Methyl 2-naphthyl ether

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

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:

other: OECD TG 443: Extended One-Generation Reproductive Toxicity Study

Principles of method if other than guideline:

According to OECD TG 443: Extended One-Generation Reproductive Toxicity Study

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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 in the reproductive 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; (F1) Males: x-x g; Females: x-x 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

Details on route of administration:

While concentrations of the formulation were fixed for each experimental group, the dose volume for each animal was calculated on the basis of the most recently recorded body weight. Formulation was administered in a single dose through oral gavage at a constant volume to weight ratio of 4 mL/ kg body weight.

Vehicle:

corn oil

Details on oral 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

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 and within 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. The parental animals were dosed until 7 weeks.

Frequency of treatment:

Once Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control Group (G1)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Low Dose Group (G2)

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Mid Dose Group (G3)

Doses / concentrations 4

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.
- Rationale for selecting satellite groups: No Data Available
- Post-exposure recovery period in satellite groups: No Data Available
- Section schedule rationale (if not random): No Data Available
- Other: No Data Available

Positive control:

No Data Available

Examinations

Observations and examinations performed and frequency:

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.

BODY WEIGHT: Yes
- Time schedule for examinations: Parental 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.

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).
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- 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: Yes

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

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: No Data Available
- Dose groups that were examined: No Data Available

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to termination of the parental animals
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: 10 males and 10 females, randomly selected from each group of parental animals
- Parameters checked in table [1.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to termination of the parental animals
- Animals fasted: Yes (isofluorane)
- How many animals: 10 males and 10 females, randomly selected from each group of parental animals
- Parameters checked in table [1.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: During the last week of the treatment, 10 males and 10 females, randomly selected from each group of parental animals. During the last week of the treatment, 10 males and 10 females, randomly selected from each group of parental animals.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [1.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: No Data Available
- Dose groups that were examined: No Data Available
- Battery of functions tested: No Data Available

IMMUNOLOGY: Not specified
- Time schedule for examinations: No Data Available
- How many animals: No Data Available
- Dose groups that were examined: No Data Available

OTHER: Hormone Analysis: From 10 males and 10 females, randomly selected from each group of parental animals , blood was collected in the morning; serum was separated and stored under appropriate conditions (-70°C ± 5°C). All collected blood samples were assessed for serum levels of T4, TSH, Estradiol, Progesterone and Testosterone using commercially available Rat ELISA kits.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1.4)

HISTOPATHOLOGY: Yes (see table 1.4), full histopathology was carried out on the preserved organs and tissues of all parental animals in the control and high dose group of both sexes. Slides for low- and mid-dose groups were processed only if, test-chemical related pathologies were observed in the high-dose group sections.

Other examinations:

Organ Weights: At the time of termination, organs marked with asterisk (*) in table 1.4 from all parental animals were trimmed of adherent tissue/ fat and weighed, prior to preservation in fixative.

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. 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.

Results and discussion

Results of examinations

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.

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-chemical 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

Description (incidence and severity):

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.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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.

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-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.

Details on results:

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.

Effect levels

1

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:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

other: Hormone Analysis

Remarks on result:

not determinable due to absence of adverse toxic effects

2

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: Hormone Analysis

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

Critical effects observed:

no

Any other information on results incl. tables

Table 2.1: Mortality and Morbidity Record

			Sex: Male
Group	Dose (mg/kg)	No. of Animals		Observation
G1	0	30		No mortality or morbidity observed
G2	62.5	30		No mortality or morbidity observed
G3	125	30		No mortality or morbidity observed
G4	250	30		No mortality or morbidity observed

 

			Sex: Female
Group	Dose (mg/kg)	No. of Animals		Observation
G1	0	30		No mortality or morbidity observed
G2	62.5	30		No mortality or morbidity observed
G3	125	30		No mortality or morbidity observed
G4	250	30		No mortality or morbidity observed

 

Table 2.2: Clinical Signs and Symptoms Record

			Sex: Male
Group	Dose (mg/kg)	No. of Animals		Observation
G1	0	30		Normal
G2	62.5	30		Normal
G3	125	30		Normal
G4	250	30		Normal

			Sex: Female
Group	Dose (mg/kg)	No. of Animals		Observation
G1	0	30		Normal
G2	62.5	30		Normal
G3	125	30		Normal
G4	250	30		*Normal

Table 2.3 Mean Body weights (g)

									Sex: Male
Group	Dose (mg/Kg)		Day of Exposure
			1	8	15	22	29	36		43	50	57	64	71
G1	0	Mean	351.73	359.60	371.80	384.63	400.93	419.17		426.90	431.53	441.13	444.70	452.53
		SD	26.71	26.09	25.43	27.51	28.22	30.60		31.14	30.71	29.88	33.09	32.31
		N	30	30	30	30	30	30		30	30	30	30	30
G2	62.5	Mean	357.10	365.17	377.03	387.97	406.10	426.53		434.03	439.53	448.87	451.17	459.23
		SD	26.62	24.64	22.51	23.92	26.57	28.79		29.40	29.89	29.65	31.22	32.62
		N	30	30	30	30	30	30		30	30	30	30	30
G3	125	Mean	356.17	363.80	373.40	385.77	402.57	419.73		428.73	435.50	441.70	447.23	454.03
		SD	25.19	24.28	24.93	28.35	26.99	30.13		33.92	32.82	31.43	34.62	38.82
		N	30	30	30	30	30	30		30	30	30	30	30
G4	250	Mean	356.17	359.17	365.40	377.27	392.43	407.17		413.50	417.67	427.13	435.77	441.33
		SD	26.31	25.80	25.49	26.75	31.22	31.45		32.22	34.58	39.78	41.59	42.78
		N	30	30	30	30	30	30		30	30	30	30	30

 

			Pre-mating		Sex: Female
Group	Dose (mg/Kg)		Day of Exposure
			1	8		15	22
G1	0	Mean	238.07	242.10		246.67	256.50
		SD	17.01	16.63		16.35	14.89
		N	30	30		30	4
G2	62.5	Mean	237.60	242.53		248.17	272.50
		SD	20.80	20.77		19.25	16.26
		N	30	30		30	2
G3	125	Mean	241.83	244.50		247.47	244.25
		SD	19.86	20.38		21.86	14.45
		N	30	30		30	4
G4	250	Mean	240.87	241.93		245.80	258.00
		SD	16.30	15.80		14.70	22.63
		N	30	30		30	2

