Registration Dossier

Administrative data

Endpoint:
extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)
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:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Principles of method if other than guideline:
According to OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals was 2 weeks
- Basis for dose level selection : Doses were selected on the basis of previously performed GLP compliant repeated dose 28 days oral toxicity study.
- Inclusion/exclusion of extension of Cohort 1B : Cohort 1B was included and examined in the study.
- Termination time for F2
- Inclusionof developmental neurotoxicity Cohorts 2A and 2B : Cohort 2A and 2B were included and examined in the study.
- Inclusion of developmental immunotoxicity Cohort 3 : Cohort 3 were included and examined in the study.
- Route of administration
- Other considerations, e.g.
- choice of species: Rat is the commonly used species for toxicity studies and recommended by the regulatory guidelines
- strain: Wistar strain is a commonly used strain to assess the reproduction and developmental toxicity in the reproductive toxicity studies.
- vehicle: Corn Oil was selected as a vehicle because the test chemical was found to be insoluble in water whereas corn oil provided adequate suspensibility and the formulation was found stable in this vehicle. Further, corn oil is widely used as vehicle in oral toxicity study and the selected concentration is well below the acceptable limit.
- number of animals: 240 animals were used in the study to generate adequate number of pups for further examinations (Cohort 1A and 1B, Cohort 2A and 2B and Cohort 3)

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl 2-naphthyl ether
EC Number:
202-213-6
EC Name:
Methyl 2-naphthyl ether
Cas Number:
93-04-9
Molecular formula:
C11H10O
IUPAC Name:
2-methoxynaphthalene
Test material form:
solid: crystalline
Details on test material:
Name: Methyl-2-naphthyl ether
CAS No.: 93-04-9
IUPAC Name: 2-Methoxynaphthalene
Molecular Weight: 158.19 g/mol
Molecular Formula: C11-H10-O
SMILES: COc1ccc2ccccc2c1

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is the commonly used species for toxicity studies and recommended by the regulatory guidelines and Wistar strain is a commonly used strain to assess the reproduction and developmental toxicity studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The test animals were procured from a CPCSEA approved vendor.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 12-14 weeks at the start of treatment wks; (F1) 11-12 weeks at the start of treatment wks
- Weight at study initiation: (P) Males: 291-413 g; Females: 198-277 g;
- Fasting period before study: No Data Available
- Housing: Two to four rats were housed in each polycarbonate cage. During mating, one male and one female rat were housed in a single cage. Pregnant and lactating females were housed individually. Sterilized corn-cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): A conventional laboratory pellet diet from approved supplier was available ad libitum.
- Water (e.g. ad libitum): Aquaguard™ filtered drinking water was available ad libitum in regularly cleaned bottles.
- Acclimation period: Animals were acclimatized for at least 5 days prior to test item administration.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.60 to 24.80°C
- Humidity (%): 34.40 to 66.70 %
- Air changes (per hr): At least 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES:
From: August 8, 2019
To: March 1, 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The vehicle used for dose administration was corn oil. Required quantity for each concentration level of test chemical was weighed and triturated in a mortar-pestle, first without vehicle and then in small volume of the vehicle. After thorough trituration, the test chemical and vehicle were transferred quantitatively into a calibrated measuring cylinder and diluted using vehicle up to the required volume of the formulation, before being transferred to the final container. Formulations were prepared on the same day or one day prior to dose administration and stored at room temperature until usage as the test item stability in solution was proved up to 24 hours. At the time of dosing, dose formulations were kept on a magnetic stirrer for maintaining homogeneity of test formulation. The dose formulation samples [of doses viz; G1 (Control), G2 (Low dose), G3 (Mid dose), and G4 (High dose)] were analyzed for homogeneity and concentration. The dose formulation analysis was carried out once on Day 1 of dose administration to animals, on day 91 and on day 182.

DIET PREPARATION
- Rate of preparation of diet (frequency): No Data Available
- Mixing appropriate amounts with (Type of food): No Data Available
- Storage temperature of food: No Data Available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn Oil was selected as a vehicle because the test chemical was found to be insoluble in water whereas corn oil provided adequate suspensibility and the formulation was found stable in this vehicle. Further, corn oil is widely used as vehicle in oral toxicity study and the selected concentration is well below the acceptable limit.
- Concentration in vehicle: 0 mg/kg bw: 0 mg/ml; 62.5 mg/kg bw: 15.63 mg/ml; 125 mg/kg bw: 31.25 mg/ml and 250 mg/kg bw: 62.5 mg/ml
- Amount of vehicle (if gavage): 4 mL/ kg
- Lot/batch no. (if required): Batch No.A1906001, N2191405, N2192016, O21904007
- Purity: No Data Available
Details on mating procedure:
- M/F ratio per cage: 1:1 ratio
- Length of cohabitation: 2 weeks until evidence of copulation was observed.
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: If mating had not occurred after 2 weeks, the animals were separated without further opportunity for mating.
- Further matings after two unsuccessful attempts: [no]
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: No Data Available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated with respect to the parameters covering specificity, linearity, limit of quantification (LOQ), precision (% RSD), Accuracy (% recovery) and Homogeneity. The analytical method was validated with respect to the following parameters as below:

1. Specificity: The result of specificity was reported either no interference observed based on visual comparison or the degree of interference does not contribute more than 20 % peak area of the target analyte at the LOQ Level.

2. Limit of Quantification: The LOQ of the method was the minimum concentration/quantity of a component, which could be quantified precisely and accurately.
The LOQ was carried out at the lowest sensitivity of the device under the same instrumental parameters used for the qualified specificity.
The LOQ of a compound for a specific method was determined by injecting in duplicate the working solution (s) made in suitable solvent fortified with test chemical / reference standard, until it produces a minimum signal to noise ratio ≥ 10:1 for precise quantification.

3. Linearity: The linearity was carried out by preparing and analyzing minimum five linear concentrations of reference standard with at least one determination. The linear calibration curve was established by plotting the analyte peak response against concentration (mg/l). The linearity was given in the form of equation of the resulting curve and correlation coefficient (r). The value of correlation coefficient (r) ≥ 0.99 was considered as an acceptable value.

4. Precision (% RSD), Accuracy (% Recovery) and Homogeneity: Precision, Accuracy was established at the level of test concentration used i.e., at low, mid and high dose level along with control. The active ingredient was diluted using sample diluent and injected onto HPLC using validated HPLC method. Minimum six determinations were made at each fortification level. The homogeneity was performed by analysing Top, Middle and Bottom layers of each fortification level. The active ingredient concentration in each replicate of each fortification level, mean of active ingredient concentration, accuracy (% recovery), SD and % RSD was calculated and reported.
The active ingredient concentration of test item in dose formulation was calculated using the following formula:
Y = bX + a

"AI Concentration (mg/L) = " "Y - a" /"b" " × D"


Where,
Y = Peak area of the sample
a = Intercept
b = Slope of the line
D = Dilution factor (if applicable)
X = A.I. Concentration (mg/L)


The accuracy (% Recovery) will be calculated using the following formula:

"Accuracy " ("% Recovery" )"= " "Obtained Concentration" /"Fortified concentration" " ×100"
The precision (% RSD) will be calculated using the formula provided as below:
"Precision (% RSD)= " "Standard Deviation" /"Mean Recovered Concentration" " ×100"

