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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: b.Commission Regulation (EC) No. 2019/1390, Annex. B.64 “Combined Repeated Dose Toxicity Study with The Reproduction/Developmental Toxicity Screening Test”

Test guideline 2

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

Test material information

Constituent 1

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 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

Details on mating procedure:

The males and females were placed in 1:1 ratio. Every morning, the vaginal smear of each female was examined for presence of sperm in the vaginal smear. The females were placed with the same male until pregnancy occurs by evidence of sperm in the vaginal smear until two weeks have elapsed. Day ‘0’ pregnancy was confirmed by the presence of sperm in the vaginal smear. Re-mating of females which were not confirmed with mating with the first male were cohabitated with proven male of same group for an additional week. The females which were still not positive for presence of sperm, such females were sacrificed after 24 days from the last day of the mating period. The females confirmed with mating but not littered were sacrificed on day 25 after gestation day ‘0’. The recovery group animals were not left for cohabitation.

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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1/Control

Doses / concentrations 2

Dose / conc.:

22.5 mg/kg bw/day (nominal)

Remarks:

G2 (low dose)

Doses / concentrations 3

Dose / conc.:

45 mg/kg bw/day (nominal)

Remarks:

G3 (mid dose)

Doses / concentrations 4

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

Parental animals: Observations and examinations:

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) and All dams at termination (lactation day 14). 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.

Oestrous cyclicity (parental animals):

Oestrus cyclicity was monitored for two weeks after five days of acclimatization to evaluate the normal oestrus cycle (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudopregnancy. The status of oestrus cyclicity of females were determined on termination day (lactation day 14). Recovery group females were not evaluated for oestrus cyclicity.

Sperm parameters (parental animals):

Parameters examined in P male parental generation: testis weight, epididymis weight. Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure - stage aware histopathological evaluation) sacrificed at termination.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups.
Hormone assessments: Two pups per litter on lactation day 4 based on the following conditions:

-two female pups in order to retain more male pups for nipple retention on PND 13.
-No pups were eliminated when the litter size dropped below 10 pups/litter.
-Only one pup was eliminated and used for blood collection for serum T4 assessments in case of availability of only one pup above the normal litter size.
Two male pups per litter at termination (lactation day 13)

Note: Serum was pooled per litter for T4 analysis on both lactation day 4 and lactation day 13. The collected blood samples were centrifuged at 5000 rpm for 10 minutes, the serum was separated and stored at frozen conditions (-80ºC) till estimation. The serum samples from adult males, dams, lactation day 4 and 13 pups were assessed for serum T4 levels.
Note: Blood from lactation day 4 pups was collected by jugular vein incision and using the retro-orbital plexus puncture method under Isoflurane anesthesia with the help of a fine capillary tube from lactation day 13 pups.

GROSS EXAMINATION OF DEAD PUPS:
The pups found dead and pups which were sacrificed on PND 4/13 were examined for gross abnormalities with particular attention to the external reproductive genitals and findings were recorded.

Postmortem examinations (parental animals):

SACRIFICE
All males were sacrificed after the completion of the post-mating period (total of 46 days of treatment) and females were sacrificed on the respective lactation day 13. Non-pregnant females were sacrificed on 24 days futher from the day using the sperm-positive vaginal smear as an evidence of 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.

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 vaginal smear of females on the day of necropsy (lactation day 14) was performed and the stage of estrous cycle was recorded. The animals were euthanized using CO2 followed by exsanguination and subjected to necropsy and gross pathological examination.
The main group males and recovery group animals were necropsied in randomized manner.


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. Testes and epididymis were preserved in modified Davidson’s fixative for 24 to 48 hrs and then transferred to 10% NBF. 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.

Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure - stage aware histopathological evaluation) sacrificed at termination. 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. The testes were sectioned at 3 to 4 micrometers and stained with Hematoxylin and eosin stain and also with Periodic Acid-Schiff (PAS) and hematoxylin stain. PAS stain aided spermatogenesis evaluation.