Keys:N: number; SD: standard deviation

 

			Gestation		Sex: Female
Group	Dose (mg/Kg)		Day of Gestation
			0	7		14	20
G1	0	Mean	253.72	280.45		310.76	374.66
		SD	17.43	19.74		22.43	29.95
		N	29	29		29	29
G2	62.5	Mean	253.36	282.14		312.32	372.75
		SD	21.86	23.49		24.03	30.58
		N	28	28		28	28
G3	125	Mean	250.93	278.55		310.45	372.38
		SD	20.57	20.32		24.60	30.02
		N	29	29		29	29
G4	250	Mean	247.04	275.42		306.04	364.27
		SD	15.24	17.44		20.50	27.77
		N	26	26		26	26

 

			Lactation			Sex: Female
Group	Dose (mg/Kg)		Day of Lactation
			0	4	7		14	21
G1	0	Mean	293.83	294.10	294.90		299.31	296.38
		SD	19.69	21.02	21.57		25.65	23.89
		N	29	29	29		29	29
G2	62.5	Mean	295.23	300.73	303.13		308.03	307.90
		SD	29.28	24.37	22.64		24.24	25.74
		N	28	28	28		28	28
G3	125	Mean	293.48	293.21	297.86		305.03	304.83
		SD	27.80	26.55	25.42		27.10	26.87
		N	29	29	29		29	29
G4	250	Mean	293.88	292.58	299.12		304.77	302.19
		SD	26.71	27.01	28.33		26.21	26.98
		N	26	26	26		26	26

Keys:N: number; SD: standard deviation

 

			Non-Parturated				Sex: Female
Group	Dose (mg/Kg)		Day of Gestation
			0	7	14	20		27	30	34
G1	0	Mean	260.00	261.00	271.00	274.00		271.00	./.	270.00
		SD	./.	./.	./.	./.		./.	./.	./.
		N	1	1	1	1		1	0	1
G2	62.5	Mean	271.50	274.50	279.50	278.50		281.50	289.00	264.00
		SD	41.72	37.48	12.02	16.26		21.92	./.	./.
		N	2	2	2	2		2	1	1
G3	125	Mean	251.00	252.00	257.00	255.00		255.00	./.	258.00
		SD	./.	./.	./.	./.		./.	./.	./.
		N	1	1	1	1		1	0	1
G4	250	Mean	254.25	268.50	272.25	270.50		274.00	./.	271.00
		SD	17.69	12.66	20.85	20.44		21.23	./.	20.85
		N	4	4	4	4		4	0	4

Keys:N: number; SD: standard deviation;./.= Not applicable

Table 2.4 Mean Body weight Change (%)

											Sex: Male
Group	Dose (mg/Kg)				Day of Exposure
					8	15	22	29		36		43		50		57	64	71
G1	0		Mean		2.28	5.81	9.46	14.11		19.26		21.47		22.83		25.59	26.57	28.83
			SD		2.19	3.17	3.81	4.23		3.92		4.07		4.89		4.99	5.55	5.66
			N		30	30	30	30		30		30		30		30	30	30
G2	62.5		Mean		2.34	5.75	8.79	13.84		19.57		21.69		23.23		25.88	26.49	28.74
			SD		2.39	3.62	3.29	3.39		4.00		4.62		4.85		5.24	5.26	5.44
			N		30	30	30	30		30		30		30		30	30	30
G3	125		Mean		2.20	4.92	8.35	13.13		17.89		20.38		22.32		24.10	25.62	27.49
			SD		2.35	3.59	3.79	4.14		3.85		4.37		4.57		4.89	5.22	6.30
			N		30	30	30	30		30		30		30		30	30	30
G4	250		Mean		0.87↓	2.65↓↓↓	5.99↓↓↓	10.20↓↓↓		14.38↓↓↓		16.17↓↓↓		17.32↓↓↓		19.96↓↓↓	22.36↓↓	23.93↓↓
			SD		1.38	2.43	2.86	3.68		4.38		4.73		5.21		7.03	7.22	7.63
			N		30	30	30	30		30		30		30		30	30	30
				Pre-mating									Sex: Female
Group		Dose (mg/Kg)		Day of Exposure
				8					15						22
G1		0	Mean	1.73					3.71						8.14
			SD	2.12					3.96						2.73
			N	30					30						4
G2		62.5	Mean	2.12					4.60						11.64
			SD	2.46					4.08						4.00
			N	30					30						2
G3		125	Mean	1.11					2.33						7.00
			SD	1.84					3.07						4.96
			N	30					30						4
G4		250	Mean	0.46					2.12						5.38
			SD	1.05					2.25						4.44
			N	30					30						2

Key: Body weight change (%) with respect to Day 1 of treatment; ↓: decreased as compared to control (P<0.05); ↓↓: decreased as compared to control (P<0.01); ↓↓↓: decreased as compared to control (P<0.001)

			Gestation		Sex: Female
Group	Dose (mg/Kg)		Day of Gestation
			7	14		20
G1	0	Mean	10.63	22.69		47.92
		SD	5.04	7.67		10.38
		N	29	29		29
G2	62.5	Mean	11.42	23.41		47.30
		SD	2.70	3.34		6.02
		N	28	28		28
G3	125	Mean	11.14	23.83		48.53
		SD	3.65	4.62		6.04
		N	29	29		29
G4	250	Mean	11.50	23.92		47.47
		SD	2.09	4.45		7.00
		N	26	26		26

 Key: Body weight change (%) with respect to Day 0 of Gestation

			Lactation		Sex: Female
Group	Dose (mg/Kg)		Day of Lactation
			4	7		14	21
G1	0	Mean	0.18	0.46		1.95	1.07
		SD	4.9	5.5		6.85	7.89
		N	29	29		29	29
G2	62.5	Mean	2.15	3.04		4.73	4.67
		SD	4.83	5.44		6.86	6.86
		N	28	28		28	28
G3	125	Mean	0.04	1.71		4.17	4.09
		SD	4.35	5.34		6.45	6.2
		N	29	29		29	29
G4	250	Mean	-0.33	1.93		3.97	3.22
		SD	5.12	6.24		6.91	8.61
		N	26	26		26	26

