

two valid OECD TG 422 studies, one dosed up to 500 mg SVS/kg bw/d and one up to 1000 mg SVC/kg bw/d

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) of a similar multi-constituent substance solution containing 25.4 % sodium ethylene sulphonate 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 ethylene sulphonate, 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 behaviour was observed in offspring. 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 offspring, 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 ethylene sulphonate, the obtained NOAEL was greater than 2000 mg/kg bw/day for the tested solution. The derived repeated dose NOAEL for 100% sodium ethylene sulphonate was greater than 500 mg/kg bw/day. No specific target organ was identified.

As this study was not dosed up to 1000 mg/kg bw/d active substance (sodium vinyl sulphonate SVS), ECHA requested a new OECD 422 study to be performed, dosed up to 1000 mg/kg bw/d active substance. This study was performed and is reported here too. Likewise, in this new study, following the same protocol as the one described above, but dosed up to 1000 mg SVS/kg bw/d no effects were noted for repeat dose toxicity or reproductive parameters, confirming the findings of the study dosed up to 500 mg SVS/kg bw/d. However, in this study no increase on auditory function (acoustic startle) was noted. Thus, the NOAEL for repeat dose toxicity, fertility and developmental toxicity in this study was set to the highest dose tested, i.e. 1000 mg/kg bw/d.

 

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

Remark: A subchronic oral toxicity study is in preparation and once result become available, the endpoint summary here will be re-visited.

Justification for classification or non-classification

Based on the results of two repeated dose toxicity studies, 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).