Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Weight of evidence approach based on the data of the test chemicals.
Cross-referenceopen allclose all
Reason / purpose:
read-across source
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
To evaluations of general and reproduction/ developmental toxicity endpoints associated with administration of repeated doses of the test chemical in Wistar rats.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): methyl phenylacetate
-Molecular Formula: C9H10O2
-Molecular Weight: 150.176 g/mole
- Substance type: Organic
- Physical state: Liquid
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Source : In-House Bred at sa-FORD, Animal Facility (CPCSEA Registration No. 1256/bc/09/CPCSEA)
Health Status :Healthy young adult animals were used for the study. Females were nulliparous and non-pregnant.
Body weight of animals :
Male: Minimum: 240g; Maximum: 315 g
Female: Minimum: 210g; Maximum: 260 g (Individual body weights were within ± 20% of mean body weight, prior to treatment)
Age: 12-13 weeks at the start of Oestrous Cycle evaluation.
Acclimatisation: Animals were acclimatised to the test conditions for 20 days prior to test item administration
Housing: Before the animals are brought in, the study room and cages were cleaned and disinfected. During the study, the floor of the experimental room and work tops were swept and mopped with disinfectant solution every day or as on requirement. Cages were cleaned at regular intervals.
A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20 [cm]). Cage rotation was carried out weekly during study period except during mating for males and females both and during gestation and lactation for females.
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.
Bedding material of batch No. SPAR-30/2015 (Sparconn Life Sciences Bangalore) was used in this study and a copy of report of microbial and chemical contaminants analysed periodically by manufacturer of bedding material are incorporated in the raw data.

Environmental conditions
The room temperature was maintained at 18.30 to 22.70 °C and the relative humidity was kept between 43.90 to 67.60%. Artificial light was set to give a cycle of 12 hours light and 12 hours dark. Air changes were about minimum 12 times per hour and filtered adequately.

Diet :A conventional laboratory pelleted diet of batch no. 004915, 041215 and 041015 from approved supplier (Nutrivet Life Sciences, Pune) was offered ad libitum. The copy of composition, microbial and chemical contaminant reports analysed periodically by manufacturer are incorporated in the raw data.

Water : Aqua guard filtered drinking water in bottles was offered ad libitum. Samples of the drinking water was subjected periodically to bacteriological tests and to chemical contaminant analysis. The latest test results are included in the raw data.
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item. The details of dose formulation preparation is maintained in raw data.

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was used as a vehicle based on the solubility testing
- Concentration in vehicle: 0, 308, 556 and 1000 mg/kg bw
- Amount of vehicle (if gavage): 0.5 ml/kg
- Lot/batch no. (if required): MR301015, MR161215
- Purity:
Details on mating procedure:
- M/F ratio per cage:1:1
- Length of cohabitation:
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: The day of detection of sperm positive vaginal smear was considered as day "0" of gestation.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.:Re-mating of unsuccessfully paired female was done with proven male of the same group.
- Further matings after two unsuccessful attempts: [no / yes (explain)] : No data
- After successful mating each pregnant female was caged (how): housed individually
- Any other deviations from standard protocol:No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and Stability of dose formulation by analysing the sample at different time points (Stability was determined by sampling and analyzing the aliquots from the sample stored at 25 ± 2°C at the time points of 0, 2 and 6 hours).Two replications was analyzed at each time point.The dose formulation analysis were carried out at the start (on the day 1) of treatment, on day 21 and day 40 during the study period.
Duration of treatment / exposure:
Male: 47 days
Female : 63 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
308 mg/kg bw/day
Dose / conc.:
556 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
Total: 124 animals
0 mg/kg bw: 13 male, 13 female
308 mg/kg bwm: 13 male, 13 female
556 mg/kg bwm: 13 male, 13 female
1000 mg/kg bwm: 13 male, 13 female
Control recovery: 5 male, 5 female
1000 mg/kg bw recovery: 5 male, 5 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: No Data Available
- Rationale for animal assignment (if not random): Vaginal smear of all females was evaluated for regular cyclicity before treatment (14 days). At the time of randomization females not showing regular cycle were euthanised and discarded
- Other:
Positive control:
No Data Available
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (morning and evening)
- Cage side observations checked in table [No.?] were included. : morbidity and mortality were examined.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, preferably at the same time each day considering the peak period of anticipated effects after dosing.
Detailed clinical examinations were carried out once before the first treatment (to allow for within-subject comparisons) and weekly thereafter.
The detailed clinical examinations were made outside the home cage, at approximately the same time, on each occasion.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), day 4 post-partum and before terminal sacrifice.

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

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

OTHER:
Hematology and Clinical Biochemistry were examined.
Oestrous cyclicity (parental animals):
Estrous cyclicity was monitored for two weeks from begining of the treatment period till cohabitation and thereafter until the evidence of mating.
Sperm parameters (parental animals):
Spermatogenesis were examined.
Litter observations:
Number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities.
Postmortem examinations (parental animals):
Organ Weight, Gross Pathology and Histopathology were examined.
Postmortem examinations (offspring):
Gross Pathology and Histopathology were examined.
Statistics:
Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks.
Reproductive indices:
Survival Index of pups, Pregnancy index and Fertility index were examined.
Offspring viability indices:
yes, viability on day 0 and 4 were examined.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period.
Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period.
Statistically significant decrease was observed in number of rears of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male on pre-treatment as compared to control G1 (0 mg/kg body weight). The statistically significant increase was observed in number of urine pools of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of fecal bolus of G3 (556 mg/kg body weight) male at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of rears of G4 (1000 mg/kg body weight) male at week 4 as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of urine pools of G3 (556 mg/kg body weight) male at week 6 as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of rears of G2 (308 mg/kg body weight), G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) female at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of fecal bolus of G4 (1000 mg/kg body weight) female at week 5 as compared to control G1 (0 mg/kg body weight).
The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence considered as incidental and not attributed to the effect of test item administration.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No mortality or morbidity was observed in any animal of the control and treatment groups throughout the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant decrease was observed in body weight of G4 (1000 mg/kg body weight) male on day 30 as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in body weight of G4 (1000 mg/kg body weight) female on day 20 of gestation as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in body weight of G4-R (1000 mg/kg body weight) male on day 29, 36, 41 as compared to control G1-R (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male on day 1-8, 1-14 whereas statistically significant decrease was observed in percent body weight change of G4 (1000 mg/kg body weight) male on day 1-21, 1-28, 1-30, 1-37, 1-44, 1-46 as compared to control G1-R (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change during gestation period of G4 (1000 mg/kg body weight) female on day 0-14, 0-20 as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change of G4-R (1000 mg/kg body weight) male on day 1-8, 1-15, 1-22, 1-29 as compared to control G1-R (0 mg/kg body weight).
Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group.
These changes observed were inconsistent, hence not considered as effect of the test item administration.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decrease in feed consumption was observed in G4 (1000 mg/kg body weight) female on gestation day 14-20 as compared to the control group G1. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group.
Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration.
Food efficiency:
not specified
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):
All hematological parameters in animals of different treated groups of both the sexes were comparable to their respective control groups, except statistically significant decrease observed for MCHC, WBC in males of G4 (1000 mg/kg body weight) as compared to G1, statistically significant increase observed for aPTT in males of G4 (1000 mg/kg body weight) and G3 (556 mg/kg body weight) as compared to G1. Statistically significant decrease observed for RBC, HCT, HGB, WBC in males of G4-R (1000 mg/kg body weight) as compared to G1-R. Statistically significant decrease observed for PT in females of G3 (556 mg/kg body weight) as compared to G1. Statistically significant decrease observed for MCHC and statistically significant increase observed for RBC, HCT, HGB in females of G4-R (1000 mg/kg body weight) as compared to G1-R.
The above changes were inconsistent, not related to the test item and may be due to the preanalytical and analytical variables.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
All clinical chemistry parameters in animals of different treated groups of both the sexes were comparable to their respective control groups, except statistically significant increase observed for ALT and statistically significant decrease observed for Sodium (Na) in males of G4 (1000 mg/kg Body weight) as compared to G1. Statistically significant increase observed for Creatinine in males of G2 (308 mg/kg Body weight) as compared to G1. Statistically significant decrease observed for Total Protein and statistically significant increase observed for A/G ratio in females of G3 (556 mg/kg Body weight) as compared to G1.
The above changes were inconsistent, not dose dependent hence considered as incidental in nature.
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes.
Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups except a statistically significant decrease was observed in hindlimb foot splay in G4-R (1000 mg/kg body weight) male as compared to the repective control group G1-R.
The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence, considered as incidental and not attributed to the effect of test item administration.
Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group except statistically significant decrease was observed in ST=Stereotypic time in G2, G3 and G4 male as compared to control group G1 and G4-R in female as compared to G1-R.
The above changes observed were inconsistent, hence considered as incidental and not attributed to the effect of test item administration.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality.
Microscopic examination of control group and rats treated at 308, 556 and 1000 mg/kg revealed varying degree of pathological changes in different organs. This includes Liver: focal to multifocal minimal lymphocytic infiltration (Male: G1:1/5, G4:2/5; Female: G1: 1/5; G4: 2/5); focal minimal necrosis (Male: G1:1/5; Female: G1: 1/5); Kidneys: focal minimal lymphocytic infiltration (Male: G1:2/5; Female: G4:1/5); focal mild mineralization (Female: G1:1/5); Lungs: multifocal minimal lymphocytic infiltration (Male:G1:1/5, G4:1/5; Female: G1: 2/5, G4: 3/5); focal minimal histiocyte infiltration (Female: G1: 1/5, G4: 1/5); Heart: focal minimal lymphocytic infiltration (Male: G1:1/5, G4:1/5); Aorta: focal minimal aneurysm (Male:G1:1/5, G4:1/5); Mandibular Lymph Node: focal moderate cystic dilation of cortex (Female: G4:1/5); Stomach: focal mild squamous epithelium hyperplasia (Female: G1: 1/5); Mesenteric lymph node: focal moderate cystic dilation of cortex (Female:G1:1/5); Spleen: focal to diffuse minimal to mild extramedullary hematopoesis (Female: G1: 2/5, G4: 3/5); Thymus: mild to moderate atrophy (Female: G1:3/5, G4:4/5); focal mild cystic epithelial dilation (Male: G4:1/5; Female: G1: 1/5, G4:1/5); Trachea: focal to multifocal minimal to moderate Neutrophilic/lymphocytic infiltration (Male: G1:3/5, G4:3/5; Female: G1: 2/5, G4:1/5); Adrenals: unilateral accessory adrenocortical tissue (Male: G1:1/5, G4:1/5); Testes: focal to multifocal minimal to mild retention of mature sperm (Male: G1:4/13, G2:8/13, G3:8/13, G4:8/13); focal minimal to mild degeneration of seminiferous tubules (Male: G1:2/13, G2:1/13, G3:1/13, G4:1/13); focal to multifocal minimal sloughing of Pachytene Spermatocyte (Male: G1:2/13, G2:2/13, G3:2/13, G4:2/13); focal minimal sloughing of round spermatid (Male: G1:1/13, G2:1/13, G3:1/13, G4:1/13); focal mild infiltration of multinucleated giant cells (Male: G1:1/13); Seminal Vesicles: multifocal mild neutrophilic/lymphocytic infiltration (Male: G1:1/13); Prostate: focal moderate necrotic debris in lumen (Male: G2:1/13); Uterus: multifocal to diffuse mild reduction of stromal cells (Female: G1:1/13; G4:2/13); focal moderate necrosis (Female: G3:1/13); multifocal mild to moderate nodular hyperplasia (Female: G1:1/13; G2:1/13; G4:1/13); Cervix: focal minimal lymphocytic infiltration (Female: G2:1/13).
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
In control group G1 and treatment group G2 all the females showed regular cyclicity i.e. 3-5 days estrous cycle; while in group G3, 3 females and in group G4, 4 females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period. In cohabitation or mating period, all females from control and treated groups showed evidence of copulation i.e. sperm positive vaginal smear. Precoital Interval was calculated, all females showed precoital interval less than 5 days, except 1, 1 and 4 females from G1, G3 and G4, respectively which showed precoital interval more than 5 days.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
No statistically significant difference between the control (G1) and treatment groups (G2, G3 and G4) in the maternal parameters i.e. Gestational length, Litter size, No. of live births, Post-implantation loss and Post-natal loss were observed.
Dose descriptor:
NOAEL
Effect level:
556 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive performance
other: No effect observed
Remarks on result:
other: Generation: no data (migrated information)
Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
No statistically significant effect were observed on No. of live births and on PND 4 of pups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no statistically significant effect were observed on pups weight at birth and PND4 as compared to control.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
At the end of treatment and recovery period, absolute and relative weight of organs of treated rats of either sex did not differ significantly except a significant increase in relative wieght of Adrenal of G4-R male group when compared to the respective control group rats.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Terminally sacrificed pups of all treated groups did not reveal any lesion of pathological significance in any of the group when compared with control group. Pups died during course of study revealed various lesions among the control and treated groups viz., external examination emaciated carcass (Male: G1:2/55, G2:1/44, G3:5/35; Female: G1:3/56, G2:1/30, G3:6/54); Cannibalism (Male: G1:3/55, G3:2/35; Female: G1:2/56, G3:3/54); Tearing of Neck Muscle (Female: G3:1/54; G4:1/18) and internal examination absence of milk in stomach (Male: G1: 6/55, G2: 6/44, G3: 12/35, G4: 3/16; Female: G1: 8/56, G3: 14/54, G4: 2/18); blood clot in thoracic cavity (Male: G1: 2/55, G2: 3/44, G3: 1/35; Female: G1: 1/56, G3: 1/54, G4: 1/18); reddish discoloration of brain (Male: G1: 1/55, G2: 1/44, G3: 1/35; Female: G1: 1/56, G3: 3/54, G4: 1/18); reddish discoloration of lungs (Male: G1: 5/55, G2: 5/44, G3: 7/35, G4: 1/16; Female: G2: 1/30, G3: 10/54, G4: 2/18); paleness of liver (Male: G1: 1/55, G2: 2/44, G3: 1/35; Female: G3: 4/54, G4: 2/18); congested intestine (Female: G1: 1/56, G3: 1/54); autolytic changes (Female: G2: 1/30, G3: 2/54, G4: 1/18).
Histopathological findings:
no effects observed
Description (incidence and severity):
Microscopic examination of thyroid of male and female pups of control group and treated group did not revealed any lesion of pathological significance.
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
558 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain
gross pathology
histopathology: non-neoplastic
other: No effect observed
Remarks on result:
other: Not Specified
Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Reproductive effects observed:
not specified
Treatment related:
not specified

