Sodium 3-nitrobenzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Reproductive Toxicity:

Based on all the available data, it was concluded that the test chemical did not have any adverse effects on the reproduction and developmental parameters at the highest dose tested i.e. 1000 mg/kg bw/day. Therefore, the test chemical is not likely to classify as 'reproductive and/or developmental toxicant' as per the CLP criteria of classifiction and labelling.

Link to relevant study records

Reference

Reference 1

Endpoint:

reproductive toxicity, other

Remarks:

Subchronic toxicity study (OECD 408 study with additional reproductive parameters)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from a study report

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)

Version / remarks:

(Adopted on September 21, 1998) Additional Reproductive parameters were also examined in this study.

Principles of method if other than guideline:

According to OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents) along with examination with additional reproductive parameters.

GLP compliance:

yes

Limit test:

no

Justification for study design:

No Data Available

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and is also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd.
- Females (if applicable) nulliparous and non-pregnant: No data
- Age at study initiation: 5-6 weeks
- Weight at study initiation: Males: 134.54 to 168.03g
Females: 114.43 to 146.06g
- Fasting period before study: No Data Available
- Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in each recovery group and sex was housed individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once aweek. During the experimental period, animals were housed in a single experimental room. Steam sterilized corn cob was used as bedding and changed along with the cage at least twice a week.
- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet – Pellet (Certified) manufactured by Envigo ad libitum
- Water (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet – Pellet (Certified) manufactured by Envigo ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: The feed and water provided to the rats was tested for contaminants. There were no known contaminants in the food, water and bedding that are expected to interfere with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 59 to 68 %
- Air changes (per hr): 12 – 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: To: 14 July 2017 - 20 December 2017

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q® water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was prepared fresh daily by mixing with Milli-Q® water and mixed well using glass rod. The final volume was made up with the vehicle to get the required final concentration of 0, 100, 300 or 1000 mg/Kg/day

Details on mating procedure:

No Data Availabke

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item in the vehicle was established at 0.7 and 125 mg/mL.
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1, during 2nd (Day
45) and 3rd (Day71) of the treatment period and analysed in-house. For each set, six composite samples were drawn from each preparation and in case of control, duplicate samples were drawn.
Dose formulations were considered acceptable as the overall mean results were within ± 10% of the claimed concentration and the overall relative standard
deviation (RSD) was less than 5%.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily

Details on study schedule:

No Data Available

Dose / conc.:

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

Remarks:

Control Group

Dose / conc.:

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

Remarks:

Control Group (Recovery)

Dose / conc.:

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

Remarks:

Low Dose Group

Dose / conc.:

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

Remarks:

Mid Dose Group

Dose / conc.:

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

Remarks:

High Dose Group

Dose / conc.:

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

Remarks:

High Dose Group (Recovery)

No. of animals per sex per dose:

Main study: 10
Recovery groups: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of 0, 100, 300 and 1000 mg/kg/day were selected for this study based on the available literature
- Rationale for animal assignment (if not random): Animals were randomly distributed to different groups by body weight stratification method using ProvantisTM software. Rats with extreme body weights were discarded. Grouping was done one day prior to start of treatment during acclimatization.
- Rationale for selecting satellite groups: The recovery groups were selected to observe the reversibility of any effects that would be obseved during the course of the study and even after the in-life phase of the study.
- Post-exposure recovery period in satellite groups: 28 days
- Section schedule rationale (if not random): No data

Positive control:

No Data Available

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each rat was observed twice daily for morbidity and mortality except during holidays wherein the observation was done once daily as there were no clinical
signs observed. Routine observations for checking general clinical signs were performed once a day during treatment (post-dose) and recovery period.
- Cage side observations checked in table [No.?] were included. Morbidity, mortality, general clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Detailed clinical examination was done prior to the test item administration on Day 1 and at weekly intervals thereafter (± 1 day) during treatment period.

During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour (e.g. self-mutilation, walking backwards).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights (g) was recorded prior to test item administration on Day 1 and weekly thereafter (± 1 day) for all groups of rats during treatment and recovery period except for week 13 wherein the animals were weighed on
day 5 of that week (Day 90). Fasting body weight was recorded prior to sacrifice for all animals.

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, The food consumption was measured at weekly intervals (± 1 day) during treatment and recovery period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations: Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination of all animals was performed with an ophthalmoscope prior to start of the treatment, at the end of the treatment period for main groups and at the end of recovery period for recovery groups. Before examination, mydriasis was induced using a 1 % solution of Tropicamide.
- Dose groups that were examined: All animals of the main study and recovery group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes , animals were fated overnight (water allowed)
- How many animals: All animals
- Parameters checked in table [No.?] were examined. RBC, HGB, MCV, MCHC, HCT, Retic, WBC, DLC, PLT, PT and APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period
- Animals fasted: Yes , animals were fated overnight (water allowed)
- How many animals: All animals
- Parameters checked in table [No.?] were examined. ALT, ALB, A/G ration, Alp, AST, BUN, BA, Ca, Cl, Creat, CK, GGT, Glob, Glu, Pi, K, Na, T. Bil, T. Chol, T. Pro, Trig, Urea, TSH, T4, Testo, Estro

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period for main groups and at the end of the recovery period for recovery groups
- Metabolism cages used for collection of urine: Yes, Each rat was placed overnight in a specially fabricated cage (water allowed)
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined. Specific gravity, nitrite, pH, proteins, glucose, ketone bodies, urobilinogen, bilirubin, appearance (colour and clarity), volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The following neurological examination was performed during 13th week (Day 86 and 87) of treatment period for main groups and towards the end of recovery period (Day 115) for recovery period.
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Home Cage Observations
Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.

Observations during Removal of Animal from Home Cage and Handling
The objective of this phase of neurological examination was to observe the subject’s response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following examinations:
- ease of removal from home cage
- handling reactivity
- palpebral closure
- eye examination
- piloerection
- lacrimation
- salivation
- skin/fur examination
- perineum wetness
- respiration
- muscle tone and
- extensor thrust response
The observations were recorded using scores/ranks.

Open Field Observation
Rat was placed (one at a time) in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each group. During this observation period, rat was evaluated as it moves about freely/unperturbed and the following observations were made, and observations were recorded using score/ranks:
- gait
- posture
- tremors
- mobility score
- arousal level
- clonic or tonic movements
- stereotypic behaviour
- bizarre behaviour
- urination
- defecation
- rearing
- abnormal vocalizations

Functional Tests
Functional testing includes motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance

Motor Activity
The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large ambulatory movement) in seconds, horizontal counts, ambulatory counts were monitored. The Opto-Varimex 4 motor activity measurement system provided the data at 1-minute interval and the data were analyzed in blocks of 10 minutes interval and the same was reported.

Sensory Reactivity Measurements
After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/ranks.
- approach response
- touch response
- click response
- tail-pinch response
- pupil response
- aerial righting reflex

Landing Hindlimbs Footsplay
The landing hind limbs foot splay was performed by dropping the rat on to a horizontal surface of the table top from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat were gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on to a SOP format, which contains the details such as Study no., Animal no, Group and Sex. A clean recording SOP format was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values are presented in the report along with the individual footsplay values.

Grip Performance
Hindlimbs and forelimbs grip performance was tested using computerized dual grip strength meter (Model: Columbus Instruments). Three trials were conducted for each rat i.e., three trials each for forelimb and hind limbs. Average of three trials for both forelimb and hindlimbs are calculated and presented in the report along with the individual grip strength values.

Physiological Observations
Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer.

IMMUNOLOGY: Not specified
- Time schedule for examinations: Not specified
- How many animals: Not specified
- Dose groups that were examined: Not specified
- Parameters checked in table [No.?] were examined. Not specified

Oestrous cyclicity (parental animals):

Vaginal smear was examined, and the stage of oestrous cycle was recorded daily for two weeks prior to necropsy for main and recovery groups. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.

Sperm parameters (parental animals):

For all male rats at termination, sperm from the right vas deferens was collected for evaluation of sperm motility using Hamilton-Thorne TOX-IVOS (version 12) sperm analyzer. Sperm smears were prepared using vas deferens sperm samples from all the males. Sperm morphology was not done as there were no abnormalities observed in sperm motility.

Litter observations:

No Data Available

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes, All rats in the study were subjected to necropsy after overnight fasting (water allowed) at the end of treatment and recovery period. Terminal body weights were recorded for all rats immediately prior to sacrifice and used in calculation of relative organ weights. The rats were euthanized by exsanguination under isoflurane anesthesia, subjected to detailed necropsy (examination of external surfaces of the body, all orifices, cranial, thoracic and abdominal cavities and their contents) and findings were recorded.