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.

Postmortem examinations (offspring):

SACRIFICE
All surviving pups were killed on PND 13. Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.

GROSS NECROPSY
The pups found dead and pups which were sacrificed on PND 4/13 were examined for gross abnormalities with particular attention to the external reproductive genitals and findings were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The thyroid along with the parathyroid was collected from one male and one female pup per litter on postnatal day 13. The thyroid along with the parathyroid from the adults and pups were preserved in 10% v/v Neutral Buffered Formalin.

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.

Reproductive indices:

Male Mating Index (%) = (No. of males with confirmed mating/ Total No. of males cohabited) X 100
Male Fertility Index (%) = (No. of males impregnating a female / Total No. of males cohabited) X 100
Female Mating Index (%) = (No. of sperm-positive females/ Total No. of females cohabited) X 100
Female Fertility Index (%) = (No. of pregnant females / No. of sperm-positive females) X 100
Gestation Index (%) = (No. of females with live born/ No. of females with evidence of pregnancy) X 100
Parturition Index (%) = (No. of females littered / No. of females with evidence of pregnancy) X 100
Pregnancy Index (%) = (No. of pregnant females / No. of females with confirmed mating) X 100

Offspring viability indices:

Post-Implantation Loss (%)= (No. of Implantations - No. of Viable pups)/No. of Implantations X 100
Post-natal loss on LD 13 (%) = (No. of pups alive on postnatal day 13/No. of live pups at birth) X 100
Live Birth Index (%) per litter = (No. of pups born alive/Total No. of pups born) X 100
Pup Survival index (%) on LD 4/7/13 = (Total No. of live pups on LD 4/7/13/No. of pups born/4/7/13) X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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)

Dermal irritation (if dermal study):

not examined

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 gestation body weight and percent change in mean gestation body weight gain in the dose groups G2 and G3 when compared with the vehicle control group. In group G3, a statistically significant decrease in mean gestation body weight was noted on gestation day 14 and 20 when compared with the vehicle control group (within the in-house historical control range). These reductions are considered as incidental and un-related to treatment as there were no such changes noted during the pre-mating period and also no effects noted in mean feed consumption during these periods at this dose level. In group G4, a decrease in mean gestation body weight and percent change in body weight gain throughout the gestation period was noted. These changes are statistically significant (p<0.001) on GD 0, 7, 14 (below the in-house historical control range) and 20 (below the in-house historical control range) for mean gestation body weight and during GD 7 to 14 (p<0.05) for percent change in body weight gain (below the in-house historical control range) when compared with the vehicle control group. These changes are considered as test item-related due to noted treatment related clinical signs of toxicity and reduced feed consumption during gestation period at this dose level. Refer: Table 14 & 15 (Summary Data)


There were no changes noted in mean lactation body weight and percent change in mean lactation body weight gain in the dose groups G2 and G3. In group G4, a statistically significant decrease (p<0.001) in mean lactation body weight on day 1, 4, 7 & 13 (below the in-house historical control range for lactation day 7 and 13) and percent change in mean lactation body weight gain during LD 1 to 4 (p<0.01), 4 to 7 (p<0.05) & 7 to 13 (p<0.001) was noted throughout the lactation period (below the in-house historical control range for all time points) when compared with the vehicle control group. These changes are considered as test item-related due to noted treatment related clinical signs of toxicity, reduced feed consumption during lactation period periods and also effects on reproductive/developmental end points at this dose level. Refer: Table 17 & 18 (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 during the gestation period in the dose groups G2 and G3 when compared with the vehicle control group. In group G4, a statistically significant decrease in mean feed consumption (p<0.001) was noted during entire gestation period measured between GD 0 to 7, 7 to 14 and 14 to 20 (below the in-house historical control range for GD 0 to 7 and 7 to 14) when compared with the vehicle control group. These changes are considered as test item-related due to noted treatment related clinical signs of toxicity, reduced mean body weight/percent change in body weight gain during gestation period at this dose level. These changes also can be correlated with test item-related reduced body weights and feed consumption continuing during the lactation period and also effects on reproductive/developmental end points at this dose level. Refer: Table 16 (Summary Data)