Key: Body weight change (%) with respect to Day 0 of Lactation

			Non-Parturated			Sex: Female
Group	Dose (mg/Kg)		Day of Gestation
			7	14	20		27	30	34
G1	0	Mean	0.38	4.23	5.38		4.23	./.	3.85
		SD	./.	./.	./.		./.	./.	./.
		N	1	1	1		1	0	1
G2	62.5	Mean	1.24	3.83	3.34		4.29	-3.99	9.09
		SD	1.75	11.53	9.89		7.95	./.	./.
		N	2	2	2		2	1	1
G3	125	Mean	0.40	2.39	1.59		1.59	./.	2.79
		SD	./.	./.	./.		./.	./.	./.
		N	1	1	1		0	1	0
G4	250	Mean	5.74	7.41	6.49		7.76	./.	6.61
		SD	3.00	10.47	5.99		3.05	./.	4.03
		N	4	4	4		4	0	4

Table 2.5 Summary of Feed Consumption (gram/animal/day)

									Sex: Male
Group	Dose (mg/Kg)		Day of Exposure
			1 to 8	8 to 15		28 to 36	36 to 43	43 to50		50 to 57	57 to 64	64 to 71
G1	0	Mean	15.82	20.01		19.78	19.10	19.74		19.57	20.54	21.00
		SD	2.90	1.52		1.66	1.33	1.47		1.10	1.78	1.58
		N	30	30		30	30	30		30	30	30
G2	62.5	Mean	19.09↑↑	19.36		20.10	19.36	19.88		19.81	20.04	21.13
		SD	1.89	1.59		1.30	1.27	1.48		1.23	1.92	1.55
		N	30	30		30	30	30		30	30	30
G3	125	Mean	18.73↑	19.18		19.71	19.32	20.31		20.31	21.37	21.90
		SD	1.48	1.73		1.48	1.53	1.78		1.23	2.11	2.10
		N	30	30		30	30	30		30	30	30
G4	250	Mean	18.03	18.88		19.04	19.07	19.59		20.02	20.91	20.08
		SD	1.39	1.42		1.73	1.28	1.33		1.76	1.83	2.95
		N	30	30		30	30	30		30	30	30

				Pre-mating		Sex: Female
Group	Dose (mg/Kg)		Day of Exposure
			1 to 8		8 to 15
G1	0	Mean	15.00		13.89
		SD	1.42		1.26
		N	30		30
G2	62.5	Mean	13.20↓↓		13.88
		SD	2.04		1.72
		N	30		30
G3	125	Mean	14.14		14.61
		SD	1.47		1.56
		N	30		30
G4	250	Mean	13.69		13.68
		SD	0.76		1.33
		N	30		30

Key: SD: Standard Deviation; N: Number of Animals; ↑: increased as compared to control (P<0.05); ↑↑: increased as compared to control (P<0.01); ↓↓: decreased as compared to control (P<0.01).

			Gestation			Sex: Female
Group	Dose (mg/Kg)		Day of Gestation
			0 to 7	7 to 14	14 to 20		20 to 22	20 to 23
G1	0	Mean	16.71	19.13	22.30		25.43	23.00
		SD	2.57	3.95	3.35		9.27	./.
		N	29	29	29		28	1
G2	62.5	Mean	15.81	19.32	22.10		21.83	18.00
		SD	2.50	1.49	3.84		7.37	1.41
		N	28	28	28		26	2
G3	125	Mean	16.93	19.68	21.95		26.39	17.33
		SD	1.97	1.98	3.13		11.30	1.41
		N	29	29	29		27	2
G4	250	Mean	16.30	19.15	20.62		25.65	./.
		SD	2.01	1.76	3.19		7.90	./.
		N	26	26	26		26	0

			Lactation		Sex: Female
Group	Dose (mg/Kg)		Day of Lactation
			0 to 4	4 to 7		7 to 14	14 to 21
G1	0	Mean	29.47	38.51		48.31	56.82
		SD	9.17	9.11		9.07	9.02
		N	29	29		29	29
G2	62.5	Mean	27.44	39.04		47.22	57.88
		SD	5.28	7.85		10.01	6.89
		N	29	29		29	29
G3	125	Mean	26.88	40.29		47.83	57.73
		SD	5.33	10.03		7.88	9.19
		N	29	29		29	29
G4	250	Mean	24.72	35.53		47.18	55.68
		SD	6.47	8.32		6.59	10.46
		N	26	26		26	26

Key: SD: Standard Deviation; N: Number of Animals

			Non-Parturated			Sex: Female
Group	Dose (mg/Kg)		Day of Gestation
			0 to 7	7 to 14	14 to 20		20 to 27	27 to 30	27 to 34
G1	0	Mean	14.86	16.29	18.5		17.71	./.	17.29
		SD	./.	./.	./.		./.	./.	./.
		N	1	1	1		1	./.	1
G2	62.5	Mean	15.22	12.5	20.42		10.5	14	11.57
		SD	7.38	3.34	5.3		9.8	./.	./.
		N	2	2	2		2	1	1
G3	125	Mean	10.43	12.57	12.83		11.86	./.	11.57
		SD	./.	./.	./.		./.	./.	./.
		N	1	1	1		1	./.	1
G4	250	Mean	15.46	15.79	13.96		18.54	./.	16
		SD	1.96	1.97	2.87		2.94	./.	1.65
		N	4	4	4		4	./.	4

Key: SD: Standard Deviation; N: Number of Animals; ./. Not applicable

Table 2.6 Feed efficiency

						Sex: Male
Group	Dose (mg/kg)		Feed Efficiency
			8	15	36		43	50	57	64	71
G1	0	Mean	0.074	0.088	0.132		0.057	0.033	0.071	0.023	0.053
		SD	0.067	0.049	0.046		0.043	0.047	0.047	0.059	0.031
		N	30	30	30		30	30	30	30	30
G2	62.5	Mean	0.061	0.086	0.144		0.055	0.039	0.067	0.015	0.054
		SD	0.063	0.055	0.047		0.043	0.039	0.040	0.053	0.040
		N	30	30	30		30	30	30	30	30
G3	125	Mean	0.058	0.071	0.122		0.065	0.049	0.044	0.036	0.044
		SD	0.06	0.051	0.087		0.061	0.037	0.036	0.046	0.066
		N	30	30	30		30	30	30	30	30
G4	250	Mean	0.023↓↓	0.047↓↓	0.110		0.047	0.030	0.064	0.059↑	0.039
		SD	0.037	0.035	0.066		0.044	0.038	0.088	0.057	0.061
		N	30	30	30		30	30	30	30	30

				Sex: Female
Group	Dose (mg/kg)		Feed Efficiency
			0		15
G1	0	Mean	0.039		0.047
		SD	0.047		0.056
		N	30		30
G2	62.5	Mean	0.057		0.059
		SD	0.070		0.057
		N	30		30
G3	125	Mean	0.026		0.028
		SD	0.044		0.035
		N	30		30
G4	250	Mean	0.011		0.039
		SD	0.026		0.037
		N	30		30

Key: SD: Standard Deviation; N: Number of Animals;↑: increased as compared to control (P<0.05);↓↓: decreased as compared to control (P<0.01).