For all male rats at termination, sperm from the right vas deferens was collected for evaluation of sperm motility using Hamilton-Thorne TOX-IVOS (version 12) sperm analyzer. Sperm smears were prepared using vas deferens sperm samples from all the males. Sperm morphology was not done as there were no abnormalities observed in sperm motility.

HISTOPATHOLOGY: Yes, On completion of the gross pathology examination, the tissues and organs noted in the following table were collected and weighed from all rats. The organ weight ratios as percentage of fasting body weight and brain weight were determined and presented in the report. Paired organs were weighed together, and combined weights were presented. The tissues were preserved in 10% Neutral Buffered Formalin (NBF) except for the testes and eyes: Adrenals, all gross lesions, aorta, bone marrow smears, Brain including cerebrum, cerebellum and medulla/pons, cecum, colon, duodenum, epididymides, eyes with optic nerves, femur bone with distal joint, heart, Ileum with Peyer’s patches, jejenum, kidneys, liver, lungs, mammary glands, Mandibular lymph nodes, Mesenteric lymph nodes, oesophagus, ovaries, oviducts, pancreasm pharnyx, pituitary, prostate. rectum, salivary glands, sciatic nerves, Seminal vesicles with coagulating glands, skelatal muscles, skin, Spinal cord at 3 levels (cervical, mid thoracic and lumber), spleen, sternum with marrow, stomach, testes, thymus, Thyroid with parathyroids, trachea, urinary bladder, uterus with cervix, vagina, Levator ani bulbocavernosus muscle complex (male), Cowper’s glands (male) and glans penis (male).

Histopathological examination was carried out on the preserved organs of the vehicle control and high dose group animals (with qualitative assessment of stages of spermatogenesis in male rats). Stages of estrus cycle in female rats was also recorded during histopathology examination. Further, none of organ/tissue were examined in the mid and low dose groups and recovery groups as there were no treatment-related microscopic changes in high dose group.

The tissues were processed for routine paraffin embedding and 4 to 5-micron sections were stained with Mayer’s Haematoxylin and Eosin stain. Testes sections from control and high dose groups were also stained with PAS and Haematoxylin stains for the assessment of stages of spermatogenesis.

Postmortem examinations (offspring):

No Data Available

Statistics:

Data captured using Provantis™ for the parameters body weight and organ weights; laboratory Investigations - Haematology (Coagulation tests PT and APTT which was entered retrospectively in ProvantisTM) and Clinical Chemistry were analyzed using built-in statistical tests.

Derived data like net body weight change, food consumption and organ weight ratios were also analyzed using above mentioned methods.

The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical
package ver.12.0. All quantitative variables like neurological observations (neuromuscular observation/body temperature/body weights) and the hormones,
viz. TSH, T4, T3, Testosterone and Estradiol were tested for normality (Shapiro- Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before performing
ANOVA. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test found to be
significant.

The data pertaining to males and female rats were evaluated separately.

All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned
tests were designated by the following symbols throughout the report:

*: Significantly higher/lower than the respective control group

Reproductive indices:

No Data Available

Offspring viability indices:

No Data Available

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed at any of the doses tested in both sexes.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No treatment-related mortality were observed at any of the doses tested in both sexes.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights and net body weight gains were not significantly different from the vehicle control group at all the tested doses in both sexes during the treatment and recovery period.

Few sporadic incidences of significantly lower net body weight gain during Days 71-78,78-85 in low dose, Days 78-85 in mid dose, Days 50-87, 71-78 and 78-85 at high dose, main toxicity group males and significantly higher net body weight gain during Days 78-85 in high dose main toxicity group females were observed. These significant differences were considered toxicologically not relevant as the absolute and percent gain at the end of 13 weeks treatment are comparable to control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The mean food consumption was not altered by the treatment in any of the tested doses either during the treatment or recovery period in both sexes. Few sporadic incidences of significantly lower food consumption were observed during Days 1-8, 8-15, and 15-22 at low dose, Days 8-15, 15-22, 29-36, 36-43, 43-50, 50-57, and 71-78 at mid dose and Days 8-15 at high dose in the main toxicity group females. In high dose recovery group females, significantly higher food consumption was observed during Days 104-111 during the recovery period. These significant differences were considered toxicologically not relevant as the mean body weights were not altered by the treatment.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmological examination carried out with an ophthalmoscope prior to start of treatment, at the end of the treatment and recovery period did not reveal any abnormalities in the eyes of the experimental rats.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in hematology parameters at all the dose levels tested.
Increased reticulocyte count at 1000 mg/kg/day dose group in females was considered as toxicologically insignificant as it was not associated with changes in hemopoietic organs.
Increased lymphocyte count at 1000 mg/kg/day dose group in females was considered as toxicologically insignificant as it was not associated with changes in lymphoid organs.
A few variations in other hematology parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.
There were no test item-related changes in coagulation parameters at all the dose levels tested.
Increased activated partial thromboplastin time at 300 mg/kg/day dose group in females was considered as toxicologically insignificant as there was no dose progression.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in clinical chemistry parameters at all the dose levels tested.

Decreased ALP activity at 1000 mg/kg/day dose group in males was considered as toxicologically insignificant as it was not associated with any microscopic changes in bones, liver and intestine.

Decreased creatine kinase activity at 300 mg/kg/day dose group in males was considered as toxicologically insignificant as there was no dose progression.

A few variations in other clinical chemistry parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.

There were no significant changes in testosterone, estrogen, thyroid stimulating hormone (TSH), and thyroxin hormone (T4) levels at all dose levels tested.
Decreased estrogen concentration at 100 mg/kg/day dose group in females was considered as toxicologically insignificant as there was no dose progression and was likely due to random biological variation.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes in urinalysis parameters at all the dose levels tested.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional Observation Battery

Home cage and Handling observations: No treatment-related abnormalities were observed in any of the tested dose groups in both sexes.
Open field observations: No treatment-related abnormalities were observed in any of the doses tested in both sexes.
Sensory observations: No treatment-related abnormalities were observed in any of the groups in both sexes.
Motor Activity: The following statistically significant variations were observed in the motor activity of rats when compared to respective vehicle control group:
Males:
Higher : Stereotypic time at interval 3 in low dose, ambulatory time at intervals 2 and 3 in low and high doses, total ambulatory time in high dose, horizontal and ambulatory counts at interval 3 in high dose.
Females:
Higher : Ambulatory time at interval 1 and total ambulatory time in mid and high doses, horizontal and ambulatory counts at interval 1 in mid and high doses, ambulatory time at interval 1 at high dose recovery
group.
The above observed statistical variations in the motor activity measurements are considered to be incidental as there were no changes observed in the home cage
or open field observations. Further, there were no clinical signs observed during daily clinical observation.

Neuromuscular observation:
Landing hind limb footsplay: No treatment-related changes were observed at all the tested doses in both sexes.
Grip strength: Significantly lower forelimb grip strength in high dose toxicity main group and significantly higher hindlimb grip strength in high dose recovery group was observed in males. Significantly higher forelimb grip strength in high dose and significantly lower hindlimb grip strength in mid dose main toxicity group was observed. These significant differences were considered toxicologically not relevant as there were no changes observed in the home cage or open field observations. Further there were no clinical signs observed during daily clinical observation.

Physiological observation:
Body temperature: No significant changes were observed at all the tested doses in both sexes.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination of animals treated at 0 and 1000 mg/kg (main study) revealed no treatment-related effects.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic changes at all the dose levels tested.

The staging of spermatogenesis in male rats did not reveal any stage specific changes. The spermatogenic cycle observed in the different seminiferous tubules of testes were complete and none of the stages of spermatogenesis were arrested in all the animals examined.

Staging of estrus cycle in female rats did not reveal any stage specific changes and all stages of estrous cycle were randomly distributed without any stage arrest.

All the single or low incidences of microscopic findings observed in different groups were considered incidental and not related to test item as they were
randomly distributed in different groups of rats.

Other effects:

no effects observed

Description (incidence and severity):

Sperm motility evaluation:
Sperm motility was unaltered by the test chemical treatment

The oral gavage administration of test article for 90 consecutive days to Wistar rats at dose levels of 0, 100, 300 and 1000 mg/kg doses did not reveal any test item-related changes in the thyroid,, testosterone and estradiol hormone levels in both the sexes at all the dose levels tested.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks prior necropsy for main and recovery groups.