All the paramaters were according to the acceptance criteria of the above test.
Duration of treatment / exposure:
Dosing in all animals was carried out on a 7-days/week basis from the treatment initiation until necropsy. All animals of both sexes were dosed 2 weeks prior to mating. Dosing was continued in both sexes during the mating period. Females were treated throughout gestation and lactation up to the day of termination after weaning. Males were treated in the same manner until termination. Dosing of selected F1 males and females begun at weaning and continue until scheduled necropsy, depending on cohort assignment.
Frequency of treatment:
Once Daily
Details on study schedule:
- F1 parental animals not mated until [12-13] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [90] days of age.
- Age at mating of the mated animals in the study: [12-13] weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group (G1)
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Remarks:
Low Dose Group (G2)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
Mid Dose Group (G3)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group (G4)
No. of animals per sex per dose:
30 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected on the basis of previously performed GLP compliant repeated dose 28 days oral toxicity study. Two deaths were observed in the highest dose (500 mg/kg bw) unrelated to the gavage error of the OECD 407 study and therefore a subsequent lower dose of 250 mg/kg bw was selected as a highest dose in current study.
- Rationale for animal assignment (if not random): During the acclimatisation phase, male and female animals were randomized into four different experimental groups, based on the most recently recorded body weights, using the “Daniel’s XL Toolbar” (http://xltoolbox.sourceforge.net/) for MS Excel. After randomisation, it was ensured that individual body weights were within ± 20% of the respective group means. Details of the randomization were documented in the study raw data. The mean body weights were analysed by One-way ANOVA and it was found that the group means were statistically comparable to each other within each sex and group.
- Fasting period before blood sampling for clinical biochemistry: Animals were overnight fasted prior to blood collection.
- Other: No Data Available
Positive control:
No Data Available

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily for cage size observation, throughout the acclimatization and study periods.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations of rats from all the groups were made once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Detailed clinical examinations were carried out for P and F1 animals (after weaning) weekly, additionally P animals were examined once before the first treatment (to allow for within-subject comparisons).

BODY WEIGHT: Yes
- Time schedule for examinations: P animals were weighed once on receipt, once during randomization, once at the start of treatment and once every week thereafter. In addition, pregnant females were weighed during gestation (GD 0, GD 7, GD 14 and GD 20) and lactation on the same days as the weighing of the pups in their litters. All F1 animals were weighed individually on PND 0, 4, 7, 14, 21 and at least weekly thereafter. Body weight was also recorded on the day when they attained puberty (completion of preputial separation or vaginal patency). All animals were weighed at sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, during the study, feed consumption was recorded weekly or on the same days as animal body weights (except during cohabitation). The food consumption of each cage of F1 animals is recorded weekly commencing with selection to a respective cohort.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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

OTHER: No Data Available
Oestrous cyclicity (parental animals):
The assessment of estrous cyclicity for P females (by vaginal cytology) started at the beginning of the treatment period and continued until confirmation of matingor the end of the 2-week mating period.
For all F1 females in cohort 1A, vaginal smear was examined daily from the onset of vaginal patency until the presence of first confirmed smear was recorded toevaluate the time interval between these two events. Estrous cycles for all F1 females in cohort 1A were monitored for a period of two weeks, commencing around PND 75.
For all F1 females in cohort 1B, estrous cycle was monitored for at least two weeks prior to mating, and during mating until the evidence of mating was found.
Sperm parameters (parental animals):
Parameters examined in [P/F1] male parental generations: Sperm parameters were measured in all P and F1 cohort 1A males from control and high dose group.
At termination, testis and epididymis weight was recorded and one of each organ was reserved for histopathological examination. The remaining epididymides were used for enumeration of cauda epididymides sperm reserves. For this same subset of males, sperm from the cauda epididymides were collected for evaluation of sperm motility and sperm morphology. For the evaluation of sperm morphology, an epididymal sperm sample (at least 200 spermatozoa per sample) was examined for any abnormality.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 5 male and 5 female pups/litter (5/sex/litter as nearly as possible); Whenever the number of pups was insufficient, partial adjustment (for example, six males and four females) was considered.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention was paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Ten male and 10 female cohort 2A animals and 10 male and 10 female cohort 2B animals, from each treatment group (for each cohort: 1 male or 1 female per litter) were used for neurotoxicity assessments. Cohort 2A animals were subjected to auditory startle test on PND 24±1 day. Animals were presented with 10 trials of each 5 different amplitudes (80, 90, 100, 110, 120 decibal) and the latency to peak as well as the peak amplitude was measured. Functional observational battery and motor activity were assessed in cohort 2A animals between PND 63 and PND 75. The observation included (but was not limited to) home cage observations (posture and convulsions), handling observations (ease of removal from cage, handling reactivity, palpebral closure, lacrimation, eye examination, piloerection, skin examination, salivation), open field observations (gait, mobility, arousal, vocalizations, rears, respiration, clonic movements, tonic movements, urination, defecation, stereotypy, bizarre behavior), sensory reactivity measurements (approach response, touch response, click response, pupil response, air righting reflex, tail pinch response), grip strength (forelimb and hind limb) and foot splay (landing hind limb). All animals from Cohort 2A and 2B were subjected to detailed neurohistopathology at termination.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: At PND 56 (±3 days), 10 male and 10 female cohort 3 animals from each treatment group (1 male or 1 female per litter) were used in a T-cell dependant antibody response assay. Each rat was injected intravenously (IV) with 300 μg KLH antigen [Hemocyanin from Megathura crenulata (keyhole limpet); Sigma-Aldrich, Batch no. 029M4886V]. Five days after KLH immunization, blood from retro orbital plexuses were collected from all the animals. Serum from all these samples were separated and stored at -70°C ± 5°C until usage. Immunotoxicity of the test chemical was evaluated by estimating KLH-specific IgM levels in serum using commercially available ELISA kit (Life Diagnostics Inc., USA) as per the manufacturer’s protocol. Exposure to the test chemical was continued until the day before collecting serum for the ELISA assay.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed as soon as possible after the last litters in P and F1 generations were produced.
- Maternal animals: All surviving animals as soon as possible after the last litters in P and F1 generations were produced.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.(Please check table 1.1 and 1.2)