				Gestation		Sex: Female
Group	Dose (mg/kg)		Feed Efficiency
			0		15		20
G1	0	Mean	0.229		0.209		0.480
		SD	0.102		0.127		0.070
		N	30		30		30
G2	62.5	Mean	0.264		0.223		0.465
		SD	0.063		0.054		0.095
		N	28		28		28
G3	125	Mean	0.236		0.231		0.478
		SD	0.078		0.058		0.084
		N	28		28		28
G4	250	Mean	0.251		0.226		0.476
		SD	0.048		0.066		0.093
		N	26		26		26

				Non Parturated Dam			Sex: Female
Group	Dose (mg/kg)		Feed Efficiency
			0		15	20		27
G1	0	Mean	0.010		0.088	0.027		-0.024
		SD	./.		./.	./.		./.
		N	1		1	1		1
G2	62.5	Mean	0.043		0.019	-0.004		0.009
		SD	0.061		0.286	0.034		0.069
		N	2		2	2		2
G3	125	Mean	0.014		0.057	-0.026		0.000
		SD	./.		./.	./.		./.
		N	1		1	1		1
G4	250	Mean	0.129		0.024	-0.029		0.024
		SD	0.045		0.160	0.153		0.069
		N	4		4	4		4

Key: SD: Standard Deviation; N: Number of Animals; ./.: not applicable

Table 2.7 Detailed clinical examination

Sex: Male

Group

Dose (mg/Kg)

Day of Exposure

1

8

15

22

29

36

43

50

57

64

71

G1

0

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

G2

62.5

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

G3

125

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

G4

250

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

Pre-mating

Sex: Female

Group

Dose (mg/Kg)

Day of Exposure

1

8

15

22

G1

0

NAD

NAD

NAD

NAD

G2

62.5

NAD

NAD

NAD

NAD

G3

125

NAD

NAD

NAD

NAD

G4

250

NAD

NAD

NAD

NAD

Gestation

Sex: Female

Group

Dos   Gestation Sex: Female e (mg/Kg)

Day of Gestation

0

7

14

20

G1

0

NAD

NAD

NAD

NAD

G2

62.5

NAD

NAD

NAD

NAD

G3

125

NAD

NAD

NAD

NAD

G4

250

NAD

NAD

NAD

NAD

Key: NAD: no abnormality detected

				Lactation			Sex: Female
Group	Dose (mg/Kg)	Day of Lactation
		0	4		7	14		21
G1	0	NAD	NAD		NAD	NAD		NAD
G2	62.5	NAD	NAD		NAD	NAD		NAD
G3	125	NAD	NAD		NAD	NAD		NAD
G4	250	NAD	NAD		NAD	NAD		NAD

				Non-Parturated				Sex: Female
Group	Dose (mg/Kg)	Day of Gestation
		0	7		14	20	27		30	34
G1	0	NAD	NAD		NAD	NAD	NAD		NAD	NAD
G2	62.5	NAD	NAD		NAD	NAD	NAD		NAD	NAD
G3	125	NAD	NAD		NAD	NAD	NAD		NAD	NAD
G4	250	NAD	NAD		NAD	NAD	NAD		NAD	NAD

Table 2.8 Mean Hormone levels (T4, TSH, Estradiol, Progesterone, Testesterone)

							Sex: Male
Group	Dose (mg/kg)			TSH(UI/ml)	Estradiol (pg/ml)	Progesterone (ng/ml)		Tetsesterone (pg/ml)	T4 (ng/ml)
G1	0	Mean		0.17	114.02	3.74		37.18	81.36
		SD		0.06	24.85	0.86		10.58	18.84
		N		10	10	10		10	10
G2	62.5	Mean		0.15	100.65	3.46		39.36	64.34
		SD		0.03	24.63	0.50		15.81	13.55
		N		10	10	10		10	10
G3	125	Mean		0.14	97.43	3.47		32.89	53.49↓↓
		SD		0.07	27.86	1.12		12.84	11.01
		N		10	10	10		10	10
G4	250	Mean		0.12	104.89	3.97		32.22	69.79
		SD		0.03	19.89	1.03		16.61	21.42
		N		10	10	10		9	10

							Sex: Female
Group	Dose (mg/kg)			TSH(UI/ml)	Estradiol (pg/ml)	Progesterone (ng/ml)		Tetsesterone (pg/ml)	T4 (ng/ml)
G1	0	Mean		0.12	104.81	6.26		52.54	49.27
		SD		0.02	27.48	1.34		10.91	8.33
		N		10	10	10		10	10
G2	62.5	Mean		0.10	97.80	5.76		65.59	51.60
		SD		0.03	24.62	2.07		16.44	6.71
		N		10	10	10		10	10
G3	125	Mean		0.12	103.23	5.10		54.71	56.61
		SD		0.03	22.84	1.56		16.05	8.09
		N		10	10	10		10	10
G4	250	Mean		0.10	84.89	3.99↓↓↓		45.70	47.63
		SD		0.02	25.31	1.56		9.51	11.29
		N		10	10	10		10	10

Key: N: number of animals, SD: standard deviation; ↓↓: decreased as compared to control (P<0.01); ↓↓↓: decreased as compared to control (P<0.001).