The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.

The calculated mean oestrous cycle length was 4.10, 4.13, 4.09 and 4.21 days in vehicle control, 100, 300 and 1000 mg/kg Bwt/day doses, respectively in main toxicity groups, 4.30 and 4.20 days in vehicle control recovery and 1000 mg/kg Bwt/day recovery groups respectively. The mean oestrous cycle length of both main and recovery groups was not significantly different from the concurrent vehicle control group.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Sperm motility was unaltered by the test chemical treatment.

Reproductive performance:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

ophthalmological examination

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Basis for effect level:

other: Not Specified

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Key result

Reproductive effects observed:

no

Treatment related:

no

Conclusions:

As there were no adverse effects observed up to the highest dose tested, the evaluated No Observed Adverse Effect Level (NOAEL) of the test chemical is considered to be 1000 mg/kg/day following oral gavage administration for 90 consecutive days to Wistar rats under the test conditions and doses employed.

Executive summary:

The purpose of this repeated dose toxicity study was to evaluate the systemic toxicity profile of the test chemical in Wistar rats when administered orally by gavage for 90 consecutive days and to assess the reversibility of any effects during a subsequent 28 days recovery period. This study was also intended to provide the information on major toxic effects, target organs and an estimation of a No Observed Adverse Effect Level (NOAEL). The test item was weighed and dissolved in vehicle i.e., Milli-Q® Water and administered to rats at the graduated dose levels of 100, 300, and 1000 mg/kg/day for low dose (G2), mid dose (G3) and high dose (G4) / high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1)/ vehicle control recovery (G1R) groups received vehicle (Milli-Q® Water) alone. The dose volume administered was 10 mL/kg body weight. Each main groups in the experiment was comprised of 10 males and 10 female rats and recovery groups comprised of 5 males and 5 female rats. The identity of the test chemical was provided by the study sponsor by a Certificate of Analysis. The stability and homogeneity of the test item in the vehicle was established at 0.7 and 125 mg/mL. Based on the results, the test item was stable and homogenous in the vehicle (Milli-Q® water) up to 48 hours when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during 2nd (Day 45) and 3rd month (Day 71) of the treatment period. The results indicated that the analysed concentrations were within ± 10% of variations from the claimed concentrations. Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and at the end of recovery period for recovery groups. The body weights and food consumption were measured during in-life phase of the experiment. Neurological examinations were conducted towards the end of treatment for main groups and towards the end of recovery period for recovery groups. Vaginal smear was examined, and the stage of oestrous cycle was recorded daily for two weeks prior to necropsy for main and recovery groups. The clinical laboratory investigations such as haematology, coagulation, clinical chemistry and urine analysis were performed at termination. Blood samples were also analyzed for Thyroid Stimulating Hormone (TSH), Thyroxin (T4), Testosterone (Testo) and Estrogen (Estradiol- All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratio were derived as percent fasting body weights. For all surviving males at termination, sperm from the right vas deferens were collected for evaluation of sperm motility using Hamilton-Thorne TOX-IVOS (version 12) sperm analyzer. Histopathological examination was carried out on the preserved organs of the vehicle control and high dose group animals (with qualitative assessment of stages of spermatogenesis in male rats). Stages of estrus cycle in female rats was also recorded during histopathology examination. There were no treatment-related microscopic changes observed in high dose group and hence, the organ/tissue in the mid and low dose groups and recovery groups were examined microscopically. During observations, there were no clinical signs or mortality observed at any of the doses tested in both sexes. Ophthalmological examination did not reveal any ocular abnormalities. No treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested. The mean body weights and food consumption were unaffected by the treatment at all the tested doses in both the sexes. No test item-related changes were observed in the haematological, coagulation, clinical chemistry and urine parameters at all the doses tested in both sexes. There were no significant changes in testosterone, estrogen, thyroid stimulating hormone (TSH), and thyroxin hormone (T4) levels at all dose levels tested. There were no test item-related changes in terminal fasting body weight and organ weights in males and females at all the dose levels tested. There were no test item-related changes in sperm motility at all the dose levels tested. There were no test item-related gross or microscopic changes at all the dose levels tested in both sexes. The staging of spermatogenesis in male rats did not reveal specific changes. Staging of  estrous cycle in female rats did not reveal any stage specific changes and all stages of estrous cycle were randomly distributed without any stage arrest. Therefore, since there were no adverse effects observed up to the highest dose tested, the evaluated No Observed Adverse Effect Level (NOAEL) of the test chemical is considered to be 1000 mg/kg/day following oral gavage administration for 90 consecutive days to Wistar rats under the test conditions and doses employed.

Reference 2

Endpoint:

fertility, other

Remarks:

Prenatal Developmental Toxicity Study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

To evaluate the embryo-fetal developmental toxicity of test chemical when administered to pregnant Wistar rats by oral route during gestation days GD 5 to GD 19.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

- Source: Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311,Gajwel Mandal,Medak District, Telangana
- Age at study initiation: 13 to 14 weeks
- Weight at study initiation: 214.952 ± 13.13 to 196.16 to 246.01
- Fasting period before study:
- Housing: Rats were housed in standard polysulfone rat cages (size: Length 425 mm x Breadth 266 mm x Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube. Additionally, polycarbonate rat huts were placed inside the cage as environmental enrichment objects which were replaced once a week.
Following was the housing pattern during different periods of the experiment:
i. Pre mating / Acclimatization: Two rats of the same sex per cage were housed per cage.
ii. Mating: Female rats were cohabited with males in a 1:1 ratio in same cage.
iii. Post-mating / Treatment: After mating confirmation, females were housed individually.
Steam sterilized corn cob was used as bedding and was changed along with the cage at least twice a week.
- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet – pellet (Certified), ad libitum
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed toUV rays in ‘Aquaguard’ on-line water filter-cum-purifier, ad libitum
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 65 – 66 %
- Air changes (per hr): adequate fresh air supply (12 to 15 air changes/hour).
- Photoperiod (hrs dark / hrs light):12 hours light and 12 hours dark cycle.

IN-LIFE DATES: From: 27 October 2017
To:29 March 2018

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Remarks:

Milli-Q®

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were freshly prepared daily before dosing and were used within the stability period. The required quantity of test item was weighed in pre-calibrated beaker*. A small volume (3 to 40 mL) of vehicle (Milli-Q® water) was added and mixed well using a glass rod. The final volume was made up with the vehicle to get the required final concentration. The volume was made up to the upper meniscus during dose formulation preparation.

*Pre-calibration of the beaker to desired volume: Milli-Q® water was measured in a graduated measuring cylinder to the final volume of the batch size (example if batch size was 70 mL, water was taken till 70 mL mark in graduated measuring cylinder). The measured water was transferred into a clean beaker (to be pre-calibrated) and upper and lower meniscus of water were marked on the beaker using a marker. Once these lines were marked, the water was discarded and the beaker was dried.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the solubility test conducted
under study, the test item is soluble in Milli-Q® water. Hence, Milli-Q® water was used as vehicle for
dose formulation preparation.
- Concentration in vehicle: 0, 100, 300 and 1000 mg/kg bw
- Lot/batch no. (if required):
- Purity:

Details on mating procedure:

- Impregnation procedure: [artificial insemination / purchased timed pregnant / cohoused]: Cohoused
- If cohoused: the female rats were cohabited with males
- M/F ratio per cage: 1:1 ratio
- Length of cohabitation: On the first day, after vaginal smear examination, all the females with proestrous and estrous stages were retained with the males; while the other females were separated from males. Subsequently females were cohabited in batches of required numbers. These females were cohabited with respective males in a 1:1 ratio. The details were recorded in raw data. This procedure was continued until there were sufficient numbers of Day 0 pregnant rats for the study.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. Not specified.
- Further matings after two unsuccessful attempts: [no / yes (explain)]Not specified.
- Verification of same strain and source of both sexes: [yes / no (explain)]: yes, vaginal smear were examination
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: day 0 of gestation
- Any other deviations from standard protocol:Not specified.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and stability of the test chemical in vehicle was established at the concentrations of 0.7 mg/mL and 125 mg/mL. Based on the results, the test item was stable and homogenous in the vehicle up to 48 hours when stored at room temperature.