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1.2] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at PND 22.
- These animals were subjected to postmortem examinations (Terminated pups were subjected to gross necropsy including an assessment of the reproductive organs as per P animals. From 10 pups per sex per group, brain, spleen, and thymus were weighed and retained under appropriate conditions.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [1.1 and 1.2] were prepared for microscopic examination and weighed, respectively.
Statistics:
The findings of this study were evaluated in terms of the observed effects, necropsy and microscopic findings. The evaluation will included the relationship between the dose of the test substance and the presence or absence, the incidence and severity, of abnormalities, including fertility, clinical abnormalities, body weight changes, effects on mortality and any other toxic effects. Raw data was processed using statistical software. The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data were checked for their homogeneity using Shapiro Wilk test. All homogenous data were analyzed using ANOVA and data showing significance in their variances were subjected to Dunnett’s t-test. All heterogeneous data was analysed using Kruskal-Wallis ANOVA on ranks and data showing significance in their variances were subjected to Dunn’s Test. P values of less than or equal to 0.05 were accepted as being statistically significant.
Reproductive indices:
Fertility Index, Mating Index, Gestational Index, Implantation Index, Resorption Index
Offspring viability indices:
Pup Viability Index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related clinical signs or symptoms were observed in any of the parent animals up to 125 mg/kg body weight of the test chemical. However, mild salivation was observed in total 13 of 30 males of G4 group (250 mg/kg), starting from the 27th day of treatment and continuing till termination. All adult animals were found normal with respect to all the parameters examined during detailed clinical examinations through the course of the study. No treatment-related symptoms were found up to the dose level of 250 mg/kg body weight in either sex.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No morbidities or mortality were recorded in adult animals of parent generation in any of the experimental groups throughout the duration of the experiment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights remained comparable among all the groups for every time point (days 8, 15, 22, 29, 36, 43, 50, 57, 64 and 71) recorded during the 70 days exposure period for males. No significant difference was found for the mean body weights of females during the two weeks pre-mating period, during mating, during gestation or during lactation period. However, the mean percent change in body weight on all the instances (days 8, 15, 22, 29, 36, 43, 50, 57, 64 and 71) with respect to the first day of exposure was significantly reduced for parental males of G4 group (250 mg/kg) (P≤0.05 to P≤0.001) as compared to control animals, whereas mean body weight change for G2 (62.5 mg/kg) and G3 (125 mg/kg) males remained comparable to control animals. Among the females, mean percent change in body weights remained statistically comparable among all groups during pre-mating, mating, gestation and lactation periods.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The mean feed consumed per animal per day significantly increased on day 8 of treatment for G2 (62.5 mg/kg) (P≤0.01) and G3 (125 mg/kg) (P≤0.05) males as compared to G1 (control) animals. However, it was found to be similar for all other time points across the groups. The mean feed consumption of treatment group (G2, G3 & G4) females remained statistically comparable to control group during pre-mating, mating, gestation and lactation period except for one instance, where mean feed consumption on day 8 of treatment was significantly reduced for G2 (62.5 mg/kg) (P≤0.01) as compared to control females. The differences found in mean feed consumption of male and female animals were neither dose-dependent nor consistent, hence it was deemed to be independent of test-item treatment.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Feed efficiency for the G4 males had statistically reduced in the first two weeks of treatment, i.e. on days 8 and 15. It was also found to have increased significantly during the penultimate week of treatment, i.e. on day 63. For all other groups among the males, the feed efficiency was statistically comparable across the groups. Among the parental females, no statistical differences were observed for feed efficiency in any of the groups.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No significant difference was observed in any of the measured haematology parameters in all the groups of both the sexes, except in the WBC count, which was found to be significantly increased in males of G4 group (P≤0.01) as compared to control males.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Significant increase was observed in Phosphorus, Sodium and Chloride levels in G4 males (P≤0.05) as compared to control males. All the other clinical chemistry parameters in males and females were found comparable in control and treatment groups.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urine parameters tested did not show up any significant changes among the experimental groups in the parental generation of either sex.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related histopathological findings are reported in this study that could arise out of test-item administration. All observed tissues were normal at the level of histology. Minimal to moderate and focal lymphocytic infiltrates were observed in some slides of liver, kidney, lung, heart, colon, thymus and adrenals with mild to moderate cyst in thyroid gland, which were distributed randomly across the experimental groups and did not show any pattern of either dose-dependency or sex-based selectivity.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
While TSH, Estradiol, Progesterone and Testosterone remained statistically comparable among the groups of parental males, the mean level of T4 was found reduced in G3 males (P<0.01) as compared to control. This is likely an incidental result, given that G4 was statistically comparable to control. The mean T4, TSH, Estradiol and testosterone levels remained comparable among the groups of parental females; whereas, Progesterone was found to be significantly reduced in G4 females (P<0.001). This could be a test-chemical related effect.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle was monitored in females for its regularity. Females of all the treatment groups (G1, G2, G3 & G4) displayed regular estrous cyclicity of 4 to 6 days, through the two weeks of pre-mating treatment period.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Enumeration of sperms and assessment of motility and morphology did not reveal abnormalities in any of the samples from the parental males.
Reproductive performance:
no effects observed
Description (incidence and severity):
Out of 30 females 29, 28, 29 and 28 females delivered pups from G1, G2, G3 and G4 groups respectively. Precoital periods (time taken from start of cohabitation to onset of pregnancy) were found to be comparable for all groups of females. The means for all groups were within the range of 3.2 to 3.6 days.

Details on results (P0)

No treatment-related clinical signs or symptoms were observed in any of the parent animals up to 125 mg/kg body weight of the test chemical. However, mild salivation was observed in total 13 of 30 males of G4 group (250 mg/kg), starting from the 27th day of treatment and continuing till termination. All adult animals were found normal with respect to all the parameters examined during detailed clinical examinations through the course of the study. No treatment-related symptoms were found up to the dose level of 250 mg/kg body weight in either sex. No morbidities or mortality were recorded in adult animals of parent generation in any of the experimental groups throughout the duration of the experiment. Mean body weights remained comparable among all the groups for every time point (days 8, 15, 22, 29, 36, 43, 50, 57, 64 and 71) recorded during the 70 days exposure period for males. No significant difference was found for the mean body weights of females during the two weeks pre-mating period, during mating, during gestation or during lactation period. However, the mean percent change in body weight on all the instances (days 8, 15, 22, 29, 36, 43, 50, 57, 64 and 71) with respect to the first day of exposure was significantly reduced for parental males of G4 group (250 mg/kg) (P≤0.05 to P≤0.001) as compared to control animals, whereas mean body weight change for G2 (62.5 mg/kg) and G3 (125 mg/kg) males remained comparable to control animals. Among the females, mean percent change in body weights remained statistically comparable among all groups during pre-mating, mating, gestation and lactation periods. The mean feed consumed per animal per day significantly increased on day 8 of treatment for G2 (62.5 mg/kg) (P≤0.01) and G3 (125 mg/kg) (P≤0.05) males as compared to G1 (control) animals. However, it was found to be similar for all other time points across the groups. The mean feed consumption of treatment group (G2, G3 & G4) females remained statistically comparable to control group during pre-mating, mating, gestation and lactation period except for one instance, where mean feed consumption on day 8 of treatment was significantly reduced for G2 (62.5 mg/kg) (P≤0.01) as compared to control females. The differences found in mean feed consumption of male and female animals were neither dose-dependent nor consistent, hence it was deemed to be independent of test-item treatment. Feed efficiency for the G4 males had statistically reduced in the first two weeks of treatment, i.e. on days 8 and 15. It was also found to have increased significantly during the penultimate week of treatment, i.e. on day 63. For all other groups among the males, the feed efficiency was statistically comparable across the groups. Among the parental females, no statistical differences were observed for feed efficiency in any of the groups. No significant difference was observed in any of the measured haematology parameters in all the groups of both the sexes, except in the WBC count, which was found to be significantly increased in males of G4 group (P≤0.01) as compared to control males. Significant increase was observed in Phosphorus, Sodium and Chloride levels in G4 males (P≤0.05) as compared to control males. All the other clinical chemistry parameters in males and females were found comparable in control and treatment groups. The urine parameters tested did not show up any significant changes among the experimental groups in the parental generation of either sex. No significant difference was observed in absolute organ weight or organ weight relative to body weight for the entire set of measured organs in parent male and female animals. However, significant reduction was observed in absolute and relative liver weight in G4 females (P<0.05) as compared to control animals. In the current study, however, the decreased liver weights with respect to body weights in G4 females does not correlate with any histological observations made, making it inconsequential from the point of view of toxic effects of test-chemical. External examination of all male and female rats of control and all treated groups did not reveal any lesion of pathological significance. Internal examination of the rats revealed reduced testicular size in two animals (Male: G2: 1/30, G4:1/30), increased prostate size in one animal (Male: G2: 1/30) and reduced prostate size in one animal (Male: G3: 1/30). Remaining animals of parent generation did not reveal any abnormality of pathological significance. No treatment-related histopathological findings are reported in this study that could arise out of test-item administration. All observed tissues were normal at the level of histology. Minimal to moderate and focal lymphocytic infiltrates were observed in some slides of liver, kidney, lung, heart, colon, thymus and adrenals with mild to moderate cyst in thyroid gland, which were distributed randomly across the experimental groups and did not show any pattern of either dose-dependency or sex-based selectivity. While TSH, Estradiol, Progesterone and Testosterone remained statistically comparable among the groups of parental males, the mean level of T4 was found reduced in G3 males (P<0.01) as compared to control. This is likely an incidental result, given that G4 was statistically comparable to control. The mean T4, TSH, Estradiol and testosterone levels remained comparable among the groups of parental females; whereas, Progesterone was found to be significantly reduced in G4 females (P<0.001). This could be a test-chemical related effect. Estrous cycle was monitored in females for its regularity. Females of all the treatment groups (G1, G2, G3 & G4) displayed regular estrous cyclicity of 4 to 6 days, through the two weeks of pre-mating treatment period. Enumeration of sperms and assessment of motility and morphology did not reveal abnormalities in any of the samples from the parental males. Out of 30 females 29, 28, 29 and 28 females delivered pups from G1, G2, G3 and G4 groups respectively. Precoital periods (time taken from start of cohabitation to onset of pregnancy) were found to be comparable for all groups of females. The means for all groups were within the range of 3.2 to 3.6 days.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
organ weights and organ / body weight ratios
other: Dose-dependent and treatment-related decrease was observed in serum levels of progesterone in parental females of 250 mg/kg group (G4) as compared to control animals.
Remarks on result:
other: Please see 'remarks'
Remarks:
There were treatment related changes observed during clinical signs examination, organ weights and progesterone (hormonal) levels.