Mortality and Morbidity

Sex: Male

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Observation During Study Period

G1

Control

0

13

NMM

G2

Low

308

13

NMM

G3

Mid

556

13

NMM

G4

High

1000

13

NMM

G1-R

Control- Recovery

0

5

NMM

G4-R

High- Recovery

1000

5

NMM

 Sex: Female

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Observation During Study Period

G1

Control

0

13

NMM

G2

Low

308

13

NMM

G3

Mid

556

13

NMM

G4

High

1000

13

NMM

G1-R

Control -Recovery

0

5

NMM

G4-R

High- Recovery

1000

5

NMM

Keys:NMM = No mortality and morbidity observed, No.= Number

Clinical Signs and Symptoms

Sex: Male

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Clinical Sign

Incidences During Study period

G1

Control

0

13

Normal

13/13

G2

Low

308

13

Normal

13/13

G3

Mid

556

13

Normal

13/13

G4

High

1000

13

Normal

13/13

G1-R

Control -Recovery

0

5

Normal

5/5

G4-R

High- Recovery

1000

5

Normal

5/5

 Sex: Female

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

 

Clinical Sign

Incidences During Study period

G1

Control

0

13

Normal

13/13

G2

Low

308

13

Normal

13/13

G3

Mid

556

13

Normal

13/13

G4

High

1000

13

Normal

13/13

G1-R

Control -Recovery

0

5

Normal

5/5

G4-R

High- Recovery

1000

5

Normal

5/5

Key:No.= Number

Mean Body Weight (g)

Sex: Male

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose

(mg/kg b. wt.)

0

308

556

1000

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Day 1

247.38

11.20

262.38

20.32

261.62

18.86

261.92

17.03

Day 8

282.31

9.33

294.92

19.40

289.31

20.12

278.69

19.98

Day 14

315.00

13.00

324.38

21.35

316.15

20.45

299.15

21.37

Day 21

333.15

15.35

339.46

25.50

336.23

22.65

316.23

18.79

Day 28

350.08

18.06

362.62

30.37

358.54

27.49

334.15

20.60

Day 30

355.77

18.46

368.00

30.44

364.54

27.22

331.62

19.88

Day 37

370.77

22.36

381.92

30.46

381.77

31.58

351.62

24.13

Day 44

393.31

26.08

407.69

34.24

402.23

34.23

368.23

26.45

Day 46

397.23

24.79

414.77

34.07

405.38

34.10

370.92

26.71

Day 47

(Fasting)

372.00

25.57

391.08

33.83

383.38

33.84

350.15

26.15

 Period: Pre-mating                                                                                                    Sex: Female

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose

(mg/kg b. wt.)

0

308

556

1000

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Day 1

226.62

6.50

231.54

7.34

230.15

6.73

228.77

7.90

Day 8

229.85

8.37

233.46

7.85

233.54

9.00

227.92

7.39

Day 14

232.77

8.32

236.92

8.23

237.00

9.65

232.62

8.01

Keys:N = Number of animals in group, g= gram, SD = Standard deviation,↓= Statistically Significant Decrease (atp<0.05)

Mean Body Weight (g) Continued

Sex: Male

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

1000

Day

Mean

SD

Mean

SD

Day 1

261.40

19.99

258.20

13.33

Day 8

294.00

18.51

270.80

17.95

Day 15

326.80

21.25

297.20

19.99

Day 22

348.20

25.65

315.20

20.20

Day 29

370.60

28.03

 328.60

19.01

Day 36

391.60

26.41

 349.40

20.94

Day 41

398.20

28.67

 351.40

20.85

Day 48

415.40

33.16

372.60

26.82

Day 54

420.40

35.56

380.80

31.00

Day 55 (Fasting)

399.20

33.88

362.80

26.88

Sex: Female

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

1000

Day

Mean

SD

Mean

SD

Day 1

227.20

13.14

228.60

7.20

Day 8

233.80

11.95

227.80

9.78

Day 15

234.00

11.55

232.00

11.34

Day 22

236.00

10.93

234.00

10.37

Day 29

239.20

10.80

237.20

10.62

Day 36

240.80

11.61

238.60

11.84

Day 41

242.40

11.84

240.40

12.38

Day 48

243.40

11.87

243.00

13.62

Day 54

245.00

11.98

245.00

14.63

Day 55 (Fasting)

230.20

11.95

229.80

15.53

Keys:N= Number of animals in group, g= gram, SD= Standard deviation,↓= Statistically Significant Decrease (atp<0.05).

Mean Body Weight Change (%)

Sex:Male      

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose (mg/kg b. wt.)