Duration of treatment / exposure:

15 days (from GD 5 to GD 19 of presumed gestation)

Frequency of treatment:

Daily

Details on study schedule:

No Data Available

Dose / conc.:

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

Remarks:

Control Group

Dose / conc.:

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

Remarks:

Low Dose Group

Dose / conc.:

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

Remarks:

Mid Dose Group

Dose / conc.:

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

Remarks:

High Dose Group

No. of animals per sex per dose:

Total: 96
0 mg/kg bw: 24 females
100 mg/kg bw: 24 females
300 mg/kg bw: 24 females
1000 mg/kg bw: 24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose range finding study was carried out at Test Facility using seven day ‘0’ pregnant rats per group at the doses of 100 (G2), 300 (G3) and 1000 (G4) mg/kg/day along with the concurrent vehicle control (G1). No effect were observed in treated femlae and Fetal examination. Therefore, Based on the results of dose range finding study doses were selected as 0, 100, 300 and 1000 mg/kg bw.

- Rationale for animal assignment (if not random): Rats were randomly distributed to different groups by body weight stratification. Based on the body weight, Day ‘0’ pregnant rats were arranged in the ascending order. These rats were then assigned to the groups starting either from control and treatment group/s in the increasing order of dose or vice-versa on GD 0. As far as possible rats were assigned equally to the study groups, the extra animals remaining were also assigned to groups. This difference was negated on subsequent day/s by assigning rats in such a way that there were equal numbers of rats in all the groups.

- Other: No Data Available

Positive control:

No Data Available

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day - pre dose and post dose (30 minutes to one hour post dose, approx.) during treatment days and once on non-treatment days
- Cage side observations checked in table [No.?] were included. : morbidity and mortality i.e., once in the nmorning and once in the afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: weighed on gestation days 0, 3, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/k mg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day # : On gestation day 20
- Organs examined: all visceral organs of dams were examined.
OTHER:

Oestrous cyclicity (parental animals):

Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Sperm parameters (parental animals):

Not speified

Litter observations:

Number of live and dead fetuses, sexed and weighed were examined.

Postmortem examinations (parental animals):

uteri weight and gross pathology were examined.

Postmortem examinations (offspring):

examined for external malformations were examined.

Statistics:

The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female were analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss, external, visceral and skeletal observations for variations were analyzed using Kruskal wallis test for group comparison.

The incidence of with and without resorptions in dams will be tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.

Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated as * throughout the report.

Reproductive indices:

Embryonic resorption index, Fetal resorption index, Implantation index (%), Live fetus index, Dead fetus index were examined.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed throughout the experimental period at any of the doses tested.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

There was no morbidity or mortality throughout the experimental period in the 100, 300 and 1000 mg/kg/ day dose groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The maternal group mean body weights and maternal body weight gain were unaffected by the administration of test chemical at the doses of 100, 300 and 1000 nmg/kg/day and were statistically comparable to vehicle control group. The corrected body weight gain was statistically significantly increased at 1000 mg/kg/day as compared to vehicle control group indicating that body weights of rats were not affected by treatment with test chemical up to the high dose of 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The maternal food consumption was comparable to vehicle control group during different periods of gestation up to the high dose of 1000 mg/kg/day.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No effect on early and late resorptions were observed in treated female rats as compared to control.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

No effect on mean numbers of corpora lutea, implantations, early and late resorptions, pre and post-implantation loss rates and dams with any resorptions were observed in treated female rat as compared to control.

There were no clinical signs observed throughout the experimental period at any of the doses tested. There was no morbidity or mortality throughout the experimental period in the 100, 300 and 1000 mg/kg/ day dose groups. The maternal group mean body weights and maternal body weight gain were unaffected by the administration of test chemical at the doses of 100, 300 and 1000 nmg/kg/day and were statistically comparable to vehicle control group. The corrected body weight gain was statistically significantly increased at 1000 mg/kg/day as compared to vehicle control group indicating that body weights of rats were not affected by treatment with test chemical up to the high dose of 1000 mg/kg/day. The maternal food consumption was comparable to vehicle control group during different periods of gestation up to the high dose of 1000 mg/kg/day. No effect on gravid uterine weights were observed in treated female rat as compared to control. There were no gross pathological findings in any of the tested doses. No effect on early and late resorptions were observed in treated female rats as compared to control. No effect on mean numbers of corpora lutea, implantations, early and late resorptions, pre and post-implantation loss rates and dams with any resorptions were observed in treated female rat as compared to control.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

reproductive function (oestrous cycle)

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

No effect on total number of fetuses were observed as compared to control.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in weight of male and female fetuses at 100, 300 and 1000 mg/kg /day.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed during external observations of fetuses of dams treated up to 1000 mg/kg/day.

Normal variants: There was no incidence of normal variant in any of the doses tested.

Minor anomalies: There was an incidence of a small fetus each in the vehicle control and low dose group. This finding was not of any toxicological significance as this is commonly observed in rat fetuses.

Major malformations: There was no incidence of major malformation in any of the doses tested.

Histopathological findings:

no effects observed

Description (incidence and severity):

No changes were observed during visceral examination of fetuses of dams treated up to 1000 mg/kg/day.

Normal Variants, Minor anomalies and Major malformations

There were no incidences of normal variants, minor anomalies and major malformations in any of the doses tested.

No treatment-related changes were observed during skeletal examination of fetuses of dams treated up to 1000 mg/kg/day.

Normal Variants: Incidences of normal variants were comparable between the vehicle control and the treatment groups.

Minor anomalies: The incidences of minor anomalies were statistically comparable to vehicle control group at all the doses tested.

Major malformations: There were no incidences of major malformations in any of the treatment groups.

Other effects:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in sex ratio of male and female fetuses at 100, 300 and 1000 mg/kg /day.

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

No effect on total number of fetuses were observed as compared to control. No treatment-related changes were observed in weight of male and female fetuses at 100, 300 and 1000 mg/kg /day. No treatment-related changes were observed during external observations of fetuses of dams treated up to 1000 mg/kg/day.
Normal variants: There was no incidence of normal variant in any of the doses tested.
Minor anomalies: There was an incidence of a small fetus each in the vehicle control and low dose group. This finding was not of any toxicological significance as this is commonly observed in rat fetuses.
Major malformations: There was no incidence of major malformation in any of the doses tested.
No changes were observed during visceral examination of fetuses of dams treated up to 1000 mg/kg/day.
Normal Variants, Minor anomalies and Major malformations
There were no incidences of normal variants, minor anomalies and major malformations in any of the doses tested.
No treatment-related changes were observed during skeletal examination of fetuses of dams treated up to 1000 mg/kg/day.
Normal Variants: Incidences of normal variants were comparable between the vehicle control and the treatment groups.
Minor anomalies: The incidences of minor anomalies were statistically comparable to vehicle control group at all the doses tested.
Major malformations: There were no incidences of major malformations in any of the treatment groups.
No treatment-related changes were observed in sex ratio of male and female fetuses at 100, 300 and 1000 mg/kg /day.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

gross pathology

histopathology: non-neoplastic

other: Sex retio

Remarks on result:

other: No effect observed

Critical effects observed:

no

Reproductive effects observed:

no

Treatment related:

no

Summary of Clinical Signs and Mortality

	Group No.	G1	G2	G3	G4
Observations	Dose (mg/kg/day)	0	100	300	1000
	Total No. of rats found sperm positive	24	24	24	24
Clinical signs		NAD	NAD	NAD	NAD
Mortality		-------------None------------

Summary of maternal group mean body weight (g)

Group No.	Dose (mg/kg/day)	No. of Rats	Group mean body weight (g) on GD
				0	3	5	8	11	14	17	20
G1	0	21	Mean	214.18	222.98	229.95	238.57	252.79	263.87	288.02	314.65
			SD	14.23	14.95	14.68	15.39	16.72	16.62	18.80	22.58
G2	100	23	Mean	216.39	226.08	234.32	242.56	256.20	267.26	291.98	320.04
			SD	15.37	17.51	18.91	20.47	22.54	24.88	27.77	32.57
G3	300	23	Mean	216.68	226.07	233.55	241.12	255.23	266.69	289.97	319.69
			SD	14.73	17.20	18.51	19.99	22.27	22.58	23.32	25.81
G4	1000	24	Mean	214.93	223.73	230.53	239.52	252.39	262.80	285.57	317.15
			SD	13.14	15.71	17.55	20.17	22.05	23.02	27.70	31.98

Summary of maternal body weight gain (g)