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs or symptoms were observed in any of the F1 generation animals up to dose level 125 mg/kg body weight of the test chemical. However, in the G4 group (250 mg/kg) of Cohort 1B (P1) mild salivation was observed in 8/20 males, starting from 56th day of treatment and continuing till termination in some animals.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No treatment-related mortality or morbidity was observed due to the test chemical administration in the P1 generation animal.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights remained comparable among all the groups for every time point (day 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85) recorded during the 87 days of exposure period for males. No significant change was found in percent change of body weight with respect to day 1 in G2, G3 and G4 group when compared with G1 group. However, significant reduction was observed in mean percent change in body weight of G3 cohort 1B (P1) male (P<0.05) on day 36 as compared to control animals. This finding was neither consistent nor dose dependent and hence, is independent of test-chemical treatment. During pre-mating, gestation and lactation period no significant difference in the mean percent change in body weight was found among all groups of Cohort 1B (P1) females.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean feed consumption (g/animal/day) remained comparable to G1 animals of the same sexes at every point of observation for Cohort 1B (P1) animals.
Food efficiency:
no effects observed
Description (incidence and severity):
Thefeed efficiency remained comparable to G1 animals of the same sexes at every point of observation for Cohort 1B (P1) animals.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No significant difference was observed in all the measured haematology parameters in all the groups of both the sexes except platelet count, which was found to be significantly decreased in males of G3 group (P<0.05) as compared to control males.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Albumin, total protein and globulin of P1 male were found significantly increased in G4 group (P≤0.05) as compared to G1 group, whereas BUN levels found significantly reduced in G3 (P≤0.01) and G4 (P≤0.05) males as compared to control group. No significant difference was observed in any other clinical chemistry parameters in P1 male. All the clinical chemistry parameters remained statistically comparable among control and treatment groups of P1 females.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urine parameters tested did not show up any significant changes among the experimental groups in the P1 animals of either sex.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The mean organ weight remained comparable among all the groups of cohort 1A male whereas significant increase was observed for relative weight of pituitary in G3 group (P<0.05) as compared to control animals. The absolute organ weight and organ weight relative to body weight remained comparable for all groups of cohort 1A Female. A statistically significant increase in absolute and relative weight of thyroid and parathyroid was observed in G3 males (P<0.05) of cohort 1B as compared to control animals. Additionaly thyroid and parthyroid weight relative to body weight was also found to be increased in G4 males (P<0.05) of cohort 1B as compared to control males. No significant difference was found for absolute and relative organ weights of control and treatment groups in cohort 1B females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
External and internal examination of cohort 1A and 1B animals did not reveal any lesion of pathological significance in either sex.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related histopathological findings are reported in this study that could arise out of test-chemical administration. All observed tissues of cohort 1A were normal at the level of histology. Minimal to moderate and focal lymphocytic infiltrates were observed in some slides of liver, kidney, lung, heart, thymus and adrenals with mild to moderate cyst in thyroid gland, which were distributed randomly across the experimental groups and did not show any pattern of either dose-dependence or sex-based selectivity.
No treatment-related histopathological findings are reported in this study that could arise out of test-item administration. All observed reproductive tissues of cohort 1B were normal at the level of histology. Minimal to moderate and focal spermatocytes were observed in testes of few animals. The follicular count of the ovarian slides did not reveal any pathological changes due to test-chemical administration.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Females of all the groups (G1, G2, G3 and G4) of both the cohorts (1A and 1B) demonstrated regular estrous cycle of 4 to 6 days. No treatment-related effect was observed in estrous cycle of cohort 1A and 1B females.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Enumeration of sperms and assessment of motility and morphology did not reveal abnormalities in any of the samples from cohort 1A males.
Reproductive performance:
no effects observed
Description (incidence and severity):
In Cohort 1B animals, out of 20 females 20, 19, 19 and 18 females delivered pups from G1, G2, G3 and G4 group respectively. Precoital periods (time taken from start of cohabitation to onset of pregnancy) were found to be comparable for all groups of females. The means for all groups were within the range of 2.5 to 2.9 days. The duration of pregnancy (days from insemination to parturition) / gestation length was also comparable among females of all the experimental groups. The means of number of corpora lutea, implantations and percentage of post-implantation loss remained comparable among females of control and treatment groups. Further, the number and percent of live births, still births, post-natal loss and percent male pup per litter remained comparable for all the groups of pregnant females. No case of dystocia (difficulty in parturition) was observed in females of any of the groups and no unusual nesting or nursing behaviour was observed as well.

Details on results (P1)

No treatment-related clinical signs or symptoms were observed in any of the F1 (Cohort 1A and 1B) generation animals up to dose level 125 mg/kg body weight of the test chemical. However, in the G4 group (250 mg/kg) of Cohort 1B (P1) mild salivation was observed in 8/20 males, starting from 56th day of treatment and continuing till termination in some animals. No treatment-related mortality or morbidity was observed due to the test chemical administration in the P1 generation animal. Mean body weights remained comparable among all the groups for every time point (day 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85) recorded during the 87 days of exposure period for males. No significant change was found in percent change of body weight with respect to day 1 in G2, G3 and G4 group when compared with G1 group. However, significant reduction was observed in mean percent change in body weight of G3 cohort 1B (P1) male (P<0.05) on day 36 as compared to control animals. This finding was neither consistent nor dose dependent and hence, is independent of test-chemical treatment. During pre-mating, gestation and lactation period no significant difference in the mean percent change in body weight was found among all groups of Cohort 1B (P1) females. The mean feed consumption (g/animal/day) remained comparable to G1 animals of the same sexes at every point of observation for Cohort 1B (P1) animals. The feed efficiency remained comparable to G1 animals of the same sexes at every point of observation for Cohort 1B (P1) animals. No significant difference was observed in all the measured haematology parameters in all the groups of both the sexes except platelet count, which was found to be significantly decreased in males of G3 group (P<0.05) as compared to control males. Albumin, total protein and globulin of P1 male were found significantly increased in G4 group (P≤0.05) as compared to G1 group, whereas BUN levels found significantly reduced in G3 (P≤0.01) and G4 (P≤0.05) males as compared to control group. No significant difference was observed in any other clinical chemistry parameters in P1 male. All the clinical chemistry parameters remained statistically comparable among control and treatment groups of P1 females. The urine parameters tested did not show up any significant changes among the experimental groups in the P1 animals of either sex. The mean organ weight remained comparable among all the groups of cohort 1A male whereas significant increase was observed for relative weight of pituitary in G3 group (P<0.05) as compared to control animals. The absolute organ weight and organ weight relative to body weight remained comparable for all groups of cohort 1A Female. A statistically significant increase in absolute and relative weight of thyroid and parathyroid was observed in G3 males (P<0.05) of cohort 1B as compared to control animals. Additionaly thyroid and parthyroid weight relative to body weight was also found to be increased in G4 males (P<0.05) of cohort 1B as compared to control males. No significant difference was found for absolute and relative organ weights of control and treatment groups in cohort 1B females. External and internal examination of cohort 1A and 1B animals did not reveal any lesion of pathological significance in either sex. No treatment-related histopathological findings are reported in this study that could arise out of test-chemical administration. All observed tissues of cohort 1A were normal at the level of histology. Minimal to moderate and focal lymphocytic infiltrates were observed in some slides of liver, kidney, lung, heart, thymus and adrenals with mild to moderate cyst in thyroid gland, which were distributed randomly across the experimental groups and did not show any pattern of either dose-dependence or sex-based selectivity.
No treatment-related histopathological findings are reported in this study that could arise out of test-item administration. All observed reproductive tissues of cohort 1B were normal at the level of histology. Minimal to moderate and focal spermatocytes were observed in testes of few animals. The follicular count of the ovarian slides did not reveal any pathological changes due to test-chemical administration. Females of all the groups (G1, G2, G3 and G4) of both the cohorts (1A and 1B) demonstrated regular estrous cycle of 4 to 6 days. No treatment-related effect was observed in estrous cycle of cohort 1A and 1B females. Enumeration of sperms and assessment of motility and morphology did not reveal abnormalities in any of the samples from cohort 1A males. In Cohort 1B animals, out of 20 females 20, 19, 19 and 18 females delivered pups from G1, G2, G3 and G4 group respectively. Precoital periods (time taken from start of cohabitation to onset of pregnancy) were found to be comparable for all groups of females. The means for all groups were within the range of 2.5 to 2.9 days. The duration of pregnancy (days from insemination to parturition) / gestation length was also comparable among females of all the experimental groups. The means of number of corpora lutea, implantations and percentage of post-implantation loss remained comparable among females of control and treatment groups. Further, the number and percent of live births, still births, post-natal loss and percent male pup per litter remained comparable for all the groups of pregnant females. No case of dystocia (difficulty in parturition) was observed in females of any of the groups and no unusual nesting or nursing behaviour was observed as well.

