5H-1,2-oxathiole 2,2-dioxide

Endpoint summary

Description of key information

Short-term repeated dose toxicity

Subacute NOAEL (male/female, rat): 45 mg/kg bw/day (OECD 422/GLP)

 

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:

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

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

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℃)

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

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

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

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.

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)

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

Critical effects observed:

no

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%.

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.

Reference 2

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Dose Range Finding Study for Combined Repeated Dose Toxicity Study with Reproduction / Developmental Toxicity Screening Test after Oral Administration in Wistar Rats (OECD 422)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 June 2020 - 26 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Modified for 14 day treatment schedule, 3 aninmals per sex per dose and no histopathology

GLP compliance:

no

Remarks:

DRF study; GLP not required

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Shijiazhuang Suntec-Chem Co., Ltd.; D1031-001
- 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 29C)

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
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: 10 weeks old, females: 10 weeks old.
- Weight at study initiation: males: 260.99 -288.19 g; females: 220.78 - 257.53 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.
- 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):19.1 to 22.9℃
- Humidity (%):43 to 64%
- 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

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 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 complete transfer of contents to the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 1, 3 and 10 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 (Sigma Aldrich)

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

A dose formulation analysis was not performed in this study.

Duration of treatment / exposure:

The test item formulations and vehicle were administered once a day for a period of 14 consecutive days.

Frequency of treatment:

7 days per week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1/Vehicle Control

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

G2/low dose

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

G3/medium dose

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G4/high dose

No. of animals per sex per dose:

3 males and 3 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

There was no information or no data available from repeated dose toxicity studies for the test item 1,3-Propene sultone (1-Propene 1,3-Sultone), CAS No. 21806-61-1. However as per this registration dossier, the LD50 of 1,3-Propene sultone in rats is between 50 to 300 mg/kg bw obtained from Acute Oral Toxicity study conducted in accordance with OECD423. Based on the above available information, the doses of 0, 10, 30 and 100 mg/kg bw/day were selected as control, low, mid and high dose groups respectively in consultation with the study monitor.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes. All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity throughout the experimental period.
All the animals were subjected to detailed clinical examinations on day 1 before treatment and weekly thereafter during treatment till termination. 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, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes; Individual animal body weight was recorded on the day of randomization, on day of treatment (Day 1) prior to treatment and weekly thereafter. Fasting body weight of all the animals were recorded at their scheduled terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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 at weekly intervals. Average feed intake per rat (g/rat/day) was calculated using the amount of food given and left over in each cage and the number of rats in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes; Ophthalmological examination was carried out once for all animals during acclimatization (Pre-treatment) before randomization and during week 2 for all the animals.

HAEMATOLOGY: Yes
Blood samples were collected from all the rats on Day 15. The animals were fasted overnight before blood collection. Water was provided ad libitum during fasting period. Blood samples were collected from the animals separately into the tubes containing K2 EDTA (2 mg/mL of blood) for hematology and 3.2% Sodium citrate tubes for Prothrombin time and activated partial thromboplastin time parameters. Blood samples were collected from retro-orbital plexus puncture under mild isoflurane anaesthesia with the help of a fine capillary tube. Hematology parameters were estimated using Advia 2120 Hematology System (Siemens Limited). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by “Opti Clot-4” coagulation analyzer [Tulip Diagnostics (P) Ltd., India]. Parameters in Table 1 were examined.

CLINICAL CHEMISTRY: Yes
Blood samples were collected from all the rats on Day 15. The animals were fasted overnight before blood collection. Water was provided ad libitum during fasting period. Blood samples were collected from the animals separately into the tubes containing sodium heparin (10 IU/mL of blood) for clinical chemistry. Blood samples were collected from retro-orbital plexus puncture under mild isoflurane anaesthesia with the help of a fine capillary tube. Clinical chemistry parameters were analysed using the Rx Daytona+ clinical chemistry analyzer (Randox Laboratories). Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Prolyte Na/K/Cl analyzer (Diamond Diagnostics). Parameters in Table 2 were examined.