Period of treatment (days of gestation)	Group No.		G1	G2	G3	G4
	Dose (mg/kg/day)		0	100	300	1000
	No. of Rats		21	23	23	24
Pre-treatment period (Days 0-5)		Mean	15.77	17.93	16.88	15.60
		SD	3.97	5.36	5.59	5.91
Treatment Period (Days 5-20)		Mean	84.70	85.72	86.13	86.62
		SD	11.05	15.85	11.20	19.02
Entire gestation (Days 0-20)		Mean	100.47	103.65	103.01	102.21
		SD	13.57	19.70	15.14	22.16

 

Summary of Corrected Body Weight and Body Weight Gain (g)

Group No.	Dose (mg/kg/day)	No. of Rats		Gestation Day 5	Terminal body wt on GD 20	Uterine Wt (g)	Carcass weight (corrected body weight on GD 20)	Corrected Bwt gain. (g)
G1	0	21	Mean	58.07	26.63	70.64	244.01	14.06
			SD	7.33	6.78	7.69	20.84	11.60
G2	100	23	Mean	57.65	28.07	64.08	255.97	21.64
			SD	11.05	6.96	12.29	25.60	11.09
G3	300	23	Mean	56.41	29.72	65.39	254.30	20.75
			SD	8.28	6.40	12.49	23.09	10.63
G4	1000	24	Mean	55.04	31.58	60.94	256.21	25.67*
			SD	13.67	7.59	20.30	23.17	9.44

  *: Significantly different from G1                                                 GD: Gestation day          

 

Corrected body weight on gestation day 20 (Carcass weight) = Terminal body weight on
day 20 - unopened uterine weight.

 

Corrected body weight gain = carcass weight - body weight on day 5.

Summary of Maternal Data

	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	100	300	1000
	No. of Rats	21	23	23	24
Gravid uterine weight (g)	Mean	70.64	64.08	65.39	60.94
	SD	7.69	12.29	12.49	20.30
Number of Corpora lutea	Mean	14.38	13.52	13.70	13.13
	SD	1.32	1.50	2.08	2.05
Number of Implantations	Mean	13.43	12.74	12.83	11.42
	SD	1.60	2.32	2.62	3.90
Early Resorptions	Mean	0.43	0.61	0.57	0.25
	SD	0.68	0.89	0.73	0.44
Late Resorptions	Mean	0.05	0.13	0.09	0.13
	SD	0.22	0.34	0.29	0.34
Pre-implantation Loss	Mean	0.95	0.78	0.87	1.71
	SD	1.32	1.31	1.01	2.26
Post-implantation Loss	Mean	0.48	0.74	0.65	0.38
	SD	0.68	0.92	0.71	0.49
Dams with any Resorption	Total	8	11	12	9

	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	100	300	1000
	No. of Rats	21	23	23	24
Early Resorptions (%)	Mean	3.02	5.12	4.20	2.10
	SD	4.76	7.26	5.35	3.77
Late Resorptions (%)	Mean	0.34	0.98	1.30	0.94
	SD	1.56	2.60	4.58	2.54
Pre-implantation Loss (%)	Mean	6.46	6.32	7.06	15.85
	SD	8.78	11.86	9.82	24.98
Post-implantation Loss (%)	Mean	3.36	6.10	5.50	3.03
	SD	4.79	7.43	6.17	4.06
Implantation Index (%)	Mean	93.54	93.68	92.94	84.15
	SD	8.78	11.86	9.82	24.98

 Summary of Litter Data

	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	100	300	1000
	No. of Rats	21	23	23	24
No. of litters		21	23	23	24
Total no. of fetuses		272	276	280	265
Mean litter size		13.0	12.0	12.2	11.0
Total live fetuses
a.  Number		272	276	280	265
b.  Weight (g)	Mean	3.57	3.48	3.49	3.66
	SD	0.24	0.21	0.29	0.47
Live male fetuses
a.  Number		146	134	142	137
b.  Weight (g)	Mean	3.66	3.56	3.58	3.73
	SD	0.26	0.23	0.30	0.48
Live female fetuses
a.  Number		126	142	138	128
b.  Weight (g)	Mean	3.46	3.41	3.41	3.52
	SD	0.22	0.21	0.30	0.23
Sex Ratio - Male : Female		1:0.86	1:1.06	1:0.97	1:0.93
(% of male fetuses)		(53.7%)	(48.6%)	(50.7%)	(51.7%)

 Summary of Gross Pathological Findings

Group No.	G1	G2	G3	G4
Parameters  Dose (mg/kg/day)	0	100	300	1000
1.  No. of rats subjected to caesarean section	24	24	24	24
2.  No. of rats pregnant at caesarean section	21	23	23	24
3.  No. of rats showing gross pathology	0	0	0	0

Summary of Fetal External Observations (Incidence and Percentage)

Group No.	G1				G2				G3				G4
Dose (mg/kg/day)	0				100				300				1000
No. of litters examined	21				23				23				24
No. of fetuses examined	272				276				280				265
	Fetus		Litter		Fetus		Litter		Fetus		Litter		Fetus		Litter
	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%
Normal Variant	None
Minor Anomalies
Small fetus	1	0.37	1	4.76	1	0.36	1	4.35	0	0.00	0	0.00	0	0.00	0	0.00
Major Malformations	None

 Summary of Fetal Visceral Observations (Incidence and Percentage)

Group No.	G1				G2				G3				G4
Dose (mg/kg/day)	0				100				300				1000
No. of litters examined	21				23				23				24
No. of fetuses examined	131				132				132				126
	Fetus		Litter		Fetus		Litter		Fetus		Litter		Fetus		Litter
	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%
Normal Variant	None
Minor Anomalies	None
Major Malformations	None

Conclusions:

Based on all the available data, it was concluded that the test chemical did not cause any maternal reproductive toxicity and fetal toxicity when pre-mated female Wistar rats were administered with the test chemical from Gestation Day 5 to 19. Therefore, the NOAEL for maternal systemic and reproductive toxicity and fetal toxicity was considered to be 1000 mg/kg bw.

Executive summary:

In a Prenatal Developmental Toxicity Study according to the OECD test guideline 414, Wistar female rats were treated with the test chemical in the concentration of 0, 100, 300 and 1000 mg/kg bw by oral route during gestation days GD 5 to GD19. No morbidity or mortality throughout the experimental period in the 100, 300 and 1000 mg/kg/day dose groups were observed as compared to control. No effect on clinical signs and body weights and maternal body weight gain were observed. The corrected body weight gain was statistically significantly increased at 1000 mg/kg/day as compared to vehicle control group indicating that body weights of rats were not affected by treatment with test item up to the high dose of 1000 mg/kg/day. The maternal food consumption was comparable to vehicle control group during different periods of gestation up to the high dose of 1000 mg/kg/day. Similarly, No effect on gravid uterine weights and gross pathology of treated rats were observed as compared to control. No reproductive effect were observed such as no effect on mean numbers of corpora lutea, implantations, early and late resorptions, pre and post-implantation loss rates and dams with any resorptions were unaffected by treatment. No treatment-related changes were observed in litter data parameters comprising of total number of fetuses, number and weight of male and female fetuses and sex ratio at 100, 300 and 1000 mg/kg /day. In addition, no gross pathological, external observations, visceral and skeletal changes were observed in fetuses of dams treated up to 1000 mg/kg/day. Therefore, based on all the available data, it was concluded that the test chemical did not cause any maternal reproductive toxicity and fetal toxicity when pre-mated female Wistar rats were administered with the test chemical from Gestation Day 5 to 19. Therefore, the NOAEL for maternal systemic and reproductive toxicity and fetal toxicity was considered to be 1000 mg/kg bw.