There were no changes noted in mean feed consumption during lactation period in the dose groups G2 and G3 when compared with the vehicle control group In group G4, a statistically significant decrease (p<0.001) in mean feed consumption was noted during the entire lactation period measured between LD 1 to 4, 4 to 7 and 7 to 13 when compared with the vehicle control group (below the in-house historical control range for LD 1 to 4 and 4 to 7).
These changes are considered as test item-related due to noted treatment related clinical signs of toxicity, reduced mean body weight/percent change in body weight gain during lactation period at this dose level.
Refer: Table 19 (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

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, ulti 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)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There were no irregularities observed in the oestrus cyclicity of females from the tested dose groups G2 and G3 during vaginal smear examination until evidence of sperm positive. The mean length of oestrus cycle (days) per dam was unaffected in the tested dose groups G2 and G3 when compared with vehicle control group.

In group G4,
-No irregularities observed in the oestrus cyclicity of 7 out of 12 females during vaginal smear examination until evidence of sperm positive and noted with normal 4 to 5 days oestrus cycle.
-A total of 2 out of 12 females (Re6475 & Re6477) were noted with irregular oestrus cycles (for these two females, a total of 6 complete oestrus cycles were recorded, among them 2 were noted with a 6-day cycle and another 4 were noted with a 5-day cycle). However, these two females were evidenced with sperm positive during the extended cohabitation period .
-A total of 3 out of 12 females were noted with irregular oestrus cycles and not confirmed with evidence of sperm positive until 21 days of the cohabitation period (Re6474: 4 out of 6 cycles noted as 5-day cycles, 1 out of 6 cycles noted as 4-day cycles & 1 out of 6 cycles noted as 6-day cyclse; Re6476: 3 out of 5 cycles noted as 5-day cycles, 2 out of 6 cycles noted as 6-day cycles; Re6481: 4 out of 5 oestrus cycles noted as 5-day cycles & 1 out of 5 oestrus cycles noted as 6-day cycle).
-The individual mean oestrus cycle (days) length of these dams was slightly increased than normal cycle length (4 to 5 days) due to the above mentioned extended cycle length. The noted increase is statistically significant (p<0.05) when compared with vehicle control group (the mean cycle length is within the historical control range).

These slight prolongations of oestrus cycle length can be considered as secondary test item-related effect induced due to stress caused bytreatment related clinical signs of toxicity, reduced mean body weight, reduced percent change in mean body weight gain, reduced mean feed consumption and effects on other reproductive/developmental end points at this dose level.
Refer: Table 13 (Summary Data)

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure - stage aware histopathological evaluation) sacrificed at termination. No abnormalities were observed.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Male Mating Index (%): A total of 11 (out of 12), 11 (out of 12), 12 (out of 12) and 7 (out of 12) males were confirmed with mating during the cohabitation period with a mating index of 91.7%, 91.7%, 100.0% and 58.3% from the tested dose groups G1, G2, G3 and G4 respectively.
In group G4, a statistically significant decrease (p<0.05) in number of males evidenced with mating was noted when compared with the vehicle control group.
This noted decrease in male mating performance is considered as a test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain, reduced feed consumption and also due to noted effects on reproductive/developmental end points at this dose level. This reduction in male mating performance is below the in-house historical control range.
Male Fertility Index (%): A total of 10 (out of 12), 9 (out of 12), 10 (out of 12) and 5 (out of 12) males were confirmed as fertile by impregnating a female or siring a litter with a fertility index of 83.3%, 75.0%, 83.3% and 41.7% from the tested dose groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for male fertility index at the tested dose groups G2 and G3 when compared with vehicle control groupIn group G4, a statistically significant decrease (p<0.05) in number of males siring a litter (confirmed as fertile) was noted when compared with the vehicle control group. This noted decrease in male fertility performance is considered as a test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain, reduced feed consumption and also due to noted effects on reproductive/developmental end points at this dose level. This reduction in male fertility performance is below the in-house historical control range.