URINALYSIS: Yes
Urine was collected and analyzed from all rats on the day of necropsy (day 15). The animals were kept in urine collection cages for overnight on day 14 and were fasted but water was provided ad libitum during this period. The overnight urine volume (mL) collected from these animals was measured. The volume of urine collected (mL), appearance and the color were recorded by physical evaluation. The urine was analyzed using “DIRUI H-500” (Dirui Industrial Compant Ltd.) Parameters in Table 3 were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On day 15, all the animals of Group G1, G2, G3 and G4 were subjected to necropsy and detailed gross pathological examination. The animals were fasted overnight (water was provided ad libitum) prior to scheduled necropsy. On the day of terminal sacrifice, the body weight of all the fasted animals was recorded prior to exsanguination. The animals were euthanized using CO2 anaesthesia followed by exsanguination. The gross pathological observations were recorded for each rat. Gross pathology examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues of each animal with special emphasis on reproductive organs was conducted.

Organ Weights: The following organs from all animals at the scheduled sacrifice were trimmed off any adherent tissue and fat, as appropriate and weighed wet as soon as possible to avoid drying (Adrenals, brain, Epididymis, Heart, Kidneys, Liver, Lungs, Ovaries, Prostate along with seminal vesicles with coagulating gland, Spleen, Testes, Thymus, Uterus with cervix).

Statistics:

The raw data was subjected to statistical analysis. After verification, the data was subjected to statistical analysis using SPSS software, version 22. Body weight, percent change in body weight with respect to day 1, hematological, clinical chemistry estimations, urinalysis parameters, absolute / relative organ weights were subjected to statistical analysis. One-way ANOVA followed by Dunnett’s post test was done for different treatment groups comparing with the control group data. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). Statistically significant changes obtained from the aforementioned tests were designated by the superscripts in the summary table throughout the report as stated below:
*: Statistically significant (P<0.05) change than the vehicle control group

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity noted in any of the animals from groups G1 (0 mg/kg bw/day), G2 (10 mg/kg bw/day) and G3 (30 mg/kg bw/day) in either sex observed throughout the experimental period. The group G4 (100 mg/kg bw/day) animals did not reveal any clinical signs of toxicity for the first two days of the treatment. However, both the males and females from this group started to reveal test item-related clinical signs of toxicity from day 3 and continued until sacrifice. All males were noted with slight perinasal staining from day 3 until sacrifice; one male was noted with lethargy in addition to slight perinasal staining from day 11 until sacrifice and another male was noted with lethargy in addition to slight perinasal staining on the day of sacrifice. All females were noted with slight perinasal staining along with lethargy from day 3 until sacrifice; one female was noted with ataxia, rough hair coat along in addition to lethargy and slight perinasal staining from day 12 until sacrifice.

There were no changes noted in any of the animals of either sex from groups G1, G2 and G3 during weekly detailed clinical examinations conducted on day 7 and 14. However, all the males from group G4 were noted with perinasal staining on day 7. On day 14, all males were noted with perinasal staining and among these, two were noted with lethargy in addition to perinasal staining. All the females from group G4 were noted with lethargy along with perinasal staining on day 7. On day 14, all females were noted with lethargy with perinasal staining with one female also noted with ataxia, rough hair coat in addition to lethargy and perinasal staining. Refer to Table 1.

Mortality:

no mortality observed

Description (incidence):

There was no mortality / morbidity noted in any test dose group or vehicle control group animals of either sex during the treatment period. Refer to Table 1

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in groups G2 and G3 when compared with the vehicle control group in either sex. In group G4, a test item-related reduction in mean body weight and percent change in body weight gain were noted in both sexes during the treatment period. These reductions are statistically significant for day 14 mean body weight of males and for week 1 and 2 percent change in body weight gain of both males (-3.25 and -5.98%) and females (-4.52 and -10.19%), when compared with the vehicle control group. These observed reductions are considered as test item-related due to noted clinical signs of toxicity at this dose level during the treatment period and also evidenced by reduced feed consumption. Refer to Tables 2 & 3.

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 at groups G2 and G3 in either sex when compared with the vehicle control group. However, a test item-related reduction in mean feed consumption was noted in group G4 males and females during the treatment period (weeks 1 and 2). These observed reductions are considered as test item-related due to noted clinical signs of toxicity and also evidenced by reduced mean body weight and reduced mean percent change in body weight gain from this dose level in both sexes during the treatment period. Refer to Table 4.