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs or symptoms were observed in any of the F1 (Cohort 1A and 1B) generation animals up to dose level 125 mg/kg body weight of the test chemical. However, in the G4 group (250 mg/kg) of Cohort 1B (P1) mild salivation was observed in 8/20 males, starting from 56th day of treatment and continuing till termination in some animals.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
No treatment-related mortality or morbidity was observed due to the test chemical administration in the P1 generation animal.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A: Mean body weights and percent change in body weight with respect to day 1 of treatment remained comparable among all the groups for every time point (day 8, 15, 22, 29, 36, 43, 50, 57, 64 and 71) recorded during the 74 days of exposure period for either sex.

Cohort 1B: Mean body weights remained comparable among all the groups for every time point (day 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85) recorded during the 87 days of exposure period for males. No significant change was found in percent change of body weight with respect to day 1 in G2, G3 and G4 group when compared with G1 group. However, significant reduction was observed in mean percent change in body weight of G3 cohort 1B male (P<0.05) on day 36 as compared to control animals. This finding was neither consistent nor dose dependent and hence, is independent of test-chemical treatment. During pre-mating, gestation and lactation period no significant difference in the mean percent change in body weight was found among all groups of Cohort 1B females.

Cohort 2A: Mean body weights remained comparable among all the groups for every time point (day 8, 15, 22, 29, 36, 43, 50 and 57) recorded during the 57 days exposure period for Cohort 2A males and females. No significant difference in body weight and percent change in body weight with respect to day 1 was found in males and Females, when G2, G3 and G4 were compared with G1.

Cohort 3: Mean body weights remained comparable among all the groups for every time point (day 8, 15, 22, 29 and 36) recorded during the 39 days exposure period for Cohort 3 males and females. Further, percent change in body weight with respect to day 1 of exposure was significantly decreased in G4 Cohort 3 male on days 15, 22 and 29 as compared to control animals. Reduced percent change in body weight was also observed on day 22 for G3 cohort 3 female (P<0.05) as compared to control females. The decreased percent change in body weight was neither consistent nor dose dependent and hence, cannot be attributable to test-chemical treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A: The mean feed consumed per animal per day for Cohort 1A male and female animals of all the groups remained comparable to control animals of the same sexes at every point of observation.

Cohort 1B: The mean feed consumption (g/animal/day) remained comparable to G1 animals of the same sexes at every point of observation for Cohort 1B animals.

Cohort 2A: The mean feed consumption (g/animal/day) remained comparable to G1 of the same sexes at every time of observation for cohort 2A animals. The mean feed efficiency significantly reduced on day 22 in G4 (P≤ 0.05) male as compared to G1 males whereas mean feed efficiency significantly increased in G3 (P≤ 0.05; P≤ 0.01) females compared with G1 on day 22 and day 57, respectively.

Cohort 3: The mean feed consumed per animal per day for Cohort 3 male and female animals of all the groups remained comparable to control animals of the same sexes at every point of observation.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A: The mean feed efficiency remained comparable for all the time points recorded for male and female of cohort 1A except on day 29, when a significant increase in feed efficiency was observed in G4 females.

Cohort 1B: The mean feed efficiency remained comparable to G1 animals of the same sexes at every point of observation for Cohort 1B animals.

Cohort 2A: The mean feed efficiency significantly reduced on day 22 in G4 (P≤ 0.05) male as compared to G1 males whereas mean feed efficiency significantly increased in G3 (P≤ 0.05; P≤ 0.01) females compared with G1 on day 22 and day 57, respectively. The mean feed efficiency remained comparable in control and treatment groups for every other time points recorded.

Cohort 3: The mean feed consumed per animal per day for Cohort 3 male and female animals of all the groups remained comparable to control animals of the same sexes at every point of observation. The mean feed efficiency of G4 male significantly reduced (P<0.01) on day 15 as compared to control males. Significantly increased feed efficiency was found in females of G2 and G3 (P<0.05) as compared to G1 on day 36. The mean feed efficiency remained comparable to control animals of the same sexes at every other time point of observation
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No significant difference was observed in all the measured haematology parameters in all the groups of both the sexes except platelet count, which was found to be significantly decreased in males of G3 group (P<0.05) as compared to control males.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Albumin, total protein and globulin of P1 male were found significantly increased in G4 group (P≤0.05) as compared to G1 group, whereas BUN levels found significantly reduced in G3 (P≤0.01) and G4 (P≤0.05) males as compared to control group. No significant difference was observed in any other clinical chemistry parameters in P1 male. All the clinical chemistry parameters remained statistically comparable among control and treatment groups of P1 females.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urine parameters tested did not show up any significant changes among the experimental groups in the P1 animals of either sex.
Sexual maturation:
no effects observed
Description (incidence and severity):
Mean age and body weight at balanopreputial separation remained comparable for all groups of males. Similarly, the mean age and body weight at the time of vaginal opening remained statistically comparable for all groups of female rats. Further, no significant difference was observed from the day of vaginal opening to appearance of first cornified smear (estrous). No treatment-related effects were observed on male and female pup sexual development markers.
Anogenital distance (AGD):
effects observed, treatment-related
Description (incidence and severity):
The mean Anogenital distance (AGD) and normalised AGD remained comparable in all groups of female pups. A significant increase was observed in AGD, both - measured and normalised values in male pups of G2 (P≤0.001) and G4 (P≤0.001) group compared to G1 group. The significantly increased AGD and normalised AGD in male pups of G4 group were assumed to be treatment-related
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
None of the male pups showed presence of nipples/areolae on PND 13.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The mean organ weight remained comparable among all the groups of cohort 1A male whereas significant increase was observed for relative weight of pituitary in G3 group (P<0.05) as compared to control animals. The absolute organ weight and organ weight relative to body weight remained comparable for all groups of cohort 1A Female. A statistically significant increase in absolute and relative weight of thyroid and parathyroid was observed in G3 males (P<0.05) of cohort 1B as compared to control animals. Additionaly thyroid and parthyroid weight relative to body weight was also found to be increased in G4 males (P<0.05) of cohort 1B as compared to control males. No significant difference was found for absolute and relative organ weights of control and treatment groups in cohort 1B females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
External and internal examination of cohort 1A and 1B animals did not reveal any lesion of pathological significance in either sex.
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A animals: No treatment-related histopathological findings were observed that could arise out of test-chemical administration. All observed tissues of cohort 1A were normal at the level of histology. Minimal to moderate and focal lymphocytic infiltrates were observed in some slides of liver, kidney, lung, heart, thymus and adrenals with mild to moderate cyst in thyroid gland, which were distributed randomly across the experimental groups and did not show any pattern of either dose-dependence or sex-based selectivity.