Female Mating Index (%): A total of 12 (out of 12), 12 (out of 12), 12 (out of 12) and 9 (out of 12) females were confirmed as sperm positive during vaginal smear examination with a mating index of 100.0%, 100.0%, 100.0% and 75.0% from groups G1, G2, G3 and G4 respectively.
In group G4, a statistically significant decrease (p<0.05) in the number of females with evidence of mating was noted when compared with the vehicle control group. This noted decrease in female mating performance is considered as a test item-related effect due to noted treatment related reduced mean body weight/percent change in body weight gain, reduced feed consumption and also due to noted effects on other reproductive/developmental end points at this dose level. This reduction in female mating performance is below the in-house historical control range.

Female Fertility Index (%): A total of 12 (out of 12), 10 (out of 12), 11 (out of 12) and 6 (out of 12) mated females were confirmed with presence of implantations / presence of live or dead fetuses / evidence of parturition with a fertility index of 100.0%, 83.3%, 91.7% and 50.0% from groups G1, G2, G3 and G4 respectively. There were no statistically significant differences noted for female fertility index at the tested dose groups G2 and G3 when compared with vehicle control group. In group G4, a statistically significant decrease (p<0.05) in number of females evidenced as fertile was noted when compared with the vehicle control group. This noted decrease in female fertility performance is considered as a test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain, reduced feed consumption and also due to noted effects on other female reproductive/developmental end points at this dose level. This reduction in female fertility performance is below the in-house historical control range.

Pre-coital Interval/Copulatory Interval/Mean time to Mating: A total of 12 pairs were left for cohabitation initially from each group. The mean
pre-coital interval was 9.4, 5.6, 6.7 and 8.4 days for groups G1, G2, G3 and G4 respectively. There were no statistically significant differences noted for this parameter in any of the dose groups when compared with the vehicle control group.

Gestation Length/Duration of Pregnancy (Days): The mean gestation length [day of confirmed as mated to day of parturition] was 22.27, 22.10, 22.90 and 22.60 days for groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the dose groups when compared with the vehicle control group.

Fecundity or Pregnancy Index: A total of 11 (out of 12), 10 (out of 12), 10 (out of 12) and 5 (out of 9) mated females were confirmed as pregnant / with evidence of implantation sites with a fecundity index of 91.7%, 83.3%, 83.3% and 55.6% from group G1, G2, G3 and G4 respectively. There were no statistically significant differences noted for this parameter at the tested dose groups G2 and G3 when compared with the vehicle control group. In group G4, a statistically significant decrease (p<0.05) in number of females confirmed with evidence of implantation sites was noted when compared with the vehicle control group.
This noted decrease in female fecundity index is considered as a test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain, reduced feed consumption during and also due to noted effects on other female reproductive/developmental end points at this dose level. This reduction in fecundity index is below the in-house historical control range.

Gestation Index (%): A total of 11 (out of 11), 11 (out of 11), 10 (out of 10) and 4 (out of 5) females were confirmed with live born pups with a gestation index of 100.0%, 100.0%, 100.0% and 80.0% from groups G1, G2, G3 and G4 respectively. In group G4, a statistically significant decrease (p<0.05) in number of females confirmed with evidence of live born pups was noted when compared with the vehicle control group. This noted decrease in gestation index is considered as a test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain, reduced feed consumption, due to noted effects on other female reproductive/developmental end points at this dose level. This reduction in gestation index is below the in-house historical control range.