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 during the opthalmoscopic examination carried out during week 2 (day 14) in all test-item dosed groups and vehicle control group animals in either sex. Refer to Table 5.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes observed in mean haematology values in any of the dose groups in either sex when compared with the vehicle control group.

However, the following statistically significant changes were noted in haematology parameters when compared with the vehicle control group:
-decrease in total leucocyte count, percent eosinophils, absolute lymphocytes, absolute eosinophils and absolute basophils in group G4 males;
-decrease in haemoglobin levels in group G4 females;
-decrease in basophils (absolute and percent) in group G2 males;
-decrease in basophils (absolute and percent) in group G3 males

Refer to Table 6.

These observed changes are considered as incidental, as these changes lackdose dependency and also the obtained values are within the in-house historical control data range of the same species and strain [(Males: Total leucocytes count (103 cells/µL) - ranges from 6.69 to 14.16; Eosinophils (%) - ranges from 0.00 to 3.40%; Absolute lymphocytes (103 cells/µL) - ranges from 4.06 to 11.64; Absolute eosinophils (103 cells/µL) - 0.04 to 0.27; Absolute basophils (103 cells/µL) - 0.01 to 0.08; Basophils (%) – 0.00 to 0.70; Absolute basophils (103 cells/µL) - 0.01 to 0.20]; [Females: Haemoglobin (g/dL) - 11.10 to 16.1)] . The presented historical control data is obtained from 15 Studies conducted as per OECD 407 TG in Sprague Dawley Rats from 2015 to 2019]

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes observed in mean clinical chemistry values in any of the dose groups in either sex when compared with the vehicle control group.

However, the following statistically significant changes were noted in clinical chemistry levels when compared with the vehicle control group:
-increase in urea and blood urea nitrogen (BUN) levels at group G2 males;
-decrease in creatinine levels at group G4 females.

These observed changes are considered as incidental, as these changes lack dose dependency and also the obtained values are within the in-house historical control data range of the same species and strain. Refer to Table 7.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no changes observed in urinalysis parameters / mean urinalysis values in any of the dose groups in either sex when compared with the vehicle control group.
Refer to Table 8.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related changes observed in both absolute and relative organ weights in any of the dose groups of either sex when compared with the vehicle control group.

However, the following statistically significant changes were noted in absolute and/or relative organ weights when compared with the vehicle control group:
-increase in relative testes, kidneys, brain and liver weights in group G4 males;
-increase in testes (absolute) weight in group G3 males;
-increase in liver (relative) weight in group G4 females.

Apart from the above specified significant changes, a test item-related reduction in mean terminal body weight and slight reduction in thymus weight was noted in group G4 males and females. These noted changes can be considered as test item-related due to noted gross pathological changes in some of the animals (reduction in thymus size in 1 out of 3 males and 1 out of 3 females) and due to test item-related reduced body weight, body weight gain and feed consumption during treatment period at this dose level. Refer to Table 9 & 10.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no external or internal gross pathological changes noted in any of the animals from both the sex at groups G1, G2 and G3 during conduct of necropsy.
However, one out of three males and one out of three females from group G4 were noted with test item-related reduced thymus size as an internal gross pathological observation during conduct of the necropsy. Refer to Table 11

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

other: Dose levels: 0, 22.5, 45 and 90 mg/kg bw/day for main study.

Effect level:

0 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Dose levels for main test identified

Critical effects observed:

no

Conclusions:

Based on the obtained results from the present dose range finding study, it is concluded that the doses 22.5, 45 and 90 mg/kg body weight/day can be selected as low, mid and high doses for the subsequent Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test of 1,3-Propenesultone by Oral Gavage in Sprague Dawley Rats.

Executive summary:

In a dose range finding study (modified OECD 407) for a combined repeated dose toxicity study with reproduction/developmental toxicity screening test (OECD 422/GLP), 1,3-Propenesultone (99.90%) was administered to 4 groups of Sprague Dawley rats (3/sex/group) by oral gavage in 0.5% (w/v) Carboxy Methyl Cellulose at dose levels of 0 (G1), 10 (G2), 30 (G3) and 100 (G4) mg/kg bw/day, 7 days per week, for a period of 14 days.