0

308

556

1000

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Day 1-8

14.20

3.21

12.55

3.76

 10.70

4.73

6.40

 3.12

Day 1-14

27.53

7.02

23.94

7.71

21.13

7.89

14.28

5.24

Day 1-21

34.87

7.85

29.71

9.59

28.91

10.07

20.98

7.42

Day 1-28

41.73

8.95

38.68

12.93

37.52

12.61

27.88

8.78

Day 1-30

44.03

9.19

40.72

12.79

39.81

12.51

26.89

8.20

Day 1-37

50.16

11.39

46.09

13.55

46.47

14.70

34.67

11.44

Day 1-44

59.32

13.26

55.96

15.08

54.42

16.81

41.10

13.16

Day 1-46

60.85

12.16

58.69

15.39

55.61

16.53

42.13

13.26

 Period: Pre-mating                                                                                                Sex: Female

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose (mg/kg b. wt.)

0

308

556

1000

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Day 1-8

1.43

2.42

0.83

0.97

1.45

1.70

-0.36

1.27

Day 1-14

2.72

2.25

2.33

1.88

2.95

2.07

1.69

1.40

 Period: Gestation                                                                                                    Sex: Female

Group (N)

G1 (12)

G2 (11)

G3 (11)

G4 (8)

Dose (mg/kg b. wt.)

0

308

556

1000

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Day 0-7

5.23

3.64

5.76

2.32

5.79

2.87

2.60

2.31

Day 0-14

15.18

6.90

13.68

2.32

15.93

6.40

7.54

3.70

Day 0-20

29.44

11.73

28.10

6.90

30.75

11.02

14.52

5.81

Keys:N = Number of animals in group, g= gram, SD = Standard deviation,↓= Statistically Significant Decrease (atp<0.05)

Mean Body Weight Change (%) Continued

 Period: Post-Partum                                                                                                             Sex: Female

Group (N)

G1 (12)

G2 (11)

G3 (11)

G4 (8)

Dose (mg/kg b. wt.)

0

308

556

1000

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Day 0/1-4

0.82

3.33

-1.21

3.07

1.52

3.21

-1.33

3.03

   Sex: Male

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

1000

Day

Mean

SD

Mean

SD

Day 1-8

12.58

3.07

4.83

2.30

Day 1-15

25.26

7.48

15.04

2.48

Day 1-22

33.51

10.23

22.03

3.05

Day 1-29

42.19

12.51

27.31

5.11

Day 1-36

50.34

13.50

35.37

6.14

Day 1-41

52.96

15.34

36.14

5.92

Day 1-48

59.63

17.61

44.33

8.14

Day 1-54

61.54

18.41

47.46

9.14

Keys:N = Number of animals in group, g= gram, SD = Standard deviation,↓= Statistically Significant Decrease (atp<0.05)

Mean Hematology Data Continued

 

Group (N)

G1-R (5)

G4-R (5)

G1-R (5)

G4-R (5)

Sex

Male

Female

Dose (mg/kg body weight)

0

1000

0

1000

Parameter↓

Mean

SD

Mean

SD

Mean

SD

Mean

SD

RBC x 106(µl)

8.70

0.07

7.81

0.46

7.83

0.34

8.29

0.22

HCT( %)

39.38

0.38

35.80

2.10

34.90

0.96

37.10

1.09

MCV (µm3 

45.26

0.73

45.80

0.98

44.56

1.21

44.68

0.73

HGB (g/dl)

15.40

0.16

14.10

0.73

13.88

0.36

14.54

0.42

MCH (pg)

17.70

0.25

18.06

0.52

17.70

0.51

17.50

0.19

MCHC (g/dl)

39.14

0.25

39.38

0.40

39.78

0.16

39.24

0.39

Platelet x 103(µl)

560.80

53.19

579.20

40.11

590.20

26.76

644.20

131.73

WBC x 103(µl)

9.20

1.06

7.10

1.61

5.80

1.49

5.50

3.20

Neutrophil (%)

17.60

2.30

14.80

3.27

14.80

4.44

14.60

2.88

Lymphocyte (%)

81.80

1.92

84.40

3.58

84.20

3.49

84.60

2.41

Monocyte (%)

0.00

0.00

0.20

0.45

0.00

0.00

0.00

0.00

Eosinophil (%)

0.60

0.55

0.60

0.55

1.00

1.00

0.80

0.84

Basophil (%)

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

PT (Sec.)

22.51

5.59

22.48

2.01

25.19

13.02

29.11

6.84

aPTT (Sec.)

25.24

12.42

29.00

9.25

20.68

18.28

34.71

4.80

Keys: SD = Standard deviation, N = Number of animals in group and NAD = No Abnormality Detected,µm3=cubic micrometer,g/dl= gram per decilitre, pg= picogram, µl= microlitre, Sec= second,↓ =statisticalsignificant decrease at 95% level of significance,↑= statisticalsignificant increase at 95% level of significance.

Mean Gestational Length

Group(N)

G1(12)

G2(11)

G3(11)

G4(8)

Dose(mg/kg bwt)

0

308

556

1000

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Gestation Length

22.17

0.39

22.55

0.82

22.00

0.45

22.75

0.89

Keys:SD= Standard Deviation, N= number of dams in a group

Mean Litter size

Group(N)

G1(12)

G2(9)

G3(11)

G4(7)

Dose(mg/kg bwt)

0

308

556

1000

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Litter size

9.33

3.37

8.22

3.35

9.18

1.72

6.71

2.36

Keys:SD= Standard Deviation, N= number of dams in a group

Mean Post-Implantation Loss (%), Post-natal Loss (%) and Pups Survival Index (%)

Group(N)

G1(12)

G2(9)

G3(11)

G4(7)

Dose(mg/kg bwt)

0

308

556

1000

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

No. of Live Births

9.33

3.37

8.22

3.35

9.18

1.72

6.71

2.36

Post-Implantation Loss

0.16

0.27

0.04

0.07

0.13

0.19

0.36

0.13

No. of alive pups at Post-natal Day 4

7.5

3.03

6.89

2.93

4.73

3.10

3.71

2.29

Post-natal Loss (%)

16.42

19.90

12.22

22.53

49.07

31.15

42.86

30.50

Fetal Survival Index at Post-natal Day 4 (%)

83.58

19.90

87.78

22.53

50.93

31.15

57.14

30.50

Keys:SD= Standard Deviation, N= number of dams in a grou

Summary of Days of Conception and Pregnancy Index (%)

Group/N

G1(13)

G2(13)

G3(13)

G4(13)

Dose (mg/kg b.wt.)

0

308

556

1000

No of females showing evidence of copulation

13

13

13

13

No of females concieving between Days 1-5 of cohabitation

12

13

12

9

No. of females concieving after Day 5 of cohabitation

1

0

1

4

Females achieving pregnancy

12

11

11

8

Pregnancy Index (%)

92.31

84.62

84.62

61.54

Key:N= number of dams in a group

 


Conclusions:
No Observed Adverse Effect Level (NOAEL) is considered to be 556 mg/kg body weight when Wistar male and female rats were orally treated with the test chemical.
Executive summary:

In a reproductive toxicity study, Wistar male and female rats were treated with the test chemical in the concentration of 0, 308, 556 and 1000 mg/kg bw orally by gavage in Corn oil for 63 days. No mortality or morbidity and apparent treatment related clinical signs were observed any of the groups of animals throughout the study period. Detailed clinical examinations like Home cage observation, handling observation and Open field observation of all animals were observed to be normal during study period. Number of rear, urine pools, fecal bolus in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. Body weight, percent body weight changes and feed consumption in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. The sensory reactivity measurements, Foot splay, fore limb and hind limb grip strength parameters and Motor activity were comparable and no treatment related changes were revealed in any of the animals of all treated groups as compare to the repective control groups. Similarly, No test item related changes in estrous cyclicity and precoital interval were observed. There was statistically significant decrease in G3 (556 mg/kg body weight) as compared to control G1 (0 mg/kg body weight). This is not dose dependent hence not considered as treatment related. There was no statistically significant difference between the control and treatment groups in the maternal and pups parameters, except markedly decreased pregnancy index / fertility index in G4 (1000 mg/kg body weight), which was considered to be treatment related. No treatment related changes were observed inhematological and clinical chemistry parameters of treated male and female rats as compared to control. In addition, no significant change in organ weight, External and visceral examination of treated and recovery groups as compared to control. Pups that died among the control and treated groups during the course of study, revealed various lesions when examined externally and internally but the observations were not considered treatment related. Emaciated carcass, Cannibalism; Tearing of Neck Muscle and internal examination Absence of milk in stomach, Thoracic cavity: Blood clot present, Reddish discoloration of brain, Reddish discoloration of lungs, Paleness of liver, Congested intestine and Autolytic changes were observed all the treated groups.Focal to multifocal minimal lymphocytic infiltration of male and female and focal minimal necrosis in male in liver, focal minimal lymphocytic infiltration of male and female and focal mild mineralization in female in kidney, multifocal minimal lymphocytic infiltration in male and female andfocal minimal histiocyte infiltration in female lungs, focal minimal lymphocytic infiltration in heart of male, focal minimal aneurysm in aorta of male, focal moderate cystic dilationof cortex of Mandibular Lymph Node in female,focal mildsquamous epitheliumhyperplasia stomach of female, focal moderate cystic dilation of cortex in Mesenteric lymph node, focalto diffuse minimal to mild extramedullary hematopoesis in spleen and mild to moderate atrophy in Thymus of female, focal to multifocal minimal to moderate Neutrophilic/lymphocytic infiltration of Trachea of male and female, unilateral accessory adrenocortical tissue of Adrenals and focal to multifocal minimal to mildretention of mature sperm,focal minimal to mild degeneration of seminiferous tubules,focal to multifocal minimalsloughing of Pachytene Spermatocyte,focal minimal sloughing of round spermatid andfocal mildinfiltration of multinucleated giant cells of Testes,multifocal mildneutrophilic/lymphocytic infiltration of Seminal Vesicles and focal moderate necrotic debris in lumen of Prostate observed in male rats, multifocal to diffuse mild reduction of stromal cells, focal moderate necrosis, multifocal mild to moderate nodular hyperplasia of Uterus and focal minimal lymphocytic infiltration of Cervix in female rats were observed. Therefore, No Observed Adverse Effect Level (NOAEL) is considered to be 556 mg/kg body weight when Wistar male and female rats were orally treated with the test chemical.