Post-Implantation Loss (%):
A mean number of 0.00, 0.20, 0.10 and 1.80 post-implantation losses with a percentage of 0.00%, 1.82%, 0.77% and 27.86% were noted from groups G1, G2, G3 and G4 respectively. However, one female each from groups G1, G3 and G4 were evidenced with implantation sites when sacrificed on gestation day 25 as these were not littered. These findings are considered as incidental without any toxicologically significance, as the occurrences were noted in control group also. There were no statistically significant changes noted for this end point in dose groups G2 and G3 when compared with the vehicle control group.
In group G4, a statistically significant increase (p<0.01) in number and percentage of post-implantation losses was noted when compared with vehicle control group. This increase is considered as a test item- related due to noted treatment related clinical signs of toxicity, reduced mean body weight/percent change in body weight gain, reduced feed consumption during pre-mating/gestation periods and due to noted effects on female reproductive/developmental end points (mating, fertility, fecundity and gestation indices) at this dose level. This reduction in post-implantation loss is above the in-house historical control range.

Postnatal Loss (%): There were no incidences of postnatal losses in all the dose groups and vehicle control group during the lactation period.

Refer to Table 21 (Summary Data)

These noted effects on reproductive performances are considered as secondary effects related to stress due to noted treatment related clinical signs of toxicity, reduced body weights, reduced body weight gain and reduced feed consumption. Also, there was no direct test item-related effects noted in any of the reproductive organs of both sexes, no gross or histopathological changes noted in any of the organs/tissues including reprodctive organs of both sexes and no effects were noted in serum T4 levels of both sexes. Based on the above considerations, these effects are concluded as secondary effects, but not direct treatment related changes.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

45 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There were no changes observed in birth parameters such as, total litter size, number of live pups born, sex ratio (m/f) and live birth index in the dose groups G2 and G3 when compared with the vehicle control group. Also, the pup survival index per litter during the lactation period was unaffected by the test item in the dose groups G2 and G3 when compared with the vehicle control group.

In group G4, a statistically significant decrease in mean litter size ((p<0.01)), mean number of live pups born (p<0.05), mean number of live male pups born (p<0.05)), mean number of live female pups born (p<0.05), mean percent live birth index (p<0.05) at birth/delivery were noted when compared with the vehicle control group (all below historical control data).

In group G4, a statistically significant decrease in mean number of total live pups during LD 1 to 4 (p<0.05)), LD 5 to 7 (p<0.01)), LD 8 to 13 (p<0.01) and mean number of male live pups during LD 1 to 4 (p<0.05), LD 5 to 7 (p<0.05) & LD 8 to 13 (p<0.05) were noted when compared with the vehicle control group.
The pup survival index was 80.0% in group G4 during LD 1 to 4 (not significant but below historical control data), whereas 100.0% for other dose groups. However, this end point was un-affected during LD 5 to 7 and 8 to 13.

The occurred changes are considered as test item-related due to noted treatment related reduced mean body weight/percent change in body weight gain during
pre-mating/gestation/lactation periods and also due to noted effects on other reproductive/developmental end points at this dose level.
Refer: Table 20 (Summary Data)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related changes noted in mean pup [both male and female] weight per litter recorded on postnatal day (PND) 1, 4, 7 and 13 in the dose groups G2 and G3 when compared with vehicle control group litters.

In group G4, a statistically significant decrease (p<0.001) in mean male live pup weight at birth and on postnatal day 4, 7 (below the in-house historical control data) and 13; a statistically significant decrease (p<0.05) in mean female live pup weight at birth and statistically significant decrease (p<0.001) on postnatal day 4, 7 and 13 was noted when compared with the vehicle control group.

These effects can be considered as test item-related due to noted treatment related effects on maternal end points such as, reduced body weights, reduced body weight gain and reduced feed consumption at this dose level during lactation period.