Table 2.9 Hematology

				Sex: Male
Group (N)	G1 (10)		G2 (10)			G3 (10)		G4 (10)
Dose (mg/kg)	0		62.5			125		250
Parameters	Mean	SD	Mean		SD	Mean	SD	Mean	SD
Total Erythrocyte Count (RBC) (X 10^12/L)	8.62	0.49	8.82		0.41	8.47	0.39	8.76	0.48
Hematocrit (HCT) (%)	43.61	1.61	44.22		2.05	42.28	2.43	43.41	1.85
Mean Corpuscular Volume (MCV) (fL)	50.69	2.78	50.11		1.37	49.90	1.39	49.57	1.97
Hemoglobin (HGB) (g/dL)	15.62	0.69	15.84		0.69	15.19	0.90	15.56	0.60
Mean Corpuscular Hemoglobin (MCH) (pg)	18.16	0.91	17.95		0.40	17.92	0.47	17.77	0.68
Mean Corpuscular Hemoglobin Concentration (MCHC) (g/dL)	35.84	0.45	35.88		0.39	35.93	0.25	35.88	0.66
Platelet Count (PLT) (X 10^9/L)	676.30	63.08	682.20		86.96	686.90	71.09	705.80	49.65
Total Leukocyte count (WBC) (X 10^9/L)	9.33	0.99	10.29		1.84	10.27	3.32	12.50↑↑	1.93
Neutrophil (%)	18.20	2.35	18.50		3.24	17.70	2.58	18.00	2.67
Lymphocyte (%)	81.30	2.26	81.00		3.16	82.00	2.40	81.10	2.42
Monocyte (%)	0.20	0.42	0.30		0.48	0.30	0.48	0.60	0.52
Eosinophil (%)	0.30	0.48	0.20		0.42	0.00	0.00	0.30	0.48
Basophil (%)	0.00	0.00	0.00		0.00	0.00	0.00	0.00	0.00
Prothrombin Time (PT) (Sec.)	22.22	2.43	23.31		1.93	22.98	2.01	22.00	1.11
Activated Partial thromboplastin Time (aPTT) (Sec.)	28.45	1.76	27.44		1.12	28.33	1.58	27.07	1.72
Reticulocytes (%)	1.00	0.07	1.00		0.05	1.05	0.10	1.00	0.05

Keys: SD = Standard Deviation; N = Number of animals; ↑↑: increased as compared to control (P<0.01).

				Sex: Female
Group (N)	G1 (10)		G2 (10)			G3 (10)		G4 (10)
Dose (mg/kg)	0		62.5			125		250
Parameters	Mean	SD	Mean		SD	Mean	SD	Mean	SD
Total Erythrocyte Count (RBC) (X 10^12/L)	7.91	0.52	7.77		0.50	7.99	0.36	7.85	0.22
Hematocrit (HCT) (%)	42.17	2.70	41.33		2.51	42.93	1.55	42.41	1.61
Mean Corpuscular Volume (MCV) (fL)	53.34	2.41	53.25		1.78	53.71	2.08	54.01	1.54
Hemoglobin (HGB) (g/dL)	14.97	0.91	14.79		0.85	15.29	0.43	15.04	0.55
Mean Corpuscular Hemoglobin (MCH) (pg)	18.95	0.66	19.06		0.49	19.14	0.61	19.16	0.61
Mean Corpuscular Hemoglobin Concentration (MCHC) (g/dL)	35.52	0.71	35.84		0.55	35.67	0.62	35.46	0.27
Platelet Count (PLT) (X 10^9/L)	718.00	82.00	739.20		82.18	718.70	81.04	719.50	58.33
Total Leukocyte count (WBC) (X 10^9/L)	9.36	3.28	11.50		5.01	12.88	3.70	13.14	3.52
Neutrophil (%)	18.30	3.30	17.60		2.84	19.40	1.78	16.40	2.01
Lymphocyte (%)	81.30	2.98	81.80		2.62	80.00	1.56	83.00	1.83
Monocyte (%)	0.20	0.42	0.30		0.48	0.40	0.52	0.40	0.52
Eosinophil (%)	0.20	0.42	0.30		0.48	0.20	0.42	0.20	0.42
Basophil (%)	0.00	0.00	0.00		0.00	0.00	0.00	0.00	0.00
Prothrombin Time (PT) (Sec.)	20.83	1.70	21.08		1.56	21.69	1.53	22.45	2.89
Activated Partial thromboplastin Time (aPTT) (Sec.)	28.39	1.53	27.18		1.24	27.17	1.37	28.25	1.42
Reticulocytes (%)	1.01	0.10	1.03		0.12	1.08	0.14	1.08	0.12

Table 3.1 Clinical Chemistry

					Sex: Male
Group (N)	G1 (10)		G2 (10)			G3 (10)		G4 (10)
Dose (mg/kg)	0		62.5			125		250
Parameters	Mean	SD	Mean	SD		Mean	SD	Mean	SD
ALT (U/l)	49.95	6.63	44.29	5.47		46.29	7.59	55.30	5.71
AST (U/l)	176.42	22.54	171.89	35.76		178.80	34.14	192.26	32.39
Albumin (g/dl)	3.85	0.14	4.00	0.17		4.00	0.20	4.07	0.36
ALP (U/l)	171.20	15.92	150.88	11.15		201.88	31.82	176.10	21.19
T.Protein (g/dl)	6.32	0.63	6.54	0.59		6.45	0.47	6.67	0.52
Urea (mg/dl)	49.76	7.90	49.48	3.88		48.13	12.59	51.20	10.64
Bile Acid (µmol/l)	21.62	11.11	25.12	10.76		27.38	9.23	23.67	11.07
Cholesterol (mg/dl)	53.21	7.92	55.99	7.83		60.08	11.93	58.38	8.37
Calcium (mg/dl)	10.11	0.31	10.12	0.60		10.30	0.25	10.37	0.51
CK- Nac (U/I)	780.10	183.19	813.90	209.05		803.40	228.85	733.80	177.29
Creatinine (mg/dl)	0.53	0.05	0.53	0.02		0.51	0.04	0.51	0.04
Glucose (mg/dl)	88.59	11.28	84.55	9.86		88.30	5.54	92.58	12.36
GGT(U/I)	4.93	1.81	5.60	2.33		5.12	2.85	5.14	2.60
LDH(U/I)	1131.39	225.25	1042.27	286.03		1028.03	238.64	1146.85	248.37
Phosphorus(mg/dl)	6.06	0.83	5.62	0.74		6.15	0.65	7.04↑	1.08
Triglyceride (mg/dl)	65.59	15.66	65.37	10.46		60.29	6.70	60.00	9.70
Total Bilirubin (mg/dl)	0.33	0.16	0.25	0.08		0.29	0.11	0.32	0.05
BUN (mg/dl)	23.25	3.69	23.10	1.84		22.50	5.88	23.45	4.92
A/G Ratio	1.65	0.39	1.67	0.50		1.68	0.30	1.63	0.42
Globulin (g/dl)	2.46	0.57	2.55	0.61		2.45	0.41	2.60	0.50
Na (mmol/l)	145.00	2.05	142.40	4.84		147.20	2.44	149.60↑	4.95
K (mmol/l)	5.87	0.79	5.99	0.93		5.85	0.52	5.76	0.44
Cl (mmol/l)	99.30	2.36	99.10	4.33		101.70	2.45	104.00↑	3.94