Reason / purpose:
read-across source
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
According to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Justification for study design:
not specified
Species:
rat
Strain:
Wistar
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
- Source: National Institute of Biosciences
- Age at study initiation: 12 - 13 weeks at the start of Oestrous Cycle evaluation
- Weight at study initiation:
- Fasting period before study
:- Housing: Cages were cleaned at regular intervals. A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20 [cm]). .Sterilized corn cob produced from pure corn, dried and free from dust, procured from approved supplier,was used as bedding material. Bedding material of batch No. 8-17 (Krishana Corncob Industries, Aurangabad) was used in this study
.- Diet (e.g. ad libitum): A conventional laboratory pelleted diet, ad libitum
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.00 to 24.40 °C
- Humidity (%): 40.50 to 64.30%
- Air changes (per hr): minimum 12 times per hour and filtered adequately
.- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark
IN-LIFE DATES: From: December 08, 2017To: April 28, 2018
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
water
Remarks:
Distilled
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (Distilled water) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. Atthe time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item

.DIET PREPARATION- Rate of preparation of diet (frequency):- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:
VEHICLE- Justification for use and choice of vehicle (if other than water): Distilled water
- Concentration in vehicle: 0, 250, 500 and 750 mg/kg bw
- Amount of vehicle (if gavage): 1.0 ml/100g body weight
- Lot/batch no. (if required):- Purity:
Details on mating procedure:
- M/F ratio per cage: one male and one female
- Length of cohabitation: until pregnancy occurs or two weeks elapsed
- Proof of pregnancy: Mating was confirmed by observation of sperm positive vaginal smear. The day of detection of sperm positive vaginal smear was considered as day "0" of gestation.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.:
- Further matings after two unsuccessful attempts: - After successful mating each pregnant female was caged (how): housed individually - Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Specificity, Linearity, Precision (%RSD), Accuracy (% Recovery) and Homogeneity were analysis by HPLC-UV
Duration of treatment / exposure:
Approx. 64 days
Frequency of treatment:
Daily
Details on study schedule:
not specified
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Total: 124
0 mg/kg bw, 13 male, 13 female
250 mg/kg bw, 13 male, 13 female
500 mg/kg bw, 13 male, 13 female
750 mg/kg bw, 13 male, 13 female

Recovery group:
0 mg/kg bw, 5 male, 5 female
750 mg/kg bw, 5 male, 5 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels 250, 500 and 750 mg/kg body weight were selected for theMain Study based on the results of Dose Range Finding (DRF).- Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing. Individual body weights were considered within ± 20% of the groups mean.- Rationale for selecting satellite groups: 0 and 750 mg/kg bw were selected as satellite groups- Post-exposure recovery period in satellite groups: 14 days- Section schedule rationale (if not random):
Positive control:
not specified
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: Twice daily (morning and evening)- Cage side observations : Morbidity and mortality, throughout theacclimatization and study period.
DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: once a day, preferably at the same time each day
BODY WEIGHT: Yes- Time schedule for examinations: Males and females were weighed during randomization, on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), on day 4 and day 13 post-partum and before terminal sacrifice
.FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kgbody weight/day: Yes- Compound intake calculated as time-weighted averages from the consumption and body weight gaindata: Yes
FOOD EFFICIENCY: No data- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weightedaverages 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
OPHTHALMOSCOPIC EXAMINATION: No data- Time schedule for examinations: No data- Dose groups that were examined: No data
HAEMATOLOGY: Yes- Time schedule for collection of blood: Just prior to necropsy.- Anaesthetic used for blood collection: Yes, Isoflurane anaesthesia- Animals fasted: Yes, fasted overnight- How many animals: five males and five females, randomly selected from each group- Parameters ] were examined. Total Erythrocyte Count (RBC), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Hemoglobin (HGB), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (PLT), Total Leukocyte count (WBC), Prothombin Time (PT) and Activated Partial Thromboplastin time (aPTT) were examined.CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: Just prior to necropsy.- Animals fasted: Yes, fasted overnight- How many animals: five males and five females, randomly selected from each group- Parameters were examined. : Glucose (Glu), Cholesterol (Chol), Triglycerides (TRIG), Alanine amino transferase (ALT), Aspartate amino transferase (AST), Calcium, Albumin (Alb), Total Protein (TP), Creatinine (Crea), Phosphorus, Urea, Sodium (Na), Potassium (K), Blood urea nitrogen (BUN), Globulin (Glob), Alb/ Glb (A:G) and Bile acids were examined.URINALYSIS: No data- Time schedule for collection of urine: No data- Metabolism cages used for collection of urine: No data- Animals fasted: No data- Parameters checked in table [No.?] were examined. No data
NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for exam
Oestrous cyclicity (parental animals):
Estrous cycle were monitored daily from beginning of the treatment period until evidence of mating. When taking vaginal smear care was taken to avoid disturbance to vaginal mucosa.
Sperm parameters (parental animals):
not specified
Litter observations:
Number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), body weight and ano-genital distance (AGD) were examined.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes, At scheduled sacrifice date, all rats of main and recovery groups were euthanized by over dose of carbon dioxide followed by exsanguination. The animals were examined externally in unopened condition. This was followed by opening of the carcasses and topographic examination of different organs. This included careful examination of the external surface of the body, all orifices, cranial, thoracic and visceral cavities and their contents.Similarly, necropsy of terminally sacrificed and found dead pups during study period were conducted andgross pathological observations were recorded.HISTOPATHOLOGY: Yes,Full histopathology was carried out on the preserved organs (ovaries, uterus, cervix with vagina, testes, epididymides, prostate, seminal vesicle with coagulating glands) of all animals and all tissues of five males and females, randomly selected from each group animals in the control and high dose groups.
Postmortem examinations (offspring):
Presence of any gross abnormalities were examined.
Statistics:
Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, FunctionalObservational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks.
Reproductive indices:
Pregnancy Index (%), Post-natal Loss (%), Gestation Length were examined.
Offspring viability indices:
Fetal Survival Index at Post-natal Day 4 (%) were examined.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No apparent treatment related clinical signs were observed in any of the animals throughout the treatment. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period. Statistically significant increase in female was observed in urine pool at Week 5 in 750 mg/kg bw as compared to control. The above changes observed were inconsistent/ biologically insignificant and not dose dependent.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No mortality or morbidity was observed in any animals of the control and treatment groups throughout the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
When treated with 500 mg/kg bw, statistically significant decrease was observed in percent body weight change on day 1-15 and day 1-28 as compared to control group.Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. These changes observed were inconsistent, hence not considered as effect of the test item administration.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment related changes were observed in treated male and female rats as compared to control.
Food efficiency:
not specified
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):
At the end of treatment period revealed statistically significant decreased was noted in WBC of females (R) at 750 mg/kg body weight. The observed variation in WBC was considered to be of no toxicological significance, of a minimal in nature and occurred in the absence of clear dose related response.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of treatment period revealed, statistically significant decrease were noted in Triglyceride in Male at 500 mg/kg body weight, Calcium in Female at 750 mg/kg body weight. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes.During treatment-free recovery period revealed statistically significant increase was noted in cholesterol in Female at 750 mg/kg body weight, A: G ratio, in Male and Female (R) at 750 mg/kg body weight while statistical significant decrease were noted in ALT and Globulin in Female at (R) 750 mg/kg body weight.The observed variations in Triglyceride, Calcium, Cholesterol, ALT, Globulin and A: G ratio were considered to be of no toxicological significance, of a minimal in nature and occurred in the absence of clear dose related response.
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The functional observation battery/neurobehavioral observation were comparable and no changes were revealed i any of the animals of all the treated groups in both the sexes.The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes.Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups. However in recovery male,statistically significant increase was noted in grip strength (hindlimb) in 750 mg/kg bw (R) when compare to control recovery group.Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group. Howerver, statistically significant increase in female were noted in Distance travelled (DT),Ambulatory time (AT), and in Horizontal counts (HC) at 750 mg/kg body weight when compare to control group.The above changes observed were not dose dependent, hence considered as incidental and not attributed to the effect of test item administration.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination of control group and rats treated at 250, 500 and 750 mg/kg revealed varying degree of pathological changes in different organs. This includes in Liver: focal to multifocal minimal to mild lymphocyte infiltration (Male: G1:2/5; Female: G1/5, G4:1/5), focal to multifocal mild degeneration (Male: G1:1/5, Female: G4/5), in Kidneys: focal mild tubular degeneration (Male: G1:2/5), focal mild lymphocyte infiltration (Female: G1/5); in Lungs: multifocal mild lymphocyte infiltration (Male: G1:1/5, G4:1/5; Adrenals: accessory adrenocortical tissue (Bilateral: Female: G4:1/5), in Testes: Male: multifocal mild cytoplasmic vacuolation at sertoli cell (Male: G1: 1/13), focal mild multinucleated giant cell infiltration (Male: G1: 1/13) focal mild seminiferous tubule degeneration (Male: G4: 2/13); focal minimal retention of mature sperm (Male: G1: 1/13, G2:1/13, G3: 1/13, G4: 1/13, G1-R: 1/5); focal minimal to mild sloughing of round spermatid (Male: G1: 1/13, G2:1/13). in Epididymis: Male: multifocal mild reduced sperm count (Male: G1: 1/13). Lesions observed in liver, kidneys, lungs, adrenals and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
In control group G1 and treatment group G2 , G3 and G4 all the females showed regular cyclicity i.e. 3-5 days estrous cycle. However in group G1 two female, group G2 one female and group G4 one female showed prolong diestrus and found to be non-pregnant. In cohabitation or mating period, all females from control and treated groups showed evidence of copulation i.e. sperm positive vaginal smear. All females showed precoital interval less than 5 days, except 1, 2 and 1 females from G2, G3 and G4, respectively which showed precoital interval more than 5 days.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no difference between the control (G1) and treatment groups (G2, G3 and G4) in the maternal and fetal parameters. Gestational length, Litter size, No. of live births, Post-implantation loss, pups weight at birth and PND14, Post-natal loss, Survival Index and weight gain for pups at PND13. Pregnancy index was found to be 84.62, 92.31, 100.00 and 92.31 in G1, G2, G3 and G4 respectively. Pups sex ratio (Male/Female) was found to be 61/57, 72/79, 80/61 and 71/56 at birth in G1, G2, G3 and G4 respectively and 56/53, 48/51, 64/54 and 69/56 at Day 4 in G1, G2, G3 and G4 respectively.
Dose descriptor:
NOAEL
Effect level:
750 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
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive performance
Remarks on result:
other: No effect observed
Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
No effect on No. of live births were observed in treated pups as compared to control.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effect on pups weight at birth and PND14 were observed in treated pups as compared to control.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Terminally sacrificed pups of all treated groups did not reveal any lesion of pathological significance in any of the group when compared with control group. Pups died during course of study revealed various lesions among the control and treated groups viz., external examination Cannibalism (Male: G1: 1/59, G2:13/60, G3: 7/81, Female:G1 :1/59, G2:14/81, G3:1/60, G4: 1/55 ; Tail absent (Anury) G3: 1/60 and internal examination Right skin swelling G1: 1/57.
Histopathological findings:
not specified
Other effects:
no effects observed
Description (incidence and severity):
No effect on Litter size and Pups sex ratio were observed as compared to control.
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain
gross pathology
other:
Remarks on result:
other: No effect observed
Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Reproductive effects observed:
not specified
Treatment related:
not specified

