Sodium 3-nitrobenzenesulphonate

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 3-nitrobenzenesulphonate

EC Number:

204-857-3

EC Name:

Sodium 3-nitrobenzenesulphonate

Cas Number:

127-68-4

Molecular formula:

C6H5NO5S.Na

IUPAC Name:

sodium 3-nitrobenzenesulphonate

Test material form:

solid: granular

Details on test material:

- Name of test material (as cited in study report): Sodium-3-nitrobenzene sulphonate
- Molecular formula: C6H4NNaO5S
- Molecular weight: 225.15 g/mol
- Substance type: Organic
- Purity: 96.83%

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control Group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Control Group (Recovery)

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Low Dose Group

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Mid Dose Group

Doses / concentrations 5

Dose / conc.:

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

Remarks:

High Dose Group

Doses / concentrations 6

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Effect levels (P0)

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

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Basis for effect level:

other: Not Specified

Remarks on result:

not measured/tested

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

no

Applicant's summary and conclusion

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.