					Sex: Female
Group (N)	G1 (10)		G2 (10)			G3 (10)		G4 (10)
Dose (mg/kg)	0		62.5			125		250
Parameters	Mean	SD	Mean	SD		Mean	SD	Mean	SD
ALT (U/l)	80.68	11.99	87.19	18.28		79.58	13.05	88.76	17.95
AST (U/l)	184.60	23.52	192.07	21.92		186.66	23.20	192.09	20.06
Albumin (g/dl)	3.94	0.35	3.78	0.31		3.88	0.28	3.78	0.33
ALP (U/l)	148.25	29.22	132.24	17.25		136.98	20.93	144.91	21.30
T.Protein (g/dl)	7.15	0.66	7.02	0.52		6.92	0.67	6.95	0.75
Urea (mg/dl)	70.14	7.13	65.41	10.72		64.40	7.58	65.18	12.35
Bile Acid (µmol/l)	55.72	13.92	52.76	19.48		50.90	20.35	50.62	17.47
Cholesterol (mg/dl)	74.43	10.30	78.96	10.13		80.41	13.27	77.15	12.86
Calcium (mg/dl)	9.52	0.30	9.68	0.51		10.02	0.41	9.58	0.51
CK- Nac (U/I)	647.00	216.82	660.10	183.83		691.60	180.56	689.30	186.26
Creatinine (mg/dl)	1.29	0.26	1.37	0.25		1.31	0.25	1.23	0.26
Glucose (mg/dl)	89.82	8.90	96.87	11.33		95.40	12.38	91.79	13.60
GGT(U/I)	9.49	2.31	8.08	2.90		8.43	1.69	8.01	1.51
LDH(U/I)	1083.00	232.85	988.32	258.29		1108.26	244.02	1120.21	279.06
Phosphorus(mg/dl)	8.01	1.03	7.70	1.08		7.87	1.35	7.88	0.58
Triglyceride (mg/dl)	61.52	19.01	65.80	12.71		63.31	9.60	67.74	10.02
Total Bilirubin (mg/dl)	0.50	0.09	0.64	0.16		0.64	0.26	0.56	0.17
BUN (mg/dl)	32.77	3.34	30.58	5.01		30.00	3.49	30.46	5.77
A/G Ratio	1.20	0.16	1.19	0.18		1.29	0.15	1.23	0.21
Globulin (g/dl)	3.28	0.56	3.23	0.43		3.04	0.45	3.17	0.62
Na (mmol/l)	139.80	1.48	139.60	0.70		139.30	2.21	138.70	2.00
K (mmol/l)	6.54	0.77	6.21	0.73		6.57	0.79	6.29	0.69
Cl (mmol/l)	99.00	2.21	99.50	2.46		100.60	2.01	100.30	2.58

Table 3.2 Urinalysis

				Sex: Male
Group (N)	G1 (10)		G2 (10)			G3 (10)		G4 (10)
Dose (mg/kg)	0		62.5			125		250
Parameters	Mean	SD	Mean		SD	Mean	SD	Mean	SD
Blood / Blood Cell (RBC/µL)	neg	./.	neg		./.	neg	./.	neg	./.
Bilirubin (mg/dL)	neg	./.	neg		./.	neg	./.	neg	./.
Urobilinogen (mg/dL)	norm	./.	norm		./.	norm	./.	norm	./.
Ketone (mg/dL)	neg	./.	neg		./.	neg	./.	neg	./.
Protein (mg/dL)	neg	./.	neg		./.	neg	./.	neg	./.
Nitrite	neg	./.	neg		./.	neg	./.	neg	./.
Glucose (mg/dL)	neg	./.	neg		./.	neg	./.	neg	./.
pH	5.90	0.74	5.85		0.47	5.80	0.48	6.00	0.62
Specific Gravity	1.022	0.005	1.017		0.006	1.020	0.006	1.019	0.007
Leukocytes (WBC/µL)	neg	./.	neg		./.	neg	./.	neg	./.
Physical Observations
Volume (mL)	16.40	3.86	16.80		3.05	15.20	2.90	16.80	3.79
Colour	Pale Yellow	./.	Pale Yellow		./.	Pale Yellow	./.	Pale Yellow	./.
Appearance	Clear	./.	Clear		./.	Clear	./.	Clear	./.

Keys: neg:negative; norm: normal

				Sex: Female
Group (N)	G1 (10)		G2 (10)			G3 (10)		G4 (10)
Dose (mg/kg)	0		62.5			125		250
Parameters	Mean	SD	Mean		SD	Mean	SD	Mean	SD
Blood / Blood Cell (RBC/µL)	neg	./.	neg		./.	neg	./.	neg	./.
Bilirubin (mg/dL)	neg	./.	neg		./.	neg	./.	neg	./.
Urobilinogen (mg/dL)	norm	./.	norm		./.	norm	./.	norm	./.
Ketone (mg/dL)	neg	./.	neg		./.	neg	./.	neg	./.
Protein (mg/dL)	neg	./.	neg		./.	neg	./.	neg	./.
Nitrite	neg	./.	neg		./.	neg	./.	neg	./.
Glucose (mg/dL)	neg	./.	neg		./.	neg	./.	neg	./.
pH	5.35	0.47	5.30		0.42	5.45	0.50	5.25	0.42
Specific Gravity	1.024	0.002	1.025		0.000	1.024	0.002	1.024	0.002
Leukocytes (WBC/µL)	neg	./.	neg		./.	neg	./.	neg	./.
Physical Observations
Volume (mL)	14.10	3.60	14.80		2.25	15.60	1.71	16.30	3.16
Colour	Pale Yellow	./.	Pale Yellow		./.	Pale Yellow	./.	Pale Yellow	./.
Appearance	Clear	./.	Clear		./.	Clear	./.	Clear	./.