Cohort 1B: No treatment-related histopathological findings were observed that could arise out of test-chemical administration. All observed reproductive tissues of cohort 1B were normal at the level of histology. Minimal to moderate and focal spermatocytes were observed in testes of few animals. The follicular count of the ovarian slides did not reveal any pathological changes due to test-chemical administration.

Cohort 2A and 2B: Microscopic examination of control group and treatment group at 250 mg/kg did not reveal any lesion of pathological significance in any of the observed organs in either sex.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Dose-dependent and treatment-related decrease was observed in serum levels of T4, TSH, estradiol and testosterone in cohort 1A females at 250 mg/kg group (G4) as compared to control animals.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Cohort 2A: Auditory startle response (latency to peak) at 120 decibel of cohort 2A males was found significantly decreased in G4 (P≤0.05) animals as compared to G1 group while the auditory startle response (latency to peak) at 120 decibel of cohort 2A females was significantly increased in G4 animals (P≤0.05) as compared to G1 females. Home cage, sensory reactivity and hind limb foot splay observations remained comparable among all the groups and in both sexes. A significant increase was observed in mean hind limb grip strength of G4 group (P<0.05) as compared to control males. The mean for limb grip strength of male as well as the mean forelimb and hind limb grip strength of female remained comparable in control and treatment groups. No significant difference was found in mean motor activity parameters (such as distance travelled, resting time, ambulatory time, burst of stereotypic movements, horizontal counts, ambulatory count, etc) in males of control and treatment groups. A significantly increased mean resting time with significantly reduced mean horizontal count and ambulatory count was observed in females of G4 group (P<0.05) as compared to control group. No significant difference was observed in any other parameters of motor activity in other groups. The neurobehavioral observations seen in G4 animals are assumed to be treatment-related. External and internal examination of animals did not reveal any lesion of pathological significance in both sexes of cohorts 2A and 2B animals.

Cohort 2A and 2B: Absolute brain weight and brain weight relative to body weight remained comparable in all the groups of cohort 2A and 2B. Microscopic examination of control group and treatment group at 250 mg/kg did not reveal any lesion of pathological significance in any of the observed organs in either sex.
Dose-dependent and treatment-related decrease was observed in serum levels of T4, TSH, estradiol and testosterone in cohort 1A females at 250 mg/kg group (G4) as compared to control animals.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
The mean T cell dependent antibody response remained comparable in all the groups of both the sexes. Microscopic examination of lymphoid organs (cohort 1A) of control group and treatment group at 250 mg/kg did not reveal any lesion of pathological significance in any of the observed organs in either sex.

Details on results (F1)

No treatment-related clinical signs or symptoms were observed in any of the F1 (Cohort 1A and 1B) generation animals up to dose level 125 mg/kg body weight of the test chemical. However, in the G4 group (250 mg/kg) of Cohort 1B (P1) mild salivation was observed in 8/20 males, starting from 56th day of treatment and continuing till termination in some animals. No treatment-related mortality or morbidity was observed due to the test chemical administration in the P1 generation animal.
Cohort 1A: Mean body weights and percent change in body weight with respect to day 1 of treatment remained comparable among all the groups for every time point (day 8, 15, 22, 29, 36, 43, 50, 57, 64 and 71) recorded during the 74 days of exposure period for either sex.

Cohort 1B: Mean body weights remained comparable among all the groups for every time point (day 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78 and 85) recorded during the 87 days of exposure period for males. No significant change was found in percent change of body weight with respect to day 1 in G2, G3 and G4 group when compared with G1 group. However, significant reduction was observed in mean percent change in body weight of G3 cohort 1B male (P<0.05) on day 36 as compared to control animals. This finding was neither consistent nor dose dependent and hence, is independent of test-chemical treatment. During pre-mating, gestation and lactation period no significant difference in the mean percent change in body weight was found among all groups of Cohort 1B females.

Cohort 2A: Mean body weights remained comparable among all the groups for every time point (day 8, 15, 22, 29, 36, 43, 50 and 57) recorded during the 57 days exposure period for Cohort 2A males and females. No significant difference in body weight and percent change in body weight with respect to day 1 was found in males and Females, when G2, G3 and G4 were compared with G1.

Cohort 3: Mean body weights remained comparable among all the groups for every time point (day 8, 15, 22, 29 and 36) recorded during the 39 days exposure period for Cohort 3 males and females. Further, percent change in body weight with respect to day 1 of exposure was significantly decreased in G4 Cohort 3 male on days 15, 22 and 29 as compared to control animals. Reduced percent change in body weight was also observed on day 22 for G3 cohort 3 female (P<0.05) as compared to control females. The decreased percent change in body weight was neither consistent nor dose dependent and hence, cannot be attributable to test-chemical treatment.
Cohort 1A: The mean feed consumed per animal per day for Cohort 1A male and female animals of all the groups remained comparable to control animals of the same sexes at every point of observation.

Cohort 1B: The mean feed consumption (g/animal/day) remained comparable to G1 animals of the same sexes at every point of observation for Cohort 1B animals.

Cohort 2A: The mean feed consumption (g/animal/day) remained comparable to G1 of the same sexes at every time of observation for cohort 2A animals. The mean feed efficiency significantly reduced on day 22 in G4 (P≤ 0.05) male as compared to G1 males whereas mean feed efficiency significantly increased in G3 (P≤ 0.05; P≤ 0.01) females compared with G1 on day 22 and day 57, respectively.

Cohort 3: The mean feed consumed per animal per day for Cohort 3 male and female animals of all the groups remained comparable to control animals of the same sexes at every point of observation.
Cohort 1A: The mean feed efficiency remained comparable for all the time points recorded for male and female of cohort 1A except on day 29, when a significant increase in feed efficiency was observed in G4 females.

Cohort 1B: The mean feed efficiency remained comparable to G1 animals of the same sexes at every point of observation for Cohort 1B animals.

Cohort 2A: The mean feed efficiency significantly reduced on day 22 in G4 (P≤ 0.05) male as compared to G1 males whereas mean feed efficiency significantly increased in G3 (P≤ 0.05; P≤ 0.01) females compared with G1 on day 22 and day 57, respectively. The mean feed efficiency remained comparable in control and treatment groups for every other time points recorded.

Cohort 3: The mean feed consumed per animal per day for Cohort 3 male and female animals of all the groups remained comparable to control animals of the same sexes at every point of observation. The mean feed efficiency of G4 male significantly reduced (P<0.01) on day 15 as compared to control males. Significantly increased feed efficiency was found in females of G2 and G3 (P<0.05) as compared to G1 on day 36. The mean feed efficiency remained comparable to control animals of the same sexes at every other time point of observation.
No significant difference was observed in all the measured haematology parameters in all the groups of both the sexes except platelet count, which was found to be significantly decreased in males of G3 group (P<0.05) as compared to control males.
Albumin, total protein and globulin of P1 male were found significantly increased in G4 group (P≤0.05) as compared to G1 group, whereas BUN levels found significantly reduced in G3 (P≤0.01) and G4 (P≤0.05) males as compared to control group. No significant difference was observed in any other clinical chemistry parameters in P1 male. All the clinical chemistry parameters remained statistically comparable among control and treatment groups of P1 females.
The urine parameters tested did not show up any significant changes among the experimental groups in the P1 animals of either sex.
Mean age and body weight at balanopreputial separation remained comparable for all groups of males. Similarly, the mean age and body weight at the time of vaginal opening remained statistically comparable for all groups of female rats. Further, no significant difference was observed from the day of vaginal opening to appearance of first cornified smear (estrous). No treatment-related effects were observed on male and female pup sexual development markers.
The mean Anogenital distance (AGD) and normalised AGD remained comparable in all groups of female pups. A significant increase was observed in AGD, both - measured and normalised values in male pups of G2 (P≤0.001) and G4 (P≤0.001) group compared to G1 group. The significantly increased AGD and normalised AGD in male pups of G4 group were assumed to be treatment-related. None of the male pups showed presence of nipples/areolae on PND 13. The mean organ weight remained comparable among all the groups of cohort 1A male whereas significant increase was observed for relative weight of pituitary in G3 group (P<0.05) as compared to control animals. The absolute organ weight and organ weight relative to body weight remained comparable for all groups of cohort 1A Female. A statistically significant increase in absolute and relative weight of thyroid and parathyroid was observed in G3 males (P<0.05) of cohort 1B as compared to control animals. Additionaly thyroid and parthyroid weight relative to body weight was also found to be increased in G4 males (P<0.05) of cohort 1B as compared to control males. No significant difference was found for absolute and relative organ weights of control and treatment groups in cohort 1B females. External and internal examination of cohort 1A and 1B animals did not reveal any lesion of pathological significance in either sex.
Cohort 1A animals: No treatment-related histopathological findings were observed that could arise out of test-chemical administration. All observed tissues of cohort 1A were normal at the level of histology. Minimal to moderate and focal lymphocytic infiltrates were observed in some slides of liver, kidney, lung, heart, thymus and adrenals with mild to moderate cyst in thyroid gland, which were distributed randomly across the experimental groups and did not show any pattern of either dose-dependence or sex-based selectivity.