No stability, homogeneity or dose formulation analysis was completed during this study.

In groups G2 (10 mg/kg bw/day) and G3 (30 mg/kg bw/day), there were no clinical signs of toxicity and no mortality or morbidity noted during the experimental period. There was no changes in weekly detailed clinical examinations in either sex. The mean body weight, percent change in body weight gain and mean feed consumption was unaffected by the test item in either sex. There were no ocular changes observed in any of the animals. The haematology, clinical chemistry and urinalysis parameters/levels did not reveal any test item-related changes in either sex analysed at termination. There were no test item-related changes noted in absolute and or relative organ weights in either sex. The gross pathological examination did not reveal any external or internal pathological changes in any of the animals in either sex.

In group G4 (100 mg/kg bw/day), test item-related clinical signs of toxicity (perinasal staining, lethargy, ataxia and rough hair coat) were noted from day 3 until sacrifice in both sexes. Test item-related reduction in mean body weight, percent change in body weight gain and mean feed consumption were noted in both sexes. Test item-related reduction in thymus weight and reduced thymus size in one male and female during gross pathological examination was noted. However, no changes in ophthalmological examination and no test item-related changes in clinical pathology parameters were noted at this dose level in either sex.

Based on the obtained results from the present dose range finding study it is concluded that, the doses 22.5, 45 and 90 mg/kg bw/day can be selected as low, mid and high doses for the subsequent Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test of 1,3-Propenesultone by Oral Gavage in Sprague Dawley Rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

45 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study was an OECD 422/GLP study and is the only study available. It was assigned a Klimisch score of 1

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

Mode of Action Analysis / Human Relevance Framework

Repeated dose toxicity:

There is a 14 day dose range finding oral study in rats and a 90 day repeated dose oral toxicity test in rats available.

Repeated dose toxicity: Dose range finding study

In a dose range finding study (modified OECD 407) for a combined repeated dose toxicity study with reproduction/developmental toxicity screening test (OECD 422/GLP), 1,3-Propenesultone (99.90%) was administered to 4 groups of Sprague Dawley rats (3/sex/group) by oral gavage in 0.5% (w/v) Carboxy Methyl Cellulose at dose levels of 0 (G1), 10 (G2), 30 (G3) and 100 (G4) mg/kg bw/day, 7 days per week, for a period of 14 days.

No stability, homogeneity or dose formulation analysis was completed during this study.

In groups G2 (10 mg/kg bw/day) and G3 (30 mg/kg bw/day), there were no clinical signs of toxicity and no mortality or morbidity noted during the experimental period. There was no changes in weekly detailed clinical examinations in either sex. The mean body weight, percent change in body weight gain and mean feed consumption was unaffected by the test item in either sex. There were no ocular changes observed in any of the animals. The haematology, clinical chemistry and urinalysis parameters/levels did not reveal any test item-related changes in either sex analysed at termination. There were no test item-related changes noted in absolute and or relative organ weights in either sex. The gross pathological examination did not reveal any external or internal pathological changes in any of the animals in either sex.

In group G4 (100 mg/kg bw/day), test item-related clinical signs of toxicity (perinasal staining, lethargy, ataxia and rough hair coat) were noted from day 3 until sacrifice in both sexes. Test item-related reduction in mean body weight, percent change in body weight gain and mean feed consumption were noted in both sexes. Test item-related reduction in thymus weight and reduced thymus size in one male and female during gross pathological examination was noted. However, no changes in ophthalmological examination and no test item-related changes in clinical pathology parameters were noted at this dose level in either sex.

Based on the obtained results from the present dose range finding study it is concluded that, the doses 22.5, 45 and 90 mg/kg bw/day can be selected as low, mid and high doses for the subsequent Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test of 1,3-Propenesultone by Oral Gavage in Sprague Dawley Rats.

Repeated dose toxicity: Main study

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.

Justification for classification or non-classification

Based on the available information in the dossier, the substance 1,3-Propenesultone (CAS No. 21806-61-1) does not need to be classified for specific target organ toxicity (repeated) when the criteria outlined in Annex I of 1272/2008/EC are applied.