Mortality and Morbidity

Sex: Male

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Observation During Study Period

G1

Control

0

13

NMM

G2

Low

250

13

NMM

G3

Mid

500

13

NMM

G4

High

750

13

NMM

G1-R

Control- Recovery

0

5

NMM

G4-R

High- Recovery

750

5

NMM

 Sex: Female

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Observation During Study Period

G1

Control

0

13

NMM

G2

Low

250

13

NMM

G3

Mid

500

13

NMM

G4

High

750

13

NMM

G1-R

Control -Recovery

0

5

NMM

G4-R

High- Recovery

750

5

NMM

Keys:NMM = No mortality and morbidity observed, No.= Number

Mean Body Weight (g)

Sex: Male

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose

(mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

1

328.62

22.46

330.46

21.52

327.92

20.07

329.08

16.28

8

343.08

26.03

348.77

26.14

350.46

24.55

350.00

20.15

14

351.62

27.09

362.92

32.04

370.00

33.60

367.46

26.28

21

364.31

26.48

380.31

33.72

385.38

39.58

379.23

30.18

28

382.77

28.60

398.77

39.39

402.62

42.57

399.00

32.01

35

397.85

31.58

407.62

37.91

412.23

44.44

407.38

32.29

40

401.15

32.82

394.92

53.26

419.54

48.74

414.69

30.05

41{fasting}

385.46

31.17

389.62

35.03

399.23

47.68

394.23

31.15

 Period: Pre-mating                                                                                                    Sex: Female

Group (N)

G1

G2

G3

G4

Dose

(mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

1

247.54

16.31

13

245.00

11.95

13

248.23

14.22

13

247.92

14.28

13

8

251.62

16.29

13

252.00

14.93

13

254.38

16.09

13

255.77

13.40

13

14

258.15

18.38

13

259.62

17.47

13

263.62

20.39

13

265.38

16.00

13

21

267.50

14.85

2

244.00

18.38

2

274.00

12.73

2

289.00

18.38

2

28

288.00

29.70

2

276.00

./.

1

./.

./.

0

295.00

./.

1

35

309.50

28.99

2

300.00

./.

1

./.

./.

0

309.00

./.

1

42

295.00

22.63

2

319.00

./.

1

./.

./.

0

307.00

./.

1

49

282.50

34.65

2

334.00

./.

1

./.

./.

0

306.00

./.

1

56

286.50

30.41

2

328.00

./.

1

./.

./.

0

303.00

./.

1

58

280.00

29.70

2

329.00

./.

1

./.

./.

0

326.00

./.

1

59(Fasting)

272.00

28.28

2

321.00

./.

1

./.

./.

0

312.00

./.

1

Period: Gestation                                                                                                  Sex: Female

Group

G1

G2

G3

G4

Dose

(mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

0

260.36

19.82

11

264.08

16.89

12

370.00

21.68

13

268.67

18.81

12

7

279.45

18.84

11

282.00

18.92

12

284.69

22.29

13

284.83

19.40

12

14

311.64

22.08

11

308.92

23.12

12

312.31

27.62

13

311.83

22.23

12

20

373.91

25.68

11

368.58

23.13

12

368.46

36.03

13

363.67

25.82

12

 Period: Post-Partum                                                                                                         Sex: Female

Group (N)

G1 (11)

G2 (12)

G3 (13)

G4 (12)

Dose

(mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

0

298.55

28.22

297.42

17.10

306.54

26.49

294.58

21.90

4

303.09

27.13

291.25

20.91

299.38

19.49

296.58

19.46

13

313.91

33.40

297.58

21.47

307.92

23.06

304.50

21.45

14(Fasting)

285.18

25.27

272.00

16.04

282.08

19.95

272.25

18.11

Sex: Male

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

750

Day

Mean

SD

Mean

SD

1

335.60

26.19

334.00

27.00

8

354.60

29.82

358.60

25.36

14

361.40

33.92

371.80

16.83

21

378.00

32.30

393.40

21.48

28

395.00

37.29

412.00

30.81

35

407.20

38.26

419.60

33.00

42

416.60

45.28

427.00

33.85

49

429.80

46.66

446.40

30.76

56

434.60

43.55

447.40

33.76

63

435.80

45.88

452.60

33.81

66

441.40

46.76

457.20

37.34

67(fasting)

421.20

45.17

436.60

34.41

Sex: Female

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

750

Day

Mean

SD

Mean

SD

1

246.60

22.73

248.60

20.13

8

255.20

19.20

257.60

23.44

14

263.20

19.68

269.60

19.63

21

270.40

20.32

276.40

22.50

28

275.60

19.93

280.00

27.96

35

279.80

20.27

278.80

25.94

42

287.00

22.44

283.80

22.70

49

292.60

25.86

287.60

23.80

56

287.80

25.97

288.00

25.56

63

291.20

26.33

287.00

23.79

66

291.00

25.66

287.40

23.04

67(fasting)

275.40

26.02

272.80

25.12

Sex:Male      

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose (mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

(1-8)

4.37

2.19

5.50

2.43

6.87

3.52

6.34

2.59

(1-15)

7.01

4.39

9.72

4.29

12.70↑

4.92

11.61

4.42

(1-28)

10.87

3.42

15.02

5.90

17.30↑

6.28

15.13

4.96

(1-29)

16.54

5.36

20.54

6.86

22.54

7.07

21.13

5.48

(1-35)

21.12

6.19

23.28

7.21

25.45

7.17

23.72

6.20

(1-40)

22.11

6.39

19.45

17.33

27.61

8.21

25.98

5.72

(1-41)

17.33

5.86

17.77

7.65

21.41

8.18

19.74

6.12

Period: Pre-mating                                                                                                Sex: Female

Group (N)

G1

G2

G3

G4

Dose (mg/kg        b. wt.)

0

250

500

750

Day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

(1-8)

1.66

1.31

13

2.83

2.41

13

2.48

2.86

13

3.20

1.19

13

(1-15)

4.28

2.24

13

5.90

3.15

13

6.10

2.68

13

7.05

2.45

13

(1-21)

9.59

2.27

2

6.02

5.39

2

9.57

2.61

2

10.53

0.44

2

(1-28)

17.89

8.07

2

17.95

./.

1

./.

./.

1

18.47

./.

1

(1-35)

26.71

7.48

2

28.21

./.

1

./.

./.

1

24.10

./.

1

(1-42)

20.81

5.07

2

36.32

./.

1

./.

./.

1

23.29

./.

1

(1-49)

15.60

10.18

2

42.74

./.

1

./.

./.

1

22.89

./.

1

(1-56)

17.27

8.38

2

40.17

./.

1

./.

./.

1

21.69

./.

1

(1-59)

14.61

8.19

2

40.60

./.

1

./.

./.

1

30.92

./.

1

Period: Gestation                                                                                                  Sex: Female

Group

G1

G2

G3

G4

Dose

(mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

(0-7)

7.43

3.09

11

6.78

2.19

12

5.85

2.40

13

6.08

3.55

12

(0-14)

19.85

5.33

11

16.97

4.42

12

16.04

3.47

13

16.17

5.54

12

(0-20)

43.90

8.24

11

39.63

3.82

12

36.88

6.74

13

35.58

8.39

12

 Period: Post-Partum                                                                                                         Sex: Female

Group (N)

G1 (11)

G2 (12)

G3 (13)

G4 (12)

Dose

(mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

(0-4)

-4.32

4.81

-8.47

4.07

-7.73

5.39

-7.34

6.33

(0-13)

5.29

7.73

0.04

4.14

0.72

6.21

3.61

7.07

(0-14)

-4.32

4.81

-8.47

4.07

-7.73

5.39

-7.34

6.33

Summary of Days of Conception and Pregnancy Index (%)

Group(N)

G1(13)

G2(13)

G3(13)

G4(13)

Dose (mg/kg b.wt.)

