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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 July 2020 - 01 March 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No. 2019/1390, Annex. B.64 “Combined Repeated Dose Toxicity Study with The Reproduction/Developmental Toxicity Screening Test”
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
5H-1,2λ⁶-oxathiole-2,2-dione
EC Number:
606-834-7
Cas Number:
21806-61-1
Molecular formula:
C3H4O3S
IUPAC Name:
5H-1,2λ⁶-oxathiole-2,2-dione
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:Shijiazhuang SuntecChem Co., Ltd;Batch NO. 200301
- Expiration date of the lot/batch: 4 March 2021
- Purity: :99.90%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29℃)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals in Bioneeds India Private Limited Devarahosahally
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: 8 - 9 weeks old, females: 8 - 9 weeks old.
- Weight at study initiation: males: 210.86 - 250.42 g; females: 200.79 - 217.64 g
- Housing:Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
During acclimatization, maximum of two animals of same sex were housed.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes ad libitum throughout the experimental period.
- Acclimation period:Healthy and young adult animals were acclimatized for five days to experimental room conditions initially.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19.8 to 22.9℃
- Humidity (%):43 to 65%
- Air changes (per hr):12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours fluorescent light and 12 hours dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% w/v
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared before dose administration on each treatment day.The required quantity of test item was weighed and grinded well in a mortar with a small quantity of vehicle until a homogenous suspension was formed and thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated many times to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 2.25, 4.5 and 9 mg/mL of test item for low, mid and high dose groups respectively.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was insoluble in water at 10 mg/mL but formed a homogeneous suspension in 0.5% w/v Carboxy Methyl Cellulose at 10 mg/mL as per in-house solubility/suspension test results.
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot/batch no. (if required): BCBN1690V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The prepared test item formulations of 1,3-Propene Sultone were stable in 0.5% w/v Carboxy Methyl Cellulose for 6 hours at room temperature and 48 hoursat 2 to 8oC with the concentrations of 1.0 mg/mL and 20 mg/mL as established by Analytical Department of Bioneeds India Private Limited (Bioneeds study no.: BIO-ANM 1645).

Homogeneity and 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 BIO-ANM 1645 and the results were presented in the Appendix 31. Sampling and analysis of formulations was performed during week 1 and week 4 of the treatment. The samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. Exact volume of test item formulation samples was included in the study report.
The prepared test item formulations were stirred using magnetic stirrer during sampling.

The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation was analyzed. The second aliquot was stored as a backup purpose at established stability conditions and were discarded as the analysis results of first set of samples were within the limits.

Formulations are considered acceptable, if mean results are within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.
Duration of treatment / exposure:
48-71 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1/Control
Dose / conc.:
22.5 mg/kg bw/day (nominal)
Remarks:
G2 (low dose)
Dose / conc.:
45 mg/kg bw/day (nominal)
Remarks:
G3 (mid-dose)
Dose / conc.:
90 mg/kg bw/day (nominal)
Remarks:
G4 (high dose)
No. of animals per sex per dose:
Main group: 24 (12 Males + 12 Females per dose)
Recovery Group: 10 (5 Males + 5 Females for G1 and G4 only)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Refer to ‘Supporting, RL1, rat/Suntec, 2020/Repeated dose toxicity: oral (DRF).001’ study record

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All the animals were observed once daily for general clinical signs of toxicity and twice daily for mortality and morbidity. All the 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 preferably at the same time. Signs noted included, but not limited to, changes in skin, fur, eyes, mucuous membranes, occurrence of secretions, and excretions and autonomic activity such as lacrimation, piloerection, pupil size and usual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group animals were weighed at receipt, on the day of randomization, on the first day of dosing, once weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during the lactation period. The recovery group animals were weighed at receipt, on the day of randomization, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; Cage wise feed consumption was measured for main group animals once a week during premating and once a week for main group males during the post mating period. Feed consumption was not measured during the mating period for main group males and females.
Feed consumption for females was also recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13.
Feed consumption was measured for recovery group animals once a week throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount of feed offered and left over in each cage and the number of rats per cage.