Reference 3

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 4

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is from a Klimisch 1 datasource and provide a robust study summary.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive toxicity:

In different studies,the test chemical has been investigated for reproductive toxicity to a greater or lesser extent. Often are the studies based on in vivo experiments in rodents, i.e. most commonly in rats for the test chemical are summarized below:

Study 1:

The purpose of this repeated dose toxicity study was to evaluate the systemic toxicity profile of the test chemical in Wistar rats when administered orally by gavage for 90 consecutive days and to assess the reversibility of any effects during a subsequent 28 days recovery period. This study was also intended to provide the information on major toxic effects, target organs and an estimation of a No Observed Adverse Effect Level (NOAEL). The test item was weighed and dissolved in vehicle i.e., Milli-Q® Water and administered to rats at the graduated dose levels of 100, 300, and 1000 mg/kg/day for low dose (G2), mid dose (G3) and high dose (G4) / high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1)/ vehicle control recovery (G1R) groups received vehicle (Milli-Q® Water) alone. The dose volume administered was 10 mL/kg body weight. Each main groups in the experiment was comprised of 10 males and 10 female rats and recovery groups comprised of 5 males and 5 female rats. The identity of the test chemical was provided by the study sponsor by a Certificate of Analysis. The stability and homogeneity of the test item in the vehicle was established at 0.7 and 125 mg/mL. Based on the results, the test item was stable and homogenous in the vehicle (Milli-Q® water) up to 48 hours when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during 2nd (Day 45) and 3rd month (Day 71) of the treatment period. The results indicated that the analysed concentrations were within ± 10% of variations from the claimed concentrations. Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and at the end of recovery period for recovery groups. The body weights and food consumption were measured during in-life phase of the experiment. Neurological examinations were conducted towards the end of treatment for main groups and towards the end of recovery period for recovery groups. Vaginal smear was examined, and the stage of oestrous cycle was recorded daily for two weeks prior to necropsy for main and recovery groups. The clinical laboratory investigations such as haematology, coagulation, clinical chemistry and urine analysis were performed at termination. Blood samples were also analyzed for Thyroid Stimulating Hormone (TSH), Thyroxin (T4), Testosterone (Testo) and Estrogen (Estradiol- All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratio were derived as percent fasting body weights. For all surviving males at termination, sperm from the right vas deferens were collected for evaluation of sperm motility using Hamilton-Thorne TOX-IVOS (version 12) sperm analyzer. Histopathological examination was carried out on the preserved organs of the vehicle control and high dose group animals (with qualitative assessment of stages of spermatogenesis in male rats). Stages of estrus cycle in female rats was also recorded during histopathology examination. There were no treatment-related microscopic changes observed in high dose group and hence, the organ/tissue in the mid and low dose groups and recovery groups were examined microscopically. During observations, there were no clinical signs or mortality observed at any of the doses tested in both sexes. Ophthalmological examination did not reveal any ocular abnormalities. No treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested. The mean body weights and food consumption were unaffected by the treatment at all the tested doses in both the sexes. No test item-related changes were observed in the haematological, coagulation, clinical chemistry and urine parameters at all the doses tested in both sexes. There were no significant changes in testosterone, estrogen, thyroid stimulating hormone (TSH), and thyroxin hormone (T4) levels at all dose levels tested. There were no test item-related changes in terminal fasting body weight and organ weights in males and females at all the dose levels tested. There were no test item-related changes in sperm motility at all the dose levels tested. There were no test item-related gross or microscopic changes at all the dose levels tested in both sexes. The staging of spermatogenesis in male rats did not reveal specific changes. Staging of  estrous cycle in female rats did not reveal any stage specific changes and all stages of estrous cycle were randomly distributed without any stage arrest. Therefore, since there were no adverse effects observed up to the highest dose tested, the evaluated No Observed Adverse Effect Level (NOAEL) of the test chemical is considered to be 1000 mg/kg/day following oral gavage administration for 90 consecutive days to Wistar rats under the test conditions and doses employed.

Study 2:

In a Prenatal Developmental Toxicity Study according to the OECD test guideline 414, Wistar female rats were treated with the test chemical in the concentration of 0, 100, 300 and 1000 mg/kg bw by oral route during gestation days GD 5 to GD19. No morbidity or mortality throughout the experimental period in the 100, 300 and 1000 mg/kg/day dose groups were observed as compared to control. No effect on clinical signs and body weights and maternal body weight gain were observed. The corrected body weight gain was statistically significantly increased at 1000 mg/kg/day as compared to vehicle control group indicating that body weights of rats were not affected by treatment with test item up to the high dose of 1000 mg/kg/day. The maternal food consumption was comparable to vehicle control group during different periods of gestation up to the high dose of 1000 mg/kg/day. Similarly, No effect on gravid uterine weights and gross pathology of treated rats were observed as compared to control. No reproductive effect were observed such as no effect on mean numbers of corpora lutea, implantations, early and late resorptions, pre and post-implantation loss rates and dams with any resorptions were unaffected by treatment. No treatment-related changes were observed in litter data parameters comprising of total number of fetuses, number and weight of male and female fetuses and sex ratio at 100, 300 and 1000 mg/kg /day. In addition, no gross pathological, external observations, visceral and skeletal changes were observed in fetuses of dams treated up to 1000 mg/kg/day. Therefore, based on all the available data, it was concluded that the test chemical did not cause any maternal reproductive toxicity and fetal toxicity when pre-mated female Wistar rats were administered with the test chemical from Gestation Day 5 to 19. Therefore, the NOAEL for maternal systemic and reproductive toxicity and fetal toxicity was considered to be 1000 mg/kg bw.

Based on all the available data, it was concluded that the test chemical did not have any adverse effects on the reproduction and developmental parameters at the highest dose tested i.e. 1000 mg/kg bw/day. Therefore, the test chemical is not likely to classify as 'reproductive and/or developmental toxicant' as per the CLP criteria of classifiction and labelling.

Effects on developmental toxicity

Description of key information

Developmental Toxicity:

Based on all the available data, it was concluded that the test chemical did not have any adverse effects on the maternal systemic or reproductive and fetal parameters at the highest dose tested i.e. 1000 mg/kg bw/day. Therefore, the test chemical is not likely to classify as 'reproductive and/or developmental toxicant' as per the CLP criteria of classifiction and labelling.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(Adopted on January 22, 2001)

Principles of method if other than guideline:

According to OECD Guideline 414 (Prenatal Developmental Toxicity Study) (Adopted on January 22, 2001)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana
- Age at study initiation: 13 to 14 weeks
- Weight at study initiation: 214.952 ± 13.13 to 196.16 to 246.01
- Fasting period before study:
- Housing: Rats were housed in standard polysulfone rat cages (size: Length 425 mm x Breadth 266 mm x Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube. Additionally, polycarbonate rat huts were placed inside the cage as environmental enrichment objects which were replaced once a week.

Following was the housing pattern during different periods of the experiment:
i. Pre mating / Acclimatization: Two rats of the same sex per cage were housed per cage.
ii. Mating: Female rats were cohabited with males in a 1:1 ratio in same cage.
iii. Post-mating / Treatment: After mating confirmation, females were housed individually.

Steam sterilized corn cob was used as bedding and was changed along with the cage at least twice a week.
- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet – pellet (Certified), ad libitum
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier, ad libitum
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 65 – 66 %
- Air changes (per hr): adequate fresh air supply (12 to 15 air changes/hour).
- Photoperiod (hrs dark / hrs light):12 hours light and 12 hours dark cycle.

IN-LIFE DATES: From: 27 October 2017
To:29 March 2018

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Remarks:

Milli-Q®

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The dose formulations were freshly prepared daily before dosing and were used within the stability period.

The required quantity of test item was weighed in pre-calibrated beaker*. A small volume (3 to 40 mL) of vehicle (Milli-Q® water) was added and mixed well using a glass rod. The final volume was made up with the vehicle to get the required final concentration. The volume was made up to the upper meniscus during dose formulation preparation.

*Pre-calibration of the beaker to desired volume: Milli-Q® water was measured in a graduated measuring cylinder to the final volume of the batch size (example if batch size was 70 mL, water was taken till 70 mL mark in graduated measuring cylinder). The measured water was transferred into a clean beaker (to be pre-calibrated) and upper and lower meniscus of water were marked on the beaker using a marker. Once these lines were marked, the water was discarded and the beaker was dried.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the solubility test conducted under study, the test item is soluble in Milli-Q® water. Hence, Milli-Q® water was used as vehicle for dose formulation preparation.
- Concentration in vehicle: 0, 100, 300 and 1000 mg/kg bw
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and stability of the test chemical in vehicle was established at the concentrations of 0.7 mg/mL and 125 mg/mL. Based on the results, the test item was stable and homogenous in the vehicle up to 48 hours when stored at room temperature.

Details on mating procedure:

- Impregnation procedure: [artificial insemination / purchased timed pregnant / cohoused]: Cohoused
- If cohoused: the female rats were cohabited with males
- M/F ratio per cage: 1:1 ratio
- Length of cohabitation: On the first day, after vaginal smear examination, all the females with proestrous and estrous stages were retained with the males; while the other females were separated from males. Subsequently females were cohabited in batches of required numbers. These females were cohabited with respective males in a 1:1 ratio. The details were recorded in raw data. This procedure was continued until there were sufficient numbers of Day 0 pregnant rats for the study.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. No Data Available
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: [yes, vaginal smear were examination]
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol: Not specified.