0

250

500

750

No. of females showed evidence of copulation

11

12

13

12

No. of females concieved between Days 1-5 of cohabitation

11

11

11

11

No. of females concieved after Day 5 of cohabitation

0

1

2

1

Females achieved pregnancy

11

12

13

12

Pregnancy Index (%)

84.62

92.31

100.00

92.31

Key:N= number of dams in a group, No. = Number

Post-natal Loss (%) and Pups Survival Index (%)

Group(N)

G1(11)

G2(12)

G3(13)

G4(12)

Dose(mg/kg bwt)

0

250

500

750

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

No. of Live Births

11.80

1.93

12.00

2.95

9.85

3.60

10.58

1.68

No. of alive pups at Post-natal Day 4

10.90

3.00

8.33

5.57

9.08

4.37

10.42

1.51

Post-natal Loss (%)

7.85

19.84

33.02

38.93

18.72

37.26

1.28

4.44

Fetal Survival Index at Post-natal Day 4 (%)

92.15

19.84

66.98

38.93

81.28

37.26

98.72

4.44

Mean Gestational Length and Litter size

Group(N)

G1(11)

G2(12)

G3(13)

G4(12)

Dose(mg/kg bwt)

0

250

500

750

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Gestation Length

22.36

0.50

22.00

0.00

22.00

0.00

22.25

0.45

Litter size

(Total No. of litter size)

10.91

3.48

12.67

2.90

10.85

2.61

10.58

1.68

Mean Pups Body Weight, Sex Ratio and Gross Observation

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean Pups Weight

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Day 0

6.34

0.57

118

6.10

0.76

151

6.24

0.86

141

6.59

0.48

127

Day 4

9.76

1.78

109

8.56

1.12

100

9.11

1.08

118

9.50

1.45

125

Day 13

24.88

3.88

87

20.70

3.00

78

22.74

2.65

86

22.05

2.90

100

Group(n)

G1(11)

G2(12)

G3(13)

G4(13)

Pups Body Weight gain

(%)

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Day 0-Day 4

47.42

20.65

109

36.29

13.99

99

41.58

13.79

118

43.66

15.12

125

Day 0-Day 13

148.89

19.84

87

143.22

25.24

78

144.89

19.45

86

135.74

22.14

100

Group

(Number of Litter size)

G1(118)

G2(151)

G3(141)

G4(127)

Sex Ratio at birth

(Male/Female)

61/57

72/79

80/61

71/56

Sex Ratio at Day 4

(Male/Female)

56/53

48/51

64/54

69/56

Gross Observations

NAD

NAD

NAD

NAD

Hormonal Analysis Data

Sex: Male (Termination)

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

4.62

0.79

13

4.34

0.96

13

4.63

0.75

13

4.18

0.73

13

TSH (uIU/mL)

3.48

2.05

13

4.44

2.52

13

3.31

1.59

13

3.97

1.62

13

Testosterone (ng/dL)

71.85

77.91

13

115.22

96.43

13

99.50

118.60

13

99.75

112.05

13

                  Sex: Female (Day 4)

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

3.54

0.56

11

3.27

0.63

12

3.60

0.74

13

3.46

0.48

12

TSH (uIU/mL)

3.10

1.31

11

3.38

1.58

12

2.69

1.41

13

3.21

1.67

12

                                                                                        Sex: Female (Day 13)

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

3.48

0.60

11

3.28

0.69

12

3.30

0.50

13

3.04

0.31

12

TSH (uIU/mL)

2.45

2.09

11

3.25

1.84

12

3.23

2.71

13

2.21

1.32

12

E2 (pg/mL)

 

33.13

11.94

11

41.09

18.51

12

29.94

15.08

13

25.25

8.02

12

Sex: Female (Non-Pregnenat)

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

5.6

3.39

2

3.90

./.

1

./.

./.

0

3.90

./.

1

TSH (uIU/mL)

5.45

1.15

2

2.62

./.

1

./.

./.

0

2.50

./.

1

E2 (pg/mL)

 

36.4

2.50

2

56.37

./.

1

./.

./.

0

39.28

./.

1

 

Sex: Male (Termination)

 

Group

G1R

G4R

Dose(mg/kg bwt)

0

750

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

2.98

0.63

5

4.76

2.18

5

TSH (uIU/mL)

5.03

1.73

5

3.91

1.65

5

Testosterone (ng/dL)

54.67

37.03

5

160.34

183.40

5

 

Sex: Female (Termination)

 

Group

G1-R

G4-R

Dose(mg/kg bwt)

0

750

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

2.82

0.11

5

2.72

0.43

5

TSH (uIU/mL)

4.69

4.74

5

2.82

2.74

5

Testosterone (ng/dL)

45.64

33.01

5

24.04

21.81

5

Day: 04 (Pups)

 

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

2.17

0.29

10

2.04

0.42

11

2.37

0.49

11

2.13

0.31

12

TSH (uIU/mL)

1.76

0.32

10

1.84

0.51

11

1.84

0.81

11

1.82

0.56

12

 

Day: 13 (Pups)

 

Group(n)

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

5.87

0.88

9

5.08

0.60

9

5.60

0.70

10

5.64

0.82

12

TSH (uIU/mL)

2.16

1.13

9

1.88

0.49

9

2.05

0.60

10

2.14

0.69

12

Conclusions:
NOAEL was considered to be 750 mg/kg bw when Wistar male and female Rats were treated with test chemical orally by gavage for more the 63 days.
Executive summary:

In a experimental study conducted according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test).The Wistar male and female rat treated with test chemical in the concentration of 0, 250, 500 and 750 mg/ kg bw orally by gavage for more than 63 days. No mortality or morbidity was observed in any animals of the control and treatment groups throughout the study period. No apparent treatment related clinical signs were observed in any of the animals throughout the treatment. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period. Statistically significant increase in female was observed in urine pool at Week 5 in 750 mg/kg bw as compared to control. The above changes observed were inconsistent/ biologically insignificant and not dose dependent. Statistically significant decrease was observed in percent body weight change on day 1-15 and day 1-28 at 500 mg/kg bw as compared to control group. Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. No treatment related changes in food consumption were observed in treated male and female rats as compared to control. Similarly, at the end of treatment period revealed statistically significant decreased was noted in WBC of females (R) at 750 mg/kg body weight. The observed variation in WBC was considered to be of no toxicological significance. At the end of treatment period revealed, statistically significant decrease were noted in Triglyceride in Male at 500 mg/kg body weight, Calcium in Female at 750 mg/kg body weight. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes. During treatment-free recovery period revealed statistically significant increase was noted in cholesterol in Female at 750 mg/kg body weight, A: G ratio, in Male and Female (R) at 750 mg/kg body weight while statistical significant decrease were noted in ALT and Globulin in Female at (R) 750 mg/kg body weight. The observed variations in Triglyceride, Calcium, Cholesterol, ALT, Globulin and A: G ratio were considered to be of no toxicological significance,

The functional observation battery/neurobehavioral observation were comparable and no changes were revealed in any of the animals of all the treated groups in both the sexes. The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups. However in recovery male, statistically significant increase was noted in grip strength (hindlimb) in 750 mg/kg bw (R) when compare to control recovery group. Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group. Howerver, statistically significant increase in female were noted in Distance travelled (DT), Ambulatory time (AT), and in Horizontal counts (HC) at 750 mg/kg body weight when compare to control group. The above changes observed were not dose dependent, hence considered as incidental and not attributed to the effect of test item administration. In addition, At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of either sex did not differ significantly when compared to respective control group except increased splenic absolute and relative to brain weight in female rats treated at 500 mg/kg, decreased splenic weight in male rats treated at 750 mg/kg during recovery and increased weight of adrenal relative to brain weight in male rats treated at 500 mg/kg with respective control group. Observed weight variations in the organs did not showed dose dependency and are minor in nature, so could be considered as spontaneous. External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality.

Microscopic examination of control group and rats treated with 250, 500 and 750 mg/kg revealed varying degree of pathological changes in different organs. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item. No effects on reproductive parameters were observed such as regular cyclicity i.e. 3-5 days estrous cycle. However in group G1 two female, group G2 one female and group G4 one female showed prolong diestrus and found to be non-pregnant. In cohabitation or mating period, all females from control and treated groups showed evidence of copulation i.e. sperm positive vaginal smear. All females showed precoital interval less than 5 days, except 1, 2 and 1 female from G2, G3 and G4, respectively which showed precoital interval more than 5 days. There was no difference between the control (G1) and treatment groups (G2, G3 and G4) in the maternal and fetal parameters. Gestational length, Litter size, No. of live births, Postimplantation loss, pups weight at birth and PND14, Post-natal loss, Survival Index and weight gain for pups at PND13. Pregnancy index and Pups sex ratio (Male/Female) respectively of treated rats as compared to control. No treatment related changes were noted in hormonal analysis (T4, TSH, Testosterone and Estrogen). Therefore, NOAEL was considered to be 750 mg/kg bw when Wistar male and female Rats were treated with test chemical orally by gavage for more the 63 days.

Reason / purpose:
read-across source
Reference
Endpoint:
reproductive toxicity, other
Remarks:
reproductive organ toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data from NTRL report
Qualifier:
equivalent or similar to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
Reproductive toxicity study of test material was performed on rats.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: CRL: COBS 'CD '(SD)BR
Sex:
male/female
Details on test animals and environmental conditions:
Details on test animals and env. conditions
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Wilmington, MA.
- Age at study initiation: 6 weeks
- Weight at study initiation: No data available
- Fasting period before study:No data available

- Housing: Animals were kept five per cage in stainless steel wire-mesh cages fitted with automatic watering nipples. Males and females were housed on separate racks. Cages containing rats of different 'dose levels were distributed randomly over the racks
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): Feed was Purina Laboratory Rodent Chow 5001, ground meal, and was available ad lib.
- Water (e.g. ad libitum): Water, ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):21.66-23.33°C
- Humidity (%):35-49%
- Air changes (per hr):No data available

- Photoperiod (hrs dark / hrs light): 12 hour light period (6 AM-6 PM).