Cohort 1B: No treatment-related histopathological findings were observed that could arise out of test-chemical administration. All observed reproductive tissues of cohort 1B were normal at the level of histology. Minimal to moderate and focal spermatocytes were observed in testes of few animals. The follicular count of the ovarian slides did not reveal any pathological changes due to test-chemical administration.

Cohort 2A and 2B: Microscopic examination of control group and treatment group at 250 mg/kg did not reveal any lesion of pathological significance in any of the observed organs in either sex.
Cohort 2A: Auditory startle response (latency to peak) at 120 decibel of cohort 2A males was found significantly decreased in G4 (P≤0.05) animals as compared to G1 group while the auditory startle response (latency to peak) at 120 decibel of cohort 2A females was significantly increased in G4 animals (P≤0.05) as compared to G1 females. Home cage, sensory reactivity and hind limb foot splay observations remained comparable among all the groups and in both sexes. A significant increase was observed in mean hind limb grip strength of G4 group (P<0.05) as compared to control males. The mean for limb grip strength of male as well as the mean forelimb and hind limb grip strength of female remained comparable in control and treatment groups. No significant difference was found in mean motor activity parameters (such as distance travelled, resting time, ambulatory time, burst of stereotypic movements, horizontal counts, ambulatory count, etc) in males of control and treatment groups. A significantly increased mean resting time with significantly reduced mean horizontal count and ambulatory count was observed in females of G4 group (P<0.05) as compared to control group. No significant difference was observed in any other parameters of motor activity in other groups. The neurobehavioral observations seen in G4 animals are assumed to be treatment-related. External and internal examination of animals did not reveal any lesion of pathological significance in both sexes of cohorts 2A and 2B animals.

Cohort 2A and 2B: Absolute brain weight and brain weight relative to body weight remained comparable in all the groups of cohort 2A and 2B. Microscopic examination of control group and treatment group at 250 mg/kg did not reveal any lesion of pathological significance in any of the observed organs in either sex.
The mean T cell dependent antibody response remained comparable in all the groups of both the sexes. Microscopic examination of lymphoid organs (cohort 1A) of control group and treatment group at 250 mg/kg did not reveal any lesion of pathological significance in any of the observed organs in either sex.

Effect levels (F1)

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Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
62.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
developmental neurotoxicity
developmental immunotoxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 3)
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental immunotoxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 2A)
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental neurotoxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 2B)
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental immunotoxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
LOAEL
Generation:
F1 (cohort 2A)
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental neurotoxicity
Remarks on result:
other: Please see 'remarks'
Remarks:
Treatment-related changes in auditory startle response was observed in male and females of cohort 2A in G4 group (250 mg/kg) as compared to control. Also, treatment-related effects were observed in motor activity parameters in cohort 2A females of G4 group (250 mg/kg) as compared to control.
Dose descriptor:
LOAEL
Generation:
F1 (cohort 1A)
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Please see 'remarks'
Remarks on result:
other: Changes in Hormonal Levels

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related signs or symptoms were noted at the time of clinical observations on PND 0, 4, 7, 14 and 21.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
No treatment-related mortality was observed due to test chemical administration.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weight of pups of both the sexes combined was significantly increased in G2 (P≤0.05) on PNDs 14 and 21 as compared to G1 group. Similarly, significant increase in pup body weight was observed on PND 21 for G4 group (P<0.001) as compared to control group. On PND 0 significant reductions in mean body weights of male pups was observed in all the treatment groups (G2, G3 and G4; P<0.05) as compared to control (G1) group. The mean body weight of male pup was significantly increased in G2 group (P<0.05) on PND 14 and in G4 group on PND 14 (P<0.05) and PND 21 (P<0.001) as compared to control group. The mean body weight of female pups on all the instances measured remained comparable among the control and treatment groups (G2, G3 and G4).
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:
no effects observed
Description (incidence and severity):
No treatment related adverse effects were observed in sexual maturation of the pups.
Anogenital distance (AGD):
effects observed, treatment-related
Description (incidence and severity):
The mean Anogenital distance (AGD) and normalised AGD remained comparable in all groups of female pups. A significant increase was observed in AGD, both - measured and normalised values in male pups of G2 (P≤0.001) and G4 (P≤0.001) group compared to G1 group. The significantly increased AGD and normalised AGD in male pups of G4 group were assumed to be treatment-related.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
None of the male pups showed presence of nipples/areolae on PND 13.
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related signs or symptoms were noted at the time of gross external observations of the pups.
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not specified

Details on results (F2)

No treatment-related signs or symptoms were noted at the time of clinical observations on PND 0, 4, 7, 14 and 21. No treatment-related mortality was observed due to test chemical administration. Mean body weight of pups of both the sexes combined was significantly increased in G2 (P≤0.05) on PNDs 14 and 21 as compared to G1 group. Similarly, significant increase in pup body weight was observed on PND 21 for G4 group (P<0.001) as compared to control group. On PND 0 significant reductions in mean body weights of male pups was observed in all the treatment groups (G2, G3 and G4; P<0.05) as compared to control (G1) group. The mean body weight of male pup was significantly increased in G2 group (P<0.05) on PND 14 and in G4 group on PND 14 (P<0.05) and PND 21 (P<0.001) as compared to control group. The mean body weight of female pups on all the instances measured remained comparable among the control and treatment groups (G2, G3 and G4). No treatment related adverse effects were observed in sexual maturation of the pups. The mean Anogenital distance (AGD) and normalised AGD remained comparable in all groups of female pups. A significant increase was observed in AGD, both - measured and normalised values in male pups of G2 (P≤0.001) and G4 (P≤0.001) group compared to G1 group. The significantly increased AGD and normalised AGD in male pups of G4 group were assumed to be treatment-related. None of the male pups showed presence of nipples/areolae on PND 13. No treatment-related signs or symptoms were noted at the time of gross external observations of the pups.

Effect levels (F2)

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Dose descriptor:
NOAEL
Generation:
F2
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
gross pathology
other: Ano-genital Distance (AGD) and Nipple Retention in Male Pups
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
LOAEL
Generation:
F2
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: Ano-Genital Distance (AGD)
Remarks on result:
other: Please see 'remarks'
Remarks:
Treatment related changes observed in body weight and body weight gain and Ano-Genital Distance (AGD) in males.