OPHTHALMOLOGY: yes; Ophthalmological examination was carried out once before treatment for all animals, at the end of the dosing period for males (shortly prior to scheduled sacrifice i.e. on day 45) and during the lactation period for females (shortly prior to scheduled sacrifice, i.e. on lactation day 13) of all the main group animals and during the last week for the recovery group animals (on day 64).

HAEMATOLOGY: Yes; haematological parameters were examined in 5 randomly selected animals from each main group per sex and for all animals of both sexes from recovery groups at termination. One day before scheduled terminal sacrifice, the animals were fasted overnight. Water was provided ad libitum during the fasting period. Blood from the abdominal aorta of the animals was collected in K3 EDTA-coated tubes. Sodium citrate (3.2%) tubes were 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. Hematology parameters were estimated using the Advia 2120 Hematology system (Siemens Limited). BlooD Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by coagulation analyzer. The haematological parameters examined are in Table a.

CLINICAL CHEMISTRY: Yes; parameters of clinical biochemistry from 5 randomly selected animals from each main group per sex and for all animals of both sexes from recovery groups at termination. 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 sodium heparin (10 IU/mL of blood) for clinical chemistry analysis. Blood samples were collected using the retro-orbital plexus puncture method under Isoflurane Anaesthesia with the help of a fine capillary tube. Clinical chemistry parameters were analyzed using Rx Daytona+ clinical chemistry analyzer (Randox Laboratories). Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using a Na/K/Cl analyzer. The parameters of clinical biochemistry examined are in Table b.

URINALYSIS: Yes; 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 parameters (Table c) were measured using qualitative indicators DIRUI H-500 (Dirui Industrial Company Ltd.).

NEUROBEHAVIOURAL EXAMINATION: Yes
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 day 45) 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 64).

The following Neurological/Functional observations were performed:
a) Home Cage Observations
b) Handling Observations
c) Open Field Observations
d) Sensory Observations
e) Neuromuscular Observations
f) Physiological Observation (Rectal temperature):
g) Grip strength assessment
h) Motor activity assessment

THYROID HORMONES: Yes; Blood samples were collected from all the main group animals for measurement of serum T4 levels on the following schedule: All adult males, at termination (after completion of 46 days of treatment). Blood samples of adult animals was collected using the retro-orbital plexus puncture method under Isoflurane anesthesia with the help of a fine capillary tube. The serum was stored at -80°C for estimation of serum levels of thyroid hormones (T4) by ELISA method using commercial assay kits. The assessment of serum T4 levels was not performed for recovery group animals.
Sacrifice and pathology:
GROSS NECROPSY
The males were sacrificed after completion of 46 days of treatment, females were sacrificed on lactation day 14 and recovery animals were sacrificed after completion of 14 days observation from the first scheduled sacrifice of dams. The animals were fasted overnight, water was provided ad libitum during fasting. The next day, the body weight of all the fasted animals was recorded prior to necropsy. The animals were euthanized using CO2 followed by exsanguination and subjected to necropsy and gross pathological examination.


HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs in Table d and e, as applicable from all animals were collected, weighed and preserved. 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 testis, epididymis and eyes. Eyes with optic nerve were preserved in modified Davidson’s fixative for 24 to 48 hrs and then transferred to 50% isopropyl alcohol. The thyroid along with the parathyroid from all the adults were weighed post fixation.

All organs and tissue samples were processed, embedded in paraffin, sectioned at a thickness of 4 to 6 micrometers and stained with hematoxylin and eosin. Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals. The investigations were not extended to the lower dose groups and recovery groups as there were no treatment related histopathological effects noted at the high dose level (target organs).