IN-LIFE DATES: From: To:
Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material soluble in corn oil and the mixture was combined with laboratory rodent chow. The
Corn oil was added to all diets at a concentration of 1.0%.
DIET PREPARATION
- Rate of preparation of diet (frequency):No data available
- Mixing appropriate amounts with (Type of food)
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water):corn oil
- Concentration in vehicle:0, 0.02%, 0.08%, 0.32 %( 9, 46, or 214 mg/kg/day, 10, 51 or 239 mg/kg/day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available
Details on mating procedure:
No data available
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Remarks:
0, 0.02%, 0.08%,0.32 %( 9, 46, or 214 mg/kg/day for male ,10,51 or 239 mg/kg/day for female )
No. of animals per sex per dose:
Total: 240 animals
For male
0 mg/kg bw/day:30
9mg/kg bw/day:30
46mg/kg bw/day:30
214mg/kg bw/day:30
For female
0 mg/kg bw/day: 30
10mg/kg bw/day: 30
51mg/kg bw/day: 30
239mg/kg bw/day: 30
Control animals:
yes
Details on study design:
The dose levels were selected on the basis of a 12 day feeding study in which rats were fed diets of 1.0, 0.1 or 0.0% of test material
Positive control:
No data available
Parental animals: Observations and examinations:
Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS: Yes

Time schedule: daily
BODY WEIGHT: Yes
Time schedule for examinations: Body weights were determined on days 0, 4, 7, and weekly thereafter
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):YesFeedconsumption was determined on days 4, 7, and twice weekly thereafter.
Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data available


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Time schedule for examinations
Oestrous cyclicity (parental animals):
No data available
Sperm parameters (parental animals):
No data available
Litter observations:
No data available
Postmortem examinations (parental animals):
Postmortem examinations (Parent Animal)
SACRIFICE : on day 90
GROSS NECROPSY: yes
HISTOPATHOLOGY / ORGAN WEIGHTS: yes
Postmortem examinations (offspring):
No data available
Statistics:
All numerical data were evaluated using the following computer generatedstatistical tests: one-way analysis of variance (ANOVA). Bartlett's test,and Duncan's multiple range test where appropriate. A significance level of p<0.05 was chosen to indicate a statistically significant difference
Reproductive indices:
No data available
Offspring viability indices:
No data available
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Red and blue discoloration of the urine under the cages of all male and female rats fed the 0.32% diets. The abnormality was seen as distinct patches of red or blue urine stained paper under the cages.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
There were no premature deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
All groups of animals gained weight although the 0.08% and 0.32% diets clearly retarded weight gain for both males and females. The 0.02% diet had no effect on body weight gain
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The 0.08% and 0.32% diets reduced (p <0.05) feed consumption for both male and female rats, although not for every time period. Feed consumption was reduced the most at the introduction of the testdiets as reflected in the data for day 4. Reduction of feed consumption wasalso greater for males than for females. Diets of 0.02% dose group generally did not alter feed consumption with fewexceptions. These exceptions (p<0.05) included decreased feed consumptionmeasured on day 73 for males and increased feed consumption for females ondays 35, 39 (90-day group), 59, 63, and 87.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No histopathology lesions in the spleen, liver, kidneys. Testes, epididymides and adipose tissue.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
not specified
The growth of the testes was depressed .Males given the 0.32% diets for 90 days had lower absolute, testes/body weight ratio, and testes/brain weight ratio. Males given the 0.32% diets for 42 days had lower absolute and testes/brain weight ratio but comparable testes/body weight ratio. Degeneration of epididymal spermatozoa indicates a higher incidence of spermatogenic effects in the 0.32% dose group (8 of 10 rats after 42 days and 13 of 20 rats after 90 days). The mid dose group (1 of 10 ratsafter 42 days) and the control group (1 of 20 rats after 90 days) had single rats with testicular atrophy and degenerative epididymal spermatozoa. The 0.02% dose group had no animals with spermatogenic lesions.
Ovarian growth was not affected by exposure to any of the test diets.
Dose descriptor:
NOAEL
Effect level:
46 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: No toxic effects were observed
Dose descriptor:
NOAEL
Effect level:
51 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: No toxic effects were observed
Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Dose descriptor:
other: not specified
Generation:
other: not specified
Based on:
not specified
Sex:
not specified
Remarks on result:
not measured/tested
Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Reproductive effects observed:
not specified
Treatment related:
not specified
Conclusions:
No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 46 mg/kg/day for male and 51mg/kg/day were considered to be the NOAEL for female. Whenmale and female rats were treated with test material orally.
Executive summary:

The reproductive toxicity study of test material was performed on male and femaleCRL: COBS 'CD '(SD)BR rats. The test material soluble in corn oil and themixture was combined with laboratory rodent chow. The Corn oil was added to all diets at a concentration of 1.0%.Estimated consumption was0, 0.02%, 0.08%,0.32 %( 9, 46, or 214 mg/kg/day for male , 10,51 or 239 mg/kg/day for female ) for 90 day. The dose levels were selected on the basis of a 12 day feeding study in which rats were fed diets of 1.0, 0.1 or 0.0% test material .Thirty males and thirty females were exposed to each dietary concentration. Ten rats of each sex at each concentration were killed approximately half-way through' the study (42 days interim groups) and twenty rats of each sex were killed after approximately 90 days. Body weights were determined on days 0, 4, 7, and weekly thereafter. Feed consumption was determined on days 4, 7, and twice weekly thereafter. Necropsies were conducted according to pathology SOP TP 180. liver, kidneys, spleen, heart, adrenal glands, ovaries, testes, and brain. Paired organs were weighed together. Organ/body weight and organ/brain weight ratios were calculated. No mortality was observed.Red and blue discoloration of the urine under the cages of all male and female rats fed the 0.32% diets. The abnormality was seen as distinct patches of red or blue urine stained paper under the cages.All groups of animals gained weight although the 0.08% and 0.32% diets clearly retarded weight gain for both males and females. The 0.02% diet had no effect on body weight gain.The 0.08% and 0.32% diets reduced (p <0.05) feed consumption for both male and female rats, although not for every timeperiod. Feed consumption was reduced the most at the introduction of the testdiets as reflected in the data for day 4. Reduction of feed consumption wasalso greater for males than for females. Diets of 0.02%dose group generallydid not alter feed consumption with fewexceptions. These exceptions(p<0.05)included decreased feed consumptionmeasured on day 73 for males and increased feed consumption for females ondays 35, 39 (90-day group), 59, 63, and 87. Ovarian growth was not affected by exposure to any of the test diets.The only target organ effects which are apparent involve enlargement of the spleen (0.08% and 0.32% diets) and atrophy of the testes (0.32% diets). The only statistically significant effects in rats given diets of 0.02% were organ to body weight ratios for the liver (90 day), brain (42 day), and adrenal gland (42 day) for females. None of the differences in rats given the 0.02% diets were of sufficient magnitude or consistency to be considered toxicologically significant.Red blood cell toxicity wascharacterized as a macrocytic, very slightly hyperchromatic anemia with Heinzand Howell-Jolly bodies and secondary lesions in the spleen, liver, and kidneydue to increased hemoglobin catabolism and increased hematopoiesis. Theseverity of red blood cell damage was dose related and was detectable at thelowest dose 9-10 mg/kg/day for 90 days (0.02% diet).Nohistopathologic lesions in the spleen, liver, kidneys. Testes, epididymides and adipose tissue.

The growth of the testes was depressed .Males given the 0.32% diets for 90 days had lower absolute, testes/body weight ratio, and testes/brain weight ratio. Degeneration of epididymal spermatozoa indicates a higher incidence of spermatogenic effects in the 0.32% dose group (8 of 10 rats after 42 days and 13 of 20 rats after 90 days). The mid dose group (1 of 10 rats after 42 days) and the control group (1 of 20 rats after 90 days) had single rats with testicular atrophy and degenerative epididymal spermatozoa. The 0.02% dose group had no animals with spermatogenic lesions. HenceNo Observed Adverse Effect Level (NOAEL) for male was considered to be 46 mg/kg/day and for female NOAEL was considered to be the 51mg/kg/day on the bases of effects observed on reproductive organ .When male and femalerats were treated withtest materialorally.

 

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2016
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018
Reference Type:
other: secondary source
Title:
Basic Toxicity of test material
Author:
NTRL report
Year:
1981
Bibliographic source:
NTRL report ,1981

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material: β-bromostyrene
- Molecular formula: C8H7Br
- Molecular weight: 183.0473 g/mole
- Substance type: Organic
- Physical state: Yellow clear liquid
- Impurities: 0.4%
- Purity: 99.6%

Test animals

Species:
other: Study 2: rat; Study 3: rat; Study 4: rat
Strain:
other: Study 2: Wistar; Study 3: Wistar; Study 4: CRL: COBS 'CD '(SD) BR
Sex:
male/female
Details on test animals and environmental conditions:
Study 2:
Source : In-House Bred at sa-FORD, Animal Facility (CPCSEA Registration No. 1256/bc/09/CPCSEA)
Health Status :Healthy young adult animals were used for the study. Females were nulliparous and non-pregnant.
Body weight of animals :
Male: Minimum: 240g; Maximum: 315 g
Female: Minimum: 210g; Maximum: 260 g (Individual body weights were within ± 20% of mean body weight, prior to treatment)
Age: 12-13 weeks at the start of Oestrous Cycle evaluation.
Acclimatisation: Animals were acclimatised to the test conditions for 20 days prior to test item administration
Housing: Before the animals are brought in, the study room and cages were cleaned and disinfected. During the study, the floor of the experimental room and work tops were swept and mopped with disinfectant solution every day or as on requirement. Cages were cleaned at regular intervals.
A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20 [cm]). Cage rotation was carried out weekly during study period except during mating for males and females both and during gestation and lactation for females.
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.
Bedding material of batch No. SPAR-30/2015 (Sparconn Life Sciences Bangalore) was used in this study and a copy of report of microbial and chemical contaminants analysed periodically by manufacturer of bedding material are incorporated in the raw data.