Keys: neg: negative; norm: normal

Table 3.3 Absolute organ weights

						Mean organ weights (g)						Sex: Male
Group	Dose		BW	Brain	Adrenals		P & SV	Testes	Ep	Heart	Liver		Kidneys	Spleen	Thymus	Pituitary	T & PT
G1	0	Mean	432.13	2.1733	0.0653		3.8409	3.2878	1.5106	1.3489	13.2371		2.8793	0.8518	0.3846	0.0148	0.0225
		SD	26.85	0.0938	0.0115		0.4526	0.2009	0.1184	0.1111	1.4264		0.4488	0.4390	0.0820	0.0021	0.0031
		N	30	30	30		30	30	30	30	30		30	30	30	30	30
G2	62.5	Mean	439.10	2.1894	0.0659		3.7032	3.3069	1.5295	1.3957	13.6968		2.9693	0.7618	0.4097	0.0146	0.0219
		SD	27.80	0.1205	0.0104		0.3724	0.2647	0.1288	0.1500	1.7457		0.1780	0.0994	0.0887	0.0017	0.0041
		N	30	30	30		30	30	30	30	30		30	30	30	30	30
G3	125	Mean	432.57	2.2122	0.0688		3.5891	3.4090	1.5136	1.3780	13.9272		3.0300	0.8931	0.4029	0.0145	0.0224
		SD	32.71	0.0822	0.0088		0.5126	0.2741	0.1457	0.1242	1.3925		0.2507	0.4175	0.0688	0.0020	0.0038
		N	30	30	30		30	30	30	30	30		30	30	30	30	30
G4	250	Mean	417.10	2.1821	0.0653		3.5503	3.2489	1.4888	1.3824	13.3019		2.9758	0.7588	0.3749	0.0138	0.0220
		SD	36.89	0.0900	0.0101		0.4892	0.4676	0.1595	0.1867	1.7709		0.3089	0.0928	0.0990	0.0016	0.0048
		N	30	30	30		30	30	30	30	30		30	30	30	30	30

						Mean organ weights (g)					Sex: Female
Group	Dose		BW	Brain	Adrenals		Ovaries	Uterus	Heart	Liver		Kidneys	Spleen	Thymus	Pituitary	T & PT
G1	0	Mean	277.13	2.0061	0.0883		0.1506	0.4639	1.0677	15.8516		2.1307	0.6678	0.2184	0.0137	0.0191
		SD	22.36	0.0991	0.0131		0.0368	0.1418	0.1691	3.0913		0.2136	0.1410	0.1030	0.0021	0.0039
		N	30	30	30		30	30	30	30		30	30	30	30	30
G2	62.5	Mean	280.60	2.0174	0.0861		0.1476	0.4310	1.0576	16.0515		2.0773	0.6261	0.2262	0.0139	0.0179
		SD	22.07	0.1135	0.0114		0.0252	0.1315	0.0815	3.4609		0.1922	0.0792	0.0813	0.0019	0.0042
		N	30	30	30		30	30	30	30		30	30	30	30	30
G3	125	Mean	278.37	1.9954	0.0873		0.1484	0.4800	1.0546	16.2290		2.0188	0.7056	0.2027	0.0137	0.0187
		SD	24.92	0.1270	0.0106		0.0308	0.1354	0.1175	3.4161		0.3792	0.4207	0.0728	0.0020	0.0036
		N	30	30	30		30	30	30	30		30	30	30	30	30
G4	250	Mean	271.83	1.9932	0.0875		0.1493	0.4865	1.0543	13.7392↓		2.0776	0.6942	0.2307	0.0146	0.0194
		SD	21.92	0.0826	0.0117		0.0227	0.1652	0.1286	2.8704		0.3441	0.2913	0.0681	0.0023	0.0032
		N	30	30	30		30	30	30	30		30	30	30	30	30

Key: BW = Body Weight; Ep = Epididymis; N = Number of samples; P & SV = Prostate and Seminal Vesicle; SD = Standard deviation; T & PT = Thyroid & Parathyroid. Dose levels are in terms of mg/kg body weight; ↓: decreased as compared to control (P<0.05).

Table 3.4 Relative organ weights

					Mean organ weight relative body weight (%)						Sex: Male
Group	Dose		Brain	Adrenals		P & SV	Testes	Ep	Heart	Liver		Kidneys	Spleen	Thymus	Pituitary	T & PT
G1	0	Mean	0.5046	0.0152		0.8920	0.7624	0.3504	0.3126	3.0619		0.6669	0.1975	0.0892	0.0034	0.0052
		SD	0.0354	0.0028		0.1175	0.0478	0.0291	0.0239	0.2536		0.1008	0.1037	0.0192	0.0004	0.0008
		N	30	30		30	30	30	30	30		30	30	30	30	30
G2	62.5	Mean	0.4999	0.0151		0.8449	0.7541	0.3487	0.3178	3.1250		0.6776	0.1740	0.0932	0.0033	0.0050
		SD	0.0321	0.0027		0.0845	0.0549	0.0255	0.0270	0.3947		0.0416	0.0239	0.0191	0.0004	0.0010
		N	30	30		30	30	30	30	30		30	30	30	30	30
G3	125	Mean	0.5141	0.0160		0.8327	0.7901	0.3509	0.3190	3.2271		0.7014	0.2081	0.0935	0.0034	0.0052
		SD	0.0411	0.0022		0.1216	0.0609	0.0336	0.0226	0.3076		0.0434	0.1036	0.0167	0.0005	0.0010
		N	30	30		30	30	30	30	30		30	30	30	30	30
G4	250	Mean	0.5266	0.0157		0.8536	0.7834	0.3589	0.3318	3.1888		0.7142	0.1831	0.0903	0.0033	0.0053
		SD	0.0451	0.0023		0.1126	0.1228	0.0442	0.0393	0.3091		0.0526	0.0258	0.0245	0.0004	0.0013
		N	30	30		30	30	30	30	30		30	30	30	30	30