Bone marrow smear (from one femur) was prepared at the time of necropsy. As there were no changes in haematology endpoints and no changes noted in histological evaluation of organs such as thymus, spleen and lymph node, the cytologic evaluation of the bone marrow was not conducted. Refer to tables d and e.

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 22.
All analysis and comparisons were evaluated at 95%, 99% and 99.9% with the level of confidence of P<0.05, P<0.01 and P<0.001 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.
** Statistically significant (P<0.01) change than the control group.
*** Statistically significant (P<0.001) change than the control group.

Note: Data of non-pregnant females, females mated but not littered and lactation data of females with total litter loss was excluded from statistical analysis.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs of toxicity noted in any of the animals of both sexes from the tested dose groups G2 (22.5 mg/kg bw/day) and G3 (45 mg/kg bw/day) throughout the experimental period. The detailed clinical examination of animals did not reveal any changes at the tested dose groups G2 and G3. In group G4/G4R (90 mg/kg bw/day), all animals of both sexes did not reveal any clinical signs until day 6 of the treatment period; the following test item-related clinical signs of toxicity were noted from day 7 onwards during daily clinical signs observations and weekly detailed clinical examinations.
-Group G4 males: lethargy, perinasal staining, rough hair coat and hair thinning.
-Group G4 females: lethargy, ataxia, perinasal staining, rough hair coat, hair thinning and vaginitis (one female only).
-Group G4R males and females: lethargy, ataxia, perinasal staining, rough hair coat and hair thinning. These observations were continued until termination for the main groups. The recovery group animals did not display clinical signs of toxicity during the recovery period. Refer: Table 1 (Summary Data)
Mortality:
no mortality observed
Description (incidence):
There were no mortalities at any dose during the study.
Body weight and weight changes:
effects observed, 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 the tested dose groups G2 and G3 in both sexes throughout the experimental period. The noted statistically significant decrease in percent change in mean body weight gain during day 1 to 14 at group G3 males when compared with vehicle control group is considered as incidental and un-relthe ated to treatment due to lack of effects on other systemic toxicity end points at this dose level.

In groups G4/G4R males and females, the following statistically significant changes were noted when compared with vehicle control groups.
-decrease in mean body weight on day 7 (p<0.05) and on days 14, 21, 28, 35 and 42 (p<0.001) in group G4 males (below the in-house historical control range on days 35 and 42);
-decrease in percent change in mean body weight gain (p<0.001) during day 1 to 7, 1 to 14, 1 to 21, 1 to 28, 1 to 35 and 1 to 42 in group G4 males (below the in-house historical control range during day 1 to 42);
-decrease in mean body weight on day 14 (p<0.01) in group G4 females (within the in-house historical control range);
-decrease in percent change in mean body weight gain (p<0.001) during day 1 to 7 and 1 to 14 in group G4 females (below the in-house historical control range on both time points);
-decrease in mean body weight on day 14 (p<0.05), 21 (p<0.05), 28 (p<0.05),
35 (p<0.01), 42 (p<0.01), 49 (p<0.01), 56 (p<0.01), 63 (p<0.01) in group G4R males (below the in-house historical control range on days 42, 49, 56 and 63);
-decrease in percent change in mean body weight gain during day 1 to 7 (p p<0.001), 1 to 14 (p<0.01), 1 to 21 (p<0.01), 1 to 28 (p<0.01), 1 to 35 (p<0.01), 1 to 42 (p<0.001), 1 to 49 (p<0.001), 1 to 56 (p<0.001) and 1 to 63 (p<0.01) in group G4R males (below the in-house historical control range during day 1 to 14, 1 to 21, 1 to 28, 1 to 35, 1 to 42, 1 to 49 and 1 to 56);
-decrease in mean body weight on day 7 (p<0.05), 14 (p<0.05), 21 (p<0.01), 28 (p<0.01), 35 (p<0.001), 42 (p<0.001), 49 (p<0.001), 56 (p<0.001) & 63 (p<0.001) in group G4R females (below the in-house historical control range on days 28, 35, 42, 49, 56 and 63);
-decrease in percent change in mean body weight gain during 1 to 7 (p<0.05), 1 to 14 (p<0.05), 1 to 21 (p<0.001), 1 to 28 (p<0.001), 1 to 35 (p<0.01), 1 to 42 (p<0.001), 1 to 49 (p<0.001), 1 to 56 (p<0.001) and 1 to 63 (p<0.001) in group G4R females (below the in-house historical control range during day 1 to 21, 1 to 28, 1 to 35, 1 to 42 and 1 to 49);