Refer: Table 23 (Summary Data)

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in mean pup [both male and female] anogenital distance measurement (mm) and ratio per litter recorded on postnatal day 4 in the dose groups G2 and G3 when compared with the vehicle control group.

In group G4, a statistically significant decrease (p<0.001) in anogenital distance measurement (mm) and ratio per litter of male pups was noted when compared with the vehicle control group. This effect can be due to noted reduction in mean pup weight when compared with vehicle control group on postnatal day 4. However, the obtained mean values for this end point are within the in-house historical control range in both sexes at all the time points.

Refer: Table 24 (Summary Data)

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no occurrences of nipples in any of the male pups examined on their postnatal day 13 from any of the dose group and vehicle control group litters.
Refer: Table 25 (Summary Data)

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes observed during necropsy in all of the pups.
Refer: Appendix 30 for Pathology Phase Report

Histopathological findings:

no effects observed

Description (incidence and severity):

The histopathological examination of thyroid collected from the pups 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.

Other effects:

no effects observed

Description (incidence and severity):

There were no test item-related changes noted in serum T4 hormone levels of postnatal day 4 pups in dose groups G2 and G3 when compared with the vehicle control group litters. The serum T4 hormone levels were not analysed for postnatal day 4 pups from group G4 litters due to unavailability of pups greater than 10 from all littered damson postnatal day 4. There were no test item-related changes noted in serum T4 hormone levels of postnatal day 13 pups in any of the dose group litters when compared with vehicle control group litters. All the obtained values are within in-house historical control range.
Refer: Table 26 (Summary Data)

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no external anomalies and no behaviour changes noted for any of the pups during daily observation from all the dose group litters during the postnatal period. All the pups were noted with normal behaviour during daily observations. Refer: Table 22 (Summary Data)

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

45 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

45 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

no

Relevant for humans:

no

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 the NOAEL (reproduction; male/female) and NOAEL (developmental) of 1,3-Propene sultone is 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 indications 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 these dose levels.

In groups G2 and G3, there were no indication of test item-related effects in any of the parameter/endpoints assessed for reproductive and developmental toxicity. Also, there was no indication of test-item related effects on endocrine disruptor (ED) relevant endpoints in both parents (oestrus cyclicity in females, relative organ weight and histopathology of gonads and accessary sex organs, thyroid weights and serum T4 levels) and pups (development of external genital organs, measurement of anogenital distance, male pup nipple retention and serum T4 levels for PND 4/13 pups) at these dose levels.

In group G4, test item-related reductions in live birth index per litter, male/female mating index, male/female fertility index, pregnancy and gestation index were noted. The increased post-implantation loss and postnatal loss were also evidenced as test item-related at this dose level. Test item-related reduced litter size, reduced live birth index per litter and increased number of dead pups at birth were also noted at this dose level. These noted effects on reproductive performances are considered as secondary effects related to stress due to noted treatment-related clinical signs of toxicity, reduced body weights, reduced body weight gain and reduced feed consumption. Also, there was no direct test item-related effects noted in any of the reproductive organs of both sexes, no gross or histopathological changes noted in the reproductive organs of both sexes and no effects were noted in serum T4 levels of both sexes. Based on the above considerations, these effects are concluded as secondary effects, but not direct treatment-related changes.

In group G4, test item-related reduced mean male pup weight per litter was noted. However, no gross pathological changes were noted in any of the surviving pups of both sexes.  In group G4, there were no test item-related effects noted for ED relevant endpoints in both parental animals and F1 pups.

The NOAEL (reproduction; male/female) is determined as 45 mg/kg bw/day, due to noted secondary test item-related effects such as reduced mating and fertility performance of both sexes, reduced pregnancy and gestation indices, reduced live birth index, increased post-implantation and postnatal losses at the dose level of 90 mg/kg bw/day. The NOAEL (developmental) is determined as 45 mg/kg bw/day, due to noted reduced mean male pup weights at the dose level of 90 mg/kg bw/day.