Environmental conditions:
The room temperature was maintained at 18.30 to 22.70 °C and the relative humidity was kept between 43.90 to 67.60%. Artificial light was set to give a cycle of 12 hours light and 12 hours dark. Air changes were about minimum 12 times per hour and filtered adequately.
Diet :A conventional laboratory pelleted diet of batch no. 004915, 041215 and 041015 from approved supplier (Nutrivet Life Sciences, Pune) was offered ad libitum. The copy of composition, microbial and chemical contaminant reports analysed periodically by manufacturer are incorporated in the raw data.
Water : Aqua guard filtered drinking water in bottles was offered ad libitum. Samples of the drinking water was subjected periodically to bacteriological tests and to chemical contaminant analysis. The latest test results are included in the raw data.

Study 3:
- Source: National Institute of Biosciences
- Age at study initiation: 12 - 13 weeks at the start of Oestrous Cycle evaluation
- Weight at study initiation:
- Fasting period before study
:- Housing: Cages were cleaned at regular intervals. A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20 [cm]). .Sterilized corn cob produced from pure corn, dried and free from dust, procured from approved supplier,was used as bedding material. Bedding material of batch No. 8-17 (Krishana Corncob Industries, Aurangabad) was used in this study
.- Diet (e.g. ad libitum): A conventional laboratory pelleted diet, ad libitum
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.00 to 24.40 °C
- Humidity (%): 40.50 to 64.30%
- Air changes (per hr): minimum 12 times per hour and filtered adequately
.- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark
IN-LIFE DATES: From: December 08, 2017To: April 28, 2018

Study 4:
Details on test animals and env. conditions
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Wilmington, MA.
- Age at study initiation: 6 weeks
- Weight at study initiation: No data available
- Fasting period before study:No data available

- Housing: Animals were kept five per cage in stainless steel wire-mesh cages fitted with automatic watering nipples. Males and females were housed on separate racks. Cages containing rats of different 'dose levels were distributed randomly over the racks
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): Feed was Purina Laboratory Rodent Chow 5001, ground meal, and was available ad lib.
- Water (e.g. ad libitum): Water, ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):21.66-23.33°C
- Humidity (%):35-49%
- Air changes (per hr):No data available

- Photoperiod (hrs dark / hrs light): 12 hour light period (6 AM-6 PM).

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
other: Study 2: oral: gavage; Study 3: oral: gavage; Study 4: oral: feed
Vehicle:
other: Study 2: corn oil; Study 3: water; Study 4: corn oil
Details on exposure:
Study 2:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item. The details of dose formulation preparation is maintained in raw data.

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was used as a vehicle based on the solubility testing
- Concentration in vehicle: 0, 308, 556 and 1000 mg/kg bw
- Amount of vehicle (if gavage): 0.5 ml/kg
- Lot/batch no. (if required): MR301015, MR161215
- Purity:

Study 3:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (Distilled water) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. Atthe time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item

.DIET PREPARATION- Rate of preparation of diet (frequency):- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:
VEHICLE- Justification for use and choice of vehicle (if other than water): Distilled water
- Concentration in vehicle: 0, 250, 500 and 750 mg/kg bw
- Amount of vehicle (if gavage): 1.0 ml/100g body weight
- Lot/batch no. (if required):- Purity:N/A

Study 4: PREPARATION OF DOSING SOLUTIONS:
The test material soluble in corn oil and the mixture was combined with laboratory rodent chow. The
Corn oil was added to all diets at a concentration of 1.0%.
DIET PREPARATION
- Rate of preparation of diet (frequency):No data available
- Mixing appropriate amounts with (Type of food)
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water):corn oil
- Concentration in vehicle:0, 0.02%, 0.08%, 0.32 %( 9, 46, or 214 mg/kg/day, 10, 51 or 239 mg/kg/day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:
Study 2:
- M/F ratio per cage:1:1
- Length of cohabitation:
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: The day of detection of sperm positive vaginal smear was considered as day "0" of gestation.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.:Re-mating of unsuccessfully paired female was done with proven male of the same group.
- Further matings after two unsuccessful attempts: [no / yes (explain)] : No data
- After successful mating each pregnant female was caged (how): housed individually
- Any other deviations from standard protocol:No data

Study 3:
- M/F ratio per cage: one male and one female
- Length of cohabitation: until pregnancy occurs or two weeks elapsed
- Proof of pregnancy: Mating was confirmed by observation of sperm positive vaginal smear. The day of detection of sperm positive vaginal smear was considered as day "0" of gestation.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.:
- Further matings after two unsuccessful attempts: - After successful mating each pregnant female was caged (how): housed individually - Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Study 2: Homogeneity and Stability of dose formulation by analysing the sample at different time points (Stability was determined by sampling and analyzing the aliquots from the sample stored at 25 ± 2°C at the time points of 0, 2 and 6 hours).Two replications was analyzed at each time point.The dose formulation analysis were carried out at the start (on the day 1) of treatment, on day 21 and day 40 during the study period.

Study 3: Specificity, Linearity, Precision (%RSD), Accuracy (% Recovery) and Homogeneity were analysis by HPLC-UV
Duration of treatment / exposure:
Study 2:
Male: 47 days
Female : 63 days

Study 3: Approx. 64 days

Study 4: 90 days
Frequency of treatment:
Study 2: Daily
Study 3: Daily
Study 4: Daily
Doses / concentrations
Remarks:
Study 2:
0, 308, 556 and 1000 mg/kg bw
Study 3:
0, 250, 500 and 750 mg/kg bw
Study 4: 0, 0.02%, 0.08%,0.32 %( 9, 46, or 214 mg/kg/day for male ,10,51 or 239 mg/kg/day for female )
No. of animals per sex per dose:
Study 2:
Total: 124 animals
0 mg/kg bw: 13 male, 13 female
308 mg/kg bwm: 13 male, 13 female
556 mg/kg bwm: 13 male, 13 female
1000 mg/kg bwm: 13 male, 13 female
Control recovery: 5 male, 5 female
1000 mg/kg bw recovery: 5 male, 5 female

Study 3:
Total: 124 animals
0 mg/kg bw, 13 male, 13 female
250 mg/kg bw, 13 male, 13 female
500 mg/kg bw, 13 male, 13 female
750 mg/kg bw, 13 male, 13 female

Recovery group:
0 mg/kg bw, 5 male, 5 female
750 mg/kg bw, 5 male, 5 female

Study 4:
Total: 240 animals
For male
0 mg/kg bw/day:30
9mg/kg bw/day:30
46mg/kg bw/day:30
214mg/kg bw/day:30
For female
0 mg/kg bw/day: 30
10mg/kg bw/day: 30
51mg/kg bw/day: 30
239mg/kg bw/day: 30
Control animals:
yes, concurrent vehicle
Details on study design:
Study 2:
- Dose selection rationale: No Data Available
- Rationale for animal assignment (if not random): Vaginal smear of all females was evaluated for regular cyclicity before treatment (14 days). At the time of randomization females not showing regular cycle were euthanised and discarded
- Other: N/A

Study 3: - Dose selection rationale: The dose levels 250, 500 and 750 mg/kg body weight were selected for theMain Study based on the results of Dose Range Finding (DRF).- Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing. Individual body weights were considered within ± 20% of the groups mean.- Rationale for selecting satellite groups: 0 and 750 mg/kg bw were selected as satellite groups- Post-exposure recovery period in satellite groups: 14 days- Section schedule rationale (if not random): N/A

Study 4: The dose levels were selected on the basis of a 12 day feeding study in which rats were fed diets of 1.0, 0.1 or 0.0% of test material
Positive control:
No Data Available

Examinations

Parental animals: Observations and examinations:
Study 2: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (morning and evening)
- Cage side observations checked in table [No.?] were included. : morbidity and mortality were examined.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, preferably at the same time each day considering the peak period of anticipated effects after dosing.
Detailed clinical examinations were carried out once before the first treatment (to allow for within-subject comparisons) and weekly thereafter.
The detailed clinical examinations were made outside the home cage, at approximately the same time, on each occasion.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), day 4 post-partum and before terminal sacrifice.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:
OTHER:
Hematology and Clinical Biochemistry were examined.

Study 3: CAGE SIDE OBSERVATIONS: Yes- Time schedule: Twice daily (morning and evening)- Cage side observations : Morbidity and mortality, throughout theacclimatization and study period.
DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: once a day, preferably at the same time each day
BODY WEIGHT: Yes- Time schedule for examinations: Males and females were weighed during randomization, on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), on day 4 and day 13 post-partum and before terminal sacrifice
.FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kgbody weight/day: Yes- Compound intake calculated as time-weighted averages from the consumption and body weight gaindata: Yes
FOOD EFFICIENCY: No data- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weightedaverages 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
OPHTHALMOSCOPIC EXAMINATION: No data- Time schedule for examinations: No data- Dose groups that were examined: No data
HAEMATOLOGY: Yes- Time schedule for collection of blood: Just prior to necropsy.- Anaesthetic used for blood collection: Yes, Isoflurane anaesthesia- Animals fasted: Yes