					Mean organ weight relative body weight (%)					Sex: Female
Group	Dose		Brain	Adrenals		Ovaries	Uterus	Heart	Liver		Kidneys	Spleen	Thymus	Pituitary	T & PT
G1	0	Mean	0.7279	0.0321		0.0545	0.1686	0.3855	5.7142		0.7716	0.2431	0.0796	0.0049	0.0069
		SD	0.0621	0.0055		0.0136	0.0532	0.0518	0.9615		0.0797	0.0590	0.0388	0.0007	0.0015
		N	30	30		30	30	30	30		30	30	30	30	30
G2	62.5	Mean	0.7224	0.0308		0.0525	0.1529	0.3777	5.7142		0.7414	0.2233	0.0809	0.0050	0.0064
		SD	0.0596	0.0040		0.0067	0.0424	0.0239	1.0950		0.0554	0.0246	0.0299	0.0008	0.0014
		N	30	30		30	30	30	30		30	30	30	30	30
G3	125	Mean	0.7208	0.0315		0.0535	0.1743	0.3793	5.8108		0.7278	0.2525	0.0731	0.0050	0.0067
		SD	0.0619	0.0037		0.0109	0.0534	0.0310	1.0410		0.1324	0.1423	0.0277	0.0009	0.0011
		N	30	30		30	30	30	30		30	30	30	30	30
G4	250	Mean	0.7371	0.0324		0.0552	0.1794	0.3877	5.0395↓		0.7652	0.2558	0.0848	0.0054	0.0072
		SD	0.0590	0.0053		0.0088	0.0600	0.0325	0.8677		0.1217	0.1115	0.0238	0.0008	0.0013
		N	30	30		30	30	30	30		30	30	30	30	30

Key: Ep = Epididymis; N = Number of samples; P & SV = Prostate and Seminal Vesicle; SD = Standard deviation; T & PT = Thyroid & Parathyroid. Dose levels are in terms of mg/kg body weight; ↓: decreased as compared to control (P<0.05).

Table 3.5 Gross Pathology

		Sex: Male
Group	G1		G2	G3	G4
Dose (mg/kg)	0		62.5	125	250
Total No. of Animals Observed	30		30	30	30
No. of animals with Abnormalities	0		2	1	1
No. of animals with No Abnormalities	30		28	29	29
Organ & Lesion
Testes
Reduced sizeTotal	0		1	0	1
Mild	0		0	0	1
Moderate	0		1	0	0
Prostate
Increased sizeTotal	0		1	0	0
Minimal	0		1	0	0
Reduced sizeTotal	0		0	1	0
Moderate	0		0	1	0

		Sex: Female
Group	G1		G2	G3	G4
Dose (mg/kg)	0		62.5	125	250
Total No. of Animals Observed	30		30	30	30
No. of animals with Abnormalities	0		0	0	0
No. of animals with No Abnormalities	30		30	30	30

Table 3.6 Individual Animal Microscopic Observations

		Sex: Male
Group	G1		G2	G3	G4
Dose (mg/kg)	0		62.5	125	250
Total No. of Animals Observed	30		30	30	30
No. of animals with Abnormalities	11		X	X	5
No. of animals with No Abnormalities	19		X	X	25
Organ & Lesion
Liver
Lymphocyte infiltrationTotal	2		X	X	1
Focal Minimal	0		X	X	1
Focal Mild	2		X	X	0
Kidneys
Tubular dilation             Total	3		X	X	0
Focal Mild	2		X	X	0
Focal Moderate	1		X	X	0
Lymphocyte infiltration    Total	1		X	X	0
Focal Mild	1		X	X	0
Lungs
Lymphocyte infiltration    Total	3		X	X	2
Multifocal Mild	2		X	X	1
Multifocal Moderate	1		X	X	1
Aorta
Aneurysm                      Total	1		X	X	1
Focal Mild	1		X	X	0
Focal Moderate	0		X	X	1
Colon
Lymphocyte infiltration    Total	1		X	X	0
Multifocal Mild	1		X	X	0
Thymus
LymphophagocytosisTotal	1		X	X	0
Multifocal Minimal	1		X	X	0
Thyroid
Ultimobranchial cystTotal	1		X	X	1
Focal Mild	1		X	X	1
Adrenals
Cytoplasmic vacuolationTotal	1		X	X	0
Diffuse Mild	1		X	X	0
Accessory adrenocortical tissueTotal	1		X	X	0
Unilateral	1		X	X	0

		Sex: Female
Group	G1		G2	G3	G4
Dose (mg/kg)	0		62.5	125	250
Total No. of Animals Observed	30		30	30	30
No. of animals with Abnormalities	12		X	X	8
No. of animals with No Abnormalities	18		X	X	22
Organ & Lesion
Liver
Lymphocyte infiltration    Total	0		X	X	1
Focal Minimal	0		X	X	1
Kidneys
Tubular dilation             Total	1		X	X	0
Multifocal Mild	1		X	X	0
Lymphocyte infiltration    Total	2		X	X	1
Focal Minimal	0		X	X	1
Focal Mild	1		X	X	0
Multifocal Mild	1		X	X	0
Lungs
Lymphocyte infiltration    Total	1		X	X	2
Focal Mild	0		X	X	1
Multifocal Mild	1		X	X	1
Alveolar histiocytosis    Total	1		X	X	1
Focal Mild	0		X	X	1
Multifocal Mild	1		X	X	0
Aorta
Aneurysm                      Total	1		X	X	0
Focal Mild	1		X	X	0
Thymus
LymphophagocytosisTotal	1		X	X	1
Multifocal Mild	1		X	X	1
Atrophy                     Total	1		X	X	0
Focal Mild	1		X	X	0
Thyroid
Ultimobranchial cystTotal	2		X	X	1
Focal Mild	2		X	X	0
Focal Moderate	0		X	X	1
Adrenals
Accessory adrenocortical tissueTotal	2		X	X	2
Unilateral	2		X	X	2

Applicant's summary and conclusion

Conclusions:

Based on all the available data, it was observed that there were no effects observed on male and female animals up to 125 mg/kg bw (Mid Dose-G3) of the test chemical administration. However, at 250 mg/kg bw (High Dose-G4) dose group, clinical signs, effects on weights of liver and effects on hormonal levels was observed, due to test chemical administration. Therefore, it was concluded that the NOAEL and LOAEL of the test chemical was 125 mg/kg bw and 250 mg/kg bw, respectively.

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. During observations, it was seen that 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 hematology 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. Based on all the available data, it was observed that there were no effects observed on male and female animals up to 125 mg/kg bw (Mid Dose-G3) of the test chemical administration. However, at 250 mg/kg bw (High Dose-G4) dose group, clinical signs, effects on weights of liver and effects on hormonal levels was observed, due to test chemical administration. Therefore, it was concluded that the NOAEL and LOAEL of the test chemical was 125 mg/kg bw and 250 mg/kg bw, respectively.