These noted changes in G4/G4R groups are considered as test item-related due to noted treatment related clinical signs of toxicity and reduced feed consumption at this dose level. However, a recovery in both mean body weight and percent change in mean body weight was noted in G4R animals of both sexes during the recovery period, i.e. on days 56 and 63. These effects are also correlated with in-house historical control data, as the obtained values from this dose level are below the in-house historical control range. Refer: Table 2 & 3 (Summary Data)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were no changes noted in mean feed consumption in the tested dose groups G2 and G3 in both sexes during pre-mating and post-mating periods when compared with the vehicle control group. The feed consumption was not measured during the mating period for main groups and measured once a week throughout the experimental period for recovery groups.
In groups G4/G4R males and females, the following statistically significant changes in mean feed consumption were noted when compared with vehicle control groups;

-decrease during week 1 and 2 (p<0.001) of pre-mating period and week 6 (p<0.001) of post-mating period in group G4 males (within the in-house historical control range);
-decrease during week 1 and 2 (p<0.001) of pre-mating period in group G4 females (within the in-house historical control range);
-decrease during week 1, 4 & 5 (p<0.05), 6 (p<0.01) and 9 (p<0.05) in group G4R males (within the in-house historical control range);
-decrease during week 1 (p<0.05), 2 & 3 (p<0.01), 4 & 5 (p<0.05),
6 (p<0.01), 7, 8 & 9 (p<0.05) in group G4R females (within the in-house historical control range);

However, a slight improvement in mean feed consumption was noted during the recovery period at groups G4R males and females.

These changes are considered as test item-related due to noted treatment-related clinical signs of toxicity, reduced mean body weight and reduced percent change in mean body weight gain at this dose level. The obtained values are within in-house historical range in both sexes, but the percent reduction was approximately 20% for both males and females when compared with vehicle control group. Refer: Table 4 (Summary Data)

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 and vehicle control groups of both main and recovery groups in both sexes during ophthalmological examination.
Refer: Table 5 (Summary Data)
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The haematological results noted with following statistically significant differences when compared with vehicle control groups:

-increase in mean total leucocyte count (p<0.05) and mean absolute lymphocytes (p<0.001) in group G3 males (within historical control range).
-decrease in mean total erythrocyte count (p<0.01), haemoglobin (p<0.05), mean corpuscular haemoglobin concentration (p<0.05), percent neutrophils (p<0.05), prothrombin time (p<0.01) in group G4 males (within historical control range).
-increase in mean corpuscular volume (p<0.001), absolute and percent reticulocyte count (p<0.01), absolute and percent lymphocytes (p<0.05) in group G4 males (within historical control range, except for absolute reticulocyte count);
-increase in mean corpuscular volume (p<0.01), mean corpuscular haemoglobin (p<0.05), percent neutrophils (p<0.05) in group G4R males (within historical control range);
-decrease in mean value of mean corpuscular haemoglobin concentration (p<0.05), percent lymphocytes (p<0.05) in group G4R males (within historical control range);
-decrease in mean value of mean corpuscular haemoglobin concentration (p<0.05) in group G4R females (within historical control range);

The above mentioned statistically significant changes can be considered as test item-related, but not adverse in group G4, as these effects are within in-house historical control data range, except for absolute reticulocytes and show a trend towards recovery during the recovery period in group G4R animals. There were no other adverse test item-related changes noted in mean haematology parameters at all the tested main and recovery groups of both sexes when compared with vehicle control group. Refer: Table 10 (Summary Data)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical chemistry results noted with following statistically significant changes in all the tested dose groups when compared with vehicle control groups:

-increase in mean triglycerides, calcium and potassium in group G3 males (within historical control range);
-increase in albumin, calcium, phosphorus and albumin globulin ratio in group G4 males (within historical control range);
-decrease in creatinine and globulin in group G4 males (within historical control range);
-increase in creatinine and decrease in total bilirubin in group G2 females (within historical control range);
-increase in triglycerides, calcium and decrease in alkaline phosphatase, chloride in group G4R males (within historical control range);
-increase in phosphorus and decrease in albumin in group G4R females (within historical control range).

The above mentioned statistically significant changes are considered as incidental and un-related to treatment as the changes did not occur in a dose dependant manner and also the mean values are within in-house historical control range of same species and strain. There were no other test item-related changes noted inmean clinical chemistry parameters at all the tested dose main and recovery groups of both sexes when compared with vehicle control group. Refer: Table 11 (Summary Data
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no changes noted in mean urinalysis parameters in all the tested main group males and recovery groups of both sexes when compared with the vehicle control groups. Refer: Table 12 (Summary Data)
Behaviour (functional findings):
effects observed, 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 the tested dose groups G2 and G3 performed towards end of the dosing period for main groups (on day 45 for males and on lactation day 13 for females/dams) and performed towards end of the recovery period for recovery groups (day 64).
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 at the tested dose groups G2 and G3 of both sexes when compared with vehicle control groups during conduct of neurological/functional examinations.
In group G4 males, a statistically significant increase in mean number of rearing, a statistically significant decrease in mean number of urine pools and defecations, a statistically significant decrease in movement counts during motor activity assessment and a statistically significant decrease in mean forelimb and hind limb grip strength were noted when compared with the vehicle control group.
In group G4 females, a statistically significant decrease in movement counts during motor activity assessment, a statistically significant decrease in mean forelimb and hind limb grip strength and a statistically significant increase in mean hind limb foot splay were noted when compared with the vehicle control group.
However, the group G4R males and females did not reveal any neurological/functional changes during the recovery period.
The noted changes in group G4 males and females are considered as secondary test item-related effects noted due to clinical signs, body weight reduction etc. The recovery group animals of the same dose level did not reveal any such changes.
Refer: Table 6, 7, 8 & 9 (Summary Data)
Immunological findings:
not examined
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 at the tested dose groups G2 and G3 of both sexes when compared with vehicle control group.

The following statistically significant changes were noted from all the tested dose group males and females when compared with vehicle control groups.
-decrease in mean absolute weight of adrenals in group G2 males;
-decrease in mean absolute weight of adrenals, epididymides and thyroid in group G4 males;
-decrease in mean absolute weight of adrenals, liver in groups G3 females;
-decrease in mean absolute weight of adrenals, heart and liver in group G4 females;
-increase in mean absolute weight of testes and liver in group G4R males;
-increase in mean relative weight of spleen, testes, kidneys, brain, liver and prostate in group G4 males;
-decrease in mean relative weight of adrenals in group G3 females;
-increase in mean relative weight of brain, pituitary and thyroid in group G4 females;
-increase in mean relative weight of adrenals, testes, epididymides, kidneys and liver in group G4R males;
-increase in mean relative weight of adrenals, thymus, spleen, heart, kidneys, brain, liver, lungs, pituitary and thyroid in group G4R females;

These changes are not considered adverse, 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, compared to vehicle controls. Also, the increased relative organ weights in group G4R males and females are due to test item related reduction in the terminal body weights. However, the obtained values are within in-house historical control range. Refer: Table 27 & 28 (Summary Data)

Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed during necropsy in all of the adult animals. Refer: Appendix 30 for Pathology Phase Report
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related histopathological findings in high dose animals of both sexes. The observed microscopic findings observed in this study, such as cyst(s) in the thymus, ultim obranchial cyst(s) in the thyroid gland 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. The histopathological examination of thyroid collected from the adults other than randomly selected animals was not conducted as there were no gross pathological changes noted and also no test item related changes were noted in absolute and or relative thyroid weights at any of the tested dose group. The microscopic investigations of other organs were not extended to the lower dose groups and recovery groups as there were no treatment-related histopathological effects noted at the high dose level (target organs). Refer: Appendix 30 for Pathology Phase Report.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no test item-related changes noted in serum T4 levels in any of the tested main and recovery groups from both sexes when compared with vehicle control groups. The statistically significant increase in mean serum T4 levels in group G4 males are considered as incidental and un-related to treatment as the changes are within the in-house historical control range of same species and strain. All the obtained values are within in-house historical control range. Refer: Tables 29 (Summary Data)

Effect levels

Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
haematology

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Homogeneity and dose formulation analysis for dose concentration verification was performed during weeks 1 and 4 of the treatment. The formulations were considered acceptable, since the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is less than 10%.

Applicant's summary and conclusion

Conclusions:
Based on the findings of this combined repeated dose and reproduction/developmental toxicity screening test in Sprague Dawley rats, the NOAEL (male/female) of 1,3-Propenesultone for general toxicity is considered to be 45 mg/kg bw/day.
Executive summary:

In a combined repeated dose and reproduction/developmental toxicity screening test (OECD 422/GLP), 1,3-Propenesultone (99.90%) in 0.5% w/v Carboxy Methyl Cellulose was administered to 4 main groups ((G1, G2, G3 and G4; 12/sex/group) and 2 recovery groups (G1R, G4R; 5/sex/group) of Sprague Dawley rats by oral gavage. The animals in the G1/G1R, G2, G3 and G4/G4R groups were administered the test item at the dose levels of 0, 22.5, 45 and 90 mg/kg bw/day, respectively, 7 days per week. The main group males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 46 days of treatment). The pregnant females from the main group were treated for a two-week pre-mating period, during cohabitation until mated, pregnancy (gestation) and up to lactation day 13. The non-pregnant females were treated for two-week pre-mating period, during cohabitation until mated and 24 days further from the day of confirmed mating. The females which were cohabitated with no evidence of mating were treated for a two-week pre-mating period, three-weeks cohabitation period and 24 days further from the day of termination of cohabitation process. The recovery group animals of both sexes were treated until the first scheduled female sacrifice (total of 49 days) and kept without treatment for a further 14-days observation.

Homogeneity and dose formulation analysis for dose concentration verification was performed during weeks 1 and 4 of the treatment. The formulations were considered acceptable, since the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was less than 10%.

There were no mortalities at any dose during the study. In groups G2 and G3, there were no indication of test item-related effects in any of the parameter/endpoints assessed for systemic toxicity. In groups G4/G4R, the animals were noted with test item-related clinical signs of toxicity such as, lethargy, perinasal staining, ataxia, rough hair coat and hair thinning and vaginitis (in one female). The animals from these dose groups were noted with test item-related reduction in body weight gain (outside historical control data) and feed consumption in both sexes. However, a recovery in both mean body weight and percent change in mean body weight was noted in G4R animals of both sexes during the recovery period. Also, secondary test item-related effects e.g. decrease in movement counts during motor activity assessment and decrease in mean forelimb and hind limb grip strength assessment in both sexes were noted but were not present in recovery animals. The changes outside historical control data in hematological values (absolute reticulocytes only) showed a trend towards recovery during the recovery period. The clinical chemistry, urinalysis and ophthalmoscopic examinations did not reveal any test item-related changes. There was no test item-related organ weight, gross pathological or histopathological changes noted at this dose level.

Based on the findings of this study the NOAEL (male/female) of 1,3-Propenesultone for general toxicity is considered to be 45 mg/kg bw/day.