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Based on the results obtained under the experimental conditions used in this study, it is hereby concluded that the No Observed Adverse Effect Level (NOAEL) and Lowest Observed Adverse Effect Level (LOAEL) of the test chemical were 125 mg/kg bw/day and 250 mg/kg bw/day, respectively for general toxicity. At 250 mg/kg bw/day of the test chemical significant increase was observed in pup body weight and ano-genital distance. Therefore, the NOAEL and LOAEL of the test chemical were 125 mg/kg bw/day and 250 mg/kg bw/day, respectively for developmental toxicity for F1 generations. At 250 mg/kg bw/day of the test chemical significant differences were observed in auditory startle response and motor activity parameters. Therefore, the NOAEL and LOAEL of the test chemical were 125 mg/kg bw/day and 250 mg/kg bw/day, respectively for developmental neurotoxicity. As there was no effect observed on T-cell dependent antibody response upto 250 mg/kg bw/day, the NOAEL of the test chemical was 250 mg/kg bw/day for developmental immunotoxicity.
Executive summary:

An Extended One-Generation Reproductive Toxicity Study was performed according to OECD TG 443 to evaluate the general, reproductive and developmental toxicological endpoints associated with the repeated oral administration of graduated doses of the test chemical in Wistar rats. A total of 240 wistar rats (120 males and 120 females) were randomized into 4 experimental groups each containing 30 animals per sex per group. The groups, viz., G1, G2, G3 and G4 received 0, 62.5, 125 and 250 mg/kg body weight of test chemical respectively. Dose administration was through an oral gavage and the vehicle used in this study was corn oil. All animals of both sexes were dosed 2 weeks prior to mating. Dosing was continued in both sexes during the mating period. Females were treated throughout gestation and lactation up to the day of termination after weaning. Males were treated in the same manner until termination. Dosing of selected F1 males and females begun at weaning and continue until scheduled necropsy, depending on cohort assignment. To ensure the accurate administration of the test chemical, the concentrations and homogeneity of the dose formulation were verified through formulation analyses on day 1, day 91 and day 182 of treatment. The results of these were found within the acceptable limits in both occasions. Observations on the animals encompassed mortality/morbidity, onset of any clinical signs/symptoms, body weight, body weight changes, feed consumption, feed efficiency, pregnancy observations, fetal developmental parameters, auditory startle response, functional observational battery/neurobehavioral observation, hematology, clinical biochemistry, urinalysis, serum hormone levels, T-cell dependent antibody response, gross pathology and histopathology. The results showed that Test chemical does not cause mortality up to doses of 250 mg/kg body weight when administered orally to wistar rats. Further, no clinical signs or symptoms were observed in animals up to doses of 125 mg/kg body weight (G3) throughout the duration of the experiment. Mild salivation was observed in some males treated at 250 mg/kg body weight (G4) from treatment day 27th in parent generation. The mean body weights of parent male and female remained comparable throughout the study period. The mean percent change in body weight on all the occasions with respect to the first day of exposure was significantly reduced for parental males of G4 group (250 mg/kg) as compared to control animals whereas mean percent change in body weights remained statistically comparable among all groups of female during pre-mating, mating, gestation and lactation periods. No treatment-related changes were observed in feed consumption and feed efficiency in any parental animal of the study. All the treated animals demonstrated regular estrous cycle of 4-6 days. Further, precoital interval, gestation length, number of corpora lutea, implantations, post-implantation loss litter size, live births, still births and percent male pup per litter remained comparable among all the groups of parent female. No anomalies were observed in pups at the time of gross observation on PND (post-natal day) 0. Analysis of mean body weights - both combined and sex-specific revealed increase in pup body weight at 250 mg/kg group (G4). Clinical chemistry parameters tested herein were inclusive of serum biochemistry, electrolytes, hematology and urine analysis. No test-chemical related changes were observed in any parameters of hematology, clinical chemistry and urinalysis. Dose-dependent and treatment-related decrease was observed in serum levels of progesterone in parental females of 250 mg/kg group (G4) as compared to control animals. Examination of external features and gross pathology during necropsy revealed no noteworthy observations attributable to test-chemical administration. Enumeration of sperms and assessment of motility and morphology did not reveal abnormalities in any of the samples from parent males. Significant reduction was observed in absolute and relative liver weight in G4 females as compared to control animals. This is assumed to be test-chemical related. No treatment-related effects were observed in other measured organ in both the sexes of parent animals. Histopathological observations were made for G1 (vehicle control) and G4 (high-dose) animals and no treatment related changes showed up in any of the observed tissues at the tested dose (250 mg/kg body weight) in either sex. The results showed that test chemical does not cause mortality up to doses of 250 mg/kg body weight when administered orally to wistar rats. Further, no clinical signs or symptoms were observed in animals up to doses of 125 mg/kg body weight (G3) throughout the duration of the experiment. Mild salivation was observed in some males treated at 250 mg/kg body weight (G4) from treatment day 51st in F1 generation. No treatment-related changes were observed in body weights or percent change in body weight with respect to the first day of exposure males and females of cohort 1A, 1B, 2A and 3. No treatment-related changes were observed in feed consumption and feed efficiency in any of the animals of cohort 1A, 1B, 2A and 3. No treatment-related effects were observed in pup sexual development markers such as baleno-preputial separation, vaginal opening and first estrous. All the treated animals of cohort 1A and 1B demonstrated regular estrous cycle of 4-6 days. Further, precoital interval, gestation length, number of corpora lutea, implantations, post-implantation loss litter size, live births, still births and percent male pup per litter remained comparable among all the groups of cohort 1B females. No anomalies were observed in pups (F2 generation) at the time of gross observation on PND (post-natal day) 0. Treatment-related increase in male pup body weight, AGD and normalized AGD was observed at 250 mg/kg group (G4). No treatment-related changes were observed in any parameters of hematology, clinical chemistry and urinalysis in cohort 1A animals. Dose-dependent and treatment-related decrease was observed in serum levels of T4, TSH, estradiol and testosterone in cohort 1A females at 250 mg/kg group (G4) as compared to control animals. Examination of external features and gross pathology during necropsy revealed no noteworthy observations attributable to test-chemical administration. Enumeration of sperms and assessment of motility and morphology did not reveal abnormalities in any of the samples from cohort 1A males. No treatment-related effects were observed organ weight and relative organ weights of cohort 1A and 1B animals. Histopathological observations were made for G1 (vehicle control) and G4 (high-dose) animals and no treatment related changes showed up in any of the observed tissues at the tested dose (250 mg/kg body weight) in either sex. A battery of observations carried out for functional and neurobehavioural assessment of animals included in-cage observations, handling response, open-field behaviour, hindlimb footsplay, sensory response, motor activity measurement, auditory startle response and grip strength measurements. Treatment-related changes in auditory startle response was observed in male and females of cohort 2A in G4 group (250 mg/kg) as compared to control. Also, treatment-related effects were observed in motor activity parameters in cohort 2A females of G4 group (250 mg/kg) as compared to control. No treatment-related changes were observed in brain weight of cohort 2A and 2B animals. Further, detailed neuro-histopathology observation did not reveal any effect of test-chemical in cohort 2A and 2B. No treatment-related changes were observed in T-cell dependent antibody response assay in both the sexes of cohort 3 animals. In addition, no treatment related lesions were observed from slides of lymphoid organs collected from 10 animals per sex per group of cohort 1A animals Thus, based on the results obtained under the experimental conditions used in this study, it is hereby concluded that the No Observed Adverse Effect Level (NOAEL) and Lowest Observed Adverse Effect Level (LOAEL) of Test chemical were 125 mg/kg bw/day and 250 mg/kg bw/day, respectively for general toxicity. At 250 mg/kg bw/day of Test chemical significant increase was observed in pup body weight and ano-genital distance. Therefore, the NOAEL and LOAEL of Test chemical were 125 mg/kg bw/day and 250 mg/kg bw/day, respectively for developmental toxicity for F1 generations. At 250 mg/kg bw/day of Test chemical significant differences were observed in auditory startle response and motor activity parameters. Therefore, the NOAEL and LOAEL of Test chemical were 125 mg/kg bw/day and 250 mg/kg bw/day, respectively for developmental neurotoxicity. AS there was no effect observed on T-cell dependent antibody response up to 250 mg/kg bw/day, the NOAEL of Test chemical was 250 mg/kg bw/day for developmental immunotoxicity.