Duration of treatment / exposure:

15 days

Frequency of treatment:

Daily

Duration of test:

From GD 5 to GD 19 of presumed gestation

Dose / conc.:

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

Remarks:

Control Group

Dose / conc.:

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

Remarks:

Low Dose Group

Dose / conc.:

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

Remarks:

Mid Dose Group

Dose / conc.:

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

Remarks:

High Dose Group

No. of animals per sex per dose:

Total: 96
0 mg/kg bw: 24 female
100 mg/kg bw: 24 female
300 mg/kg bw: 24 female
1000 mg/kg bw: 24 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose range finding study was carried out at Test Facility using seven day ‘0’ pregnant rats per group at the doses of 100 (G2), 300 (G3) and 1000 (G4) mg/kg/day along with the concurrent vehicle control (G1). No effect were observed in treated female and Fetal examination. Therefore, based on the results of dose range finding study doses were selected as 0, 100, 300 and 1000 mg/kg bw.
- Rationale for animal assignment (if not random): Rats were randomly distributed to different groups by body weight stratification. Based on the body weight, Day ‘0’ pregnant rats were arranged in the ascending order. These rats were then assigned to the groups starting either from control and treatment group/s in the increasing order of dose or vice-versa on GD 0. As far as possible rats were assigned equally to the study groups, the extra animals remaining were also assigned to groups. This difference was negated on subsequent day/s by assigning rats in such a way that there were equal numbers of rats in all the groups.
- Other: No Data Available

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day - pre dose and post dose (30 minutes to one hour post dose, approx.) during treatment days and once on non-treatment days
- Cage side observations checked in table [No.?] were included. : morbidity and mortality i.e., once in the morning and once in the afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: weighed on gestation days 0, 3, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day # : On gestation day 20
- Organs examined: all visceral organs of dams were examined.

OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Gross evaluation of placenta

Fetal examinations:

- External examinations: Yes:All the fetuses were examined
- Soft tissue examinations: Yes: Approximately one half of the fetuses from each litter were subjected to visceral
- Skeletal examinations: Yes: the remaining half of the fetuses were subjected to skeletal evaluation.
- Head examinations: No data

Statistics:

The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female were analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss, external, visceral and skeletal observations for variations were analyzed using Kruskal wallis test for group comparison.

The incidence of with and without resorptions in dams will be tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.

Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated as * throughout the report.

Indices:

Embryonic resorption index, Fetal resorption index, Implantation index (%), Live fetus index, Dead fetus index were examined.

Historical control data:

Not specified

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed throughout the experimental period at any of the doses tested.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

There was no morbidity or mortality throughout the experimental period in the 100, 300 and 1000 mg/kg/day dose groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The maternal group mean body weights and maternal body weight gain were unaffected by the administration of test chemical at the doses of 100, 300 and 1000 mg/kg/day and were statistically comparable to vehicle control group.

The corrected body weight gain was statistically significantly increased at 1000 mg/kg/day as compared to vehicle control group indicating that body weights of rats were not affected by treatment with test chemical up to the high dose of 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The maternal food consumption was comparable to vehicle control group during different periods of gestation up to the high dose of 1000 mg/kg/day.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effect on gravid uterine weights were observed in treated female rat as compared to control.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological findings in any of the tested doses.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No effect on pre and post-implantation loss rates were observed in treated female rats as compared to control.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No effect on loss rates were observed in treated female rats as compared to control.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No effect on early and late resorptions were observed in treated female rats as compared to control.

Dead fetuses:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in dead fetuses as compaed to control.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

No effect on preganacy duration were observed in treated female rats as compared to control.

Changes in number of pregnant:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

maternal abnormalities

mortality

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in weight of male and female fetuses at 100, 300 and 1000 mg/kg /day.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No reduction in the number of live male and female fetuses were observed due to the administration of the test chemical.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in sex ratio of male and female fetuses at 100, 300 and 1000 mg/kg /day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No adverse effects or changes in the mean litter sizes and weights were observed due to the administration of the test chemical.

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed during external observations of fetuses of dams treated up to 1000 mg/kg/day.

Normal variants: There was no incidence of normal variant in any of the doses tested.

Minor anomalies: There was an incidence of a small fetus each in the vehicle control and low dose group. This finding was not of any toxicological significance as this is commonly observed in rat fetuses.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed during skeletal examination of fetuses of dams treated up to 1000 mg/kg/day.

Normal Variants: Incidences of normal variants were comparable between the vehicle control and the treatment groups.

Minor anomalies: The incidences of minor anomalies were statistically comparable to vehicle control group at all the doses tested.

Major malformations: There were no incidences of major malformations in any of the treatment groups.

Visceral malformations:

no effects observed

Description (incidence and severity):

No changes were observed during visceral examination of fetuses of dams treated up to 1000 mg/kg/day.

Normal Variants, Minor anomalies and Major malformations: There were no incidences of normal variants, minor anomalies and major malformations in any of the doses tested.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Treatment related:

no

 Summary of Clinical Signs and Mortality

	Group No.	G1	G2	G3	G4
Observations	Dose (mg/kg/day)	0	100	300	1000
	Total No. of rats found sperm positive	24	24	24	24
Clinical signs		NAD	NAD	NAD	NAD
Mortality		-------------None------------

Summary of maternal group mean body weight (g)

Group No.	Dose (mg/kg/day)	No. of Rats	Group mean body weight (g) on GD
				0	3	5	8	11	14	17	20
G1	0	21	Mean	214.18	222.98	229.95	238.57	252.79	263.87	288.02	314.65
			SD	14.23	14.95	14.68	15.39	16.72	16.62	18.80	22.58
G2	100	23	Mean	216.39	226.08	234.32	242.56	256.20	267.26	291.98	320.04
			SD	15.37	17.51	18.91	20.47	22.54	24.88	27.77	32.57
G3	300	23	Mean	216.68	226.07	233.55	241.12	255.23	266.69	289.97	319.69
			SD	14.73	17.20	18.51	19.99	22.27	22.58	23.32	25.81
G4	1000	24	Mean	214.93	223.73	230.53	239.52	252.39	262.80	285.57	317.15
			SD	13.14	15.71	17.55	20.17	22.05	23.02	27.70	31.98

Summary of maternal body weight gain (g)

Period of treatment (days of gestation)	Group No.		G1	G2	G3	G4
	Dose (mg/kg/day)		0	100	300	1000
	No. of Rats		21	23	23	24
Pre-treatment period (Days 0-5)		Mean	15.77	17.93	16.88	15.60
		SD	3.97	5.36	5.59	5.91
Treatment Period (Days 5-20)		Mean	84.70	85.72	86.13	86.62
		SD	11.05	15.85	11.20	19.02
Entire gestation (Days 0-20)		Mean	100.47	103.65	103.01	102.21
		SD	13.57	19.70	15.14	22.16

 

Summary of Corrected Body Weight and Body Weight Gain (g)

Group No.	Dose (mg/kg/day)	No. of Rats		Gestation Day 5	Terminal body wt on GD 20	Uterine Wt (g)	Carcass weight (corrected body weight on GD 20)	Corrected Bwt gain. (g)
G1	0	21	Mean	58.07	26.63	70.64	244.01	14.06
			SD	7.33	6.78	7.69	20.84	11.60
G2	100	23	Mean	57.65	28.07	64.08	255.97	21.64
			SD	11.05	6.96	12.29	25.60	11.09
G3	300	23	Mean	56.41	29.72	65.39	254.30	20.75
			SD	8.28	6.40	12.49	23.09	10.63
G4	1000	24	Mean	55.04	31.58	60.94	256.21	25.67*
			SD	13.67	7.59	20.30	23.17	9.44

  *: Significantly different from G1                                                 GD: Gestation day          

 

Corrected body weight on gestation day 20 (Carcass weight) = Terminal body weight on
day 20 - unopened uterine weight.

 

Corrected body weight gain = carcass weight - body weight on day 5.

Summary of Maternal Data

	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	100	300	1000
	No. of Rats	21	23	23	24
Gravid uterine weight (g)	Mean	70.64	64.08	65.39	60.94
	SD	7.69	12.29	12.49	20.30
Number of Corpora lutea	Mean	14.38	13.52	13.70	13.13
	SD	1.32	1.50	2.08	2.05
Number of Implantations	Mean	13.43	12.74	12.83	11.42
	SD	1.60	2.32	2.62	3.90
Early Resorptions	Mean	0.43	0.61	0.57	0.25
	SD	0.68	0.89	0.73	0.44
Late Resorptions	Mean	0.05	0.13	0.09	0.13
	SD	0.22	0.34	0.29	0.34
Pre-implantation Loss	Mean	0.95	0.78	0.87	1.71
	SD	1.32	1.31	1.01	2.26
Post-implantation Loss	Mean	0.48	0.74	0.65	0.38
	SD	0.68	0.92	0.71	0.49
Dams with any Resorption	Total	8	11	12	9

	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	100	300	1000
	No. of Rats	21	23	23	24
Early Resorptions (%)	Mean	3.02	5.12	4.20	2.10
	SD	4.76	7.26	5.35	3.77
Late Resorptions (%)	Mean	0.34	0.98	1.30	0.94
	SD	1.56	2.60	4.58	2.54
Pre-implantation Loss (%)	Mean	6.46	6.32	7.06	15.85
	SD	8.78	11.86	9.82	24.98
Post-implantation Loss (%)	Mean	3.36	6.10	5.50	3.03
	SD	4.79	7.43	6.17	4.06
Implantation Index (%)	Mean	93.54	93.68	92.94	84.15
	SD	8.78	11.86	9.82	24.98

 Summary of Litter Data

	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	100	300	1000
	No. of Rats	21	23	23	24
No. of litters		21	23	23	24
Total no. of fetuses		272	276	280	265
Mean litter size		13.0	12.0	12.2	11.0
Total live fetuses
a.  Number		272	276	280	265
b.  Weight (g)	Mean	3.57	3.48	3.49	3.66
	SD	0.24	0.21	0.29	0.47
Live male fetuses
a.  Number		146	134	142	137
b.  Weight (g)	Mean	3.66	3.56	3.58	3.73
	SD	0.26	0.23	0.30	0.48
Live female fetuses
a.  Number		126	142	138	128
b.  Weight (g)	Mean	3.46	3.41	3.41	3.52
	SD	0.22	0.21	0.30	0.23
Sex Ratio - Male : Female		1:0.86	1:1.06	1:0.97	1:0.93
(% of male fetuses)		(53.7%)	(48.6%)	(50.7%)	(51.7%)

 Summary of Gross Pathological Findings

Group No.	G1	G2	G3	G4
Parameters  Dose (mg/kg/day)	0	100	300	1000
1.  No. of rats subjected to caesarean section	24	24	24	24
2.  No. of rats pregnant at caesarean section	21	23	23	24
3.  No. of rats showing gross pathology	0	0	0	0

Summary of Fetal External Observations (Incidence and Percentage)

Group No.	G1				G2				G3				G4
Dose (mg/kg/day)	0				100				300				1000
No. of litters examined	21				23				23				24
No. of fetuses examined	272				276				280				265
	Fetus		Litter		Fetus		Litter		Fetus		Litter		Fetus		Litter
	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%
Normal Variant	None
Minor Anomalies
Small fetus	1	0.37	1	4.76	1	0.36	1	4.35	0	0.00	0	0.00	0	0.00	0	0.00
Major Malformations	None

 Summary of Fetal Visceral Observations (Incidence and Percentage)

Group No.	G1				G2				G3				G4
Dose (mg/kg/day)	0				100				300				1000
No. of litters examined	21				23				23				24
No. of fetuses examined	131				132				132				126
	Fetus		Litter		Fetus		Litter		Fetus		Litter		Fetus		Litter
	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%	Inc.	%
Normal Variant	None
Minor Anomalies	None
Major Malformations	None

Conclusions:

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for Maternal toxicity and fetal developmental toxicity is 1000 mg/kg/day in Wistar rats when the test chemical was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.

Executive summary:

The objective of this study was to evaluate the embryo-fetal developmental toxicity of test chemical when administered to pregnant Wistar rats by oral route during gestation days GD 5 to GD 19. The results of this study helped to establish the No Observed Adverse Effect Level (NOAEL) of the test item for maternal and developmental toxicity in rats. In this study, each group (G1, G2, G3 and G4) consisted of 24 presumed pregnant Wistar rats (gestation day 0). Day '0' of gestation for each individual female rat in the study was considered as the day on which vaginal plug was observed or vaginal smear was found sperm positive. The test chemical was dissolved in vehicle - Milli-Q water and administered orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 100, 300 and 1000 mg/kg/day for low (G2), mid (G3) and high (G4) dose group rats, respectively. The rats in the vehicle control (G1) group received the vehicle alone. A constant dose volume of 10 mL/kg body weight was administered to all groups. The dose formulation solutions were analyzed for active ingredient concentrations at the initiation and termination of treatment. The results of analysis of formulations revealed that the analyzed concentrations were within the acceptable limits. The mated females were observed twice daily for clinical signs, mortality and morbidity. Body weights and food consumption were monitored during the different periods of gestation. The intermittent body weight gain and food consumption was calculated and presented for rats found pregnant at caesarean section. Caesarean section was performed for all the rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the dams was removed (by laparotomy) and the contents were examined. The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. The number of corpora lutea was counted on each ovary. All the fetuses were sexed, weighed and examined for external malformations. Approximately half the number of fetuses from each litter was examined for visceral malformations and the remaining half was evaluated for skeletal malformations. During observations, there were no mortalities and clinical signs at all the doses tested. The maternal body weights and food consumption were comparable to vehicle control group up to the highest dose of 1000 mg/kg/day. The maternal data parameters comprising of mean number of corpora lutea, implantations, number of early and late resorptions, pre and post implantation loss and dams with any resorptions were comparable to vehicle control group at all the doses tested. Gross evaluation of placenta revealed no findings. The litter data parameters comprising of total number of fetuses, mean weights of male and female fetuses and sex ratio were comparable to vehicle control group at all the doses tested. Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs of teratogenicity in any of the tested doses. Therefore, based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for Maternal toxicity and fetal developmental toxicity is 1000 mg/kg/day in Wistar rats when the test chemical was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is from a Klimisch 1 database and provides a robut study summary.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity:

The objective of this study was to evaluate the embryo-fetal developmental toxicity of test chemical when administered to pregnant Wistar rats by oral route during gestation days GD 5 to GD 19. The results of this study helped to establish the No Observed Adverse Effect Level (NOAEL) of the test item for maternal and developmental toxicity in rats. In this study, each group (G1, G2, G3 and G4) consisted of 24 presumed pregnant Wistar rats (gestation day 0). Day '0' of gestation for each individual female rat in the study was considered as the day on which vaginal plug was observed or vaginal smear was found sperm positive. The test chemical was dissolved in vehicle - Milli-Q water and administered orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 100, 300 and 1000 mg/kg/day for low (G2), mid (G3) and high (G4) dose group rats, respectively. The rats in the vehicle control (G1) group received the vehicle alone. A constant dose volume of 10 mL/kg body weight was administered to all groups. The dose formulation solutions were analyzed for active ingredient concentrations at the initiation and termination of treatment. The results of analysis of formulations revealed that the analyzed concentrations were within the acceptable limits. The mated females were observed twice daily for clinical signs, mortality and morbidity. Body weights and food consumption were monitored during the different periods of gestation. The intermittent body weight gain and food consumption was calculated and presented for rats found pregnant at caesarean section. Caesarean section was performed for all the rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the dams was removed (by laparotomy) and the contents were examined. The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. The number of corpora lutea was counted on each ovary. All the fetuses were sexed, weighed and examined for external malformations. Approximately half the number of fetuses from each litter was examined for visceral malformations and the remaining half was evaluated for skeletal malformations. During observations, there were no mortalities and clinical signs at all the doses tested. The maternal body weights and food consumption were comparable to vehicle control group up to the highest dose of 1000 mg/kg/day. The maternal data parameters comprising of mean number of corpora lutea, implantations, number of early and late resorptions, pre and post implantation loss and dams with any resorptions were comparable to vehicle control group at all the doses tested. Gross evaluation of placenta revealed no findings. The litter data parameters comprising of total number of fetuses, mean weights of male and female fetuses and sex ratio were comparable to vehicle control group at all the doses tested. Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs of teratogenicity in any of the tested doses. Therefore, based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for Maternal toxicity and fetal developmental toxicity is 1000 mg/kg/day in Wistar rats when the test chemical was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.

Justification for classification or non-classification

Based on the above studies, it can be concluded that the test chemical is not likely to classify for the reproductive and developmental as it does not show any effects on reproduction and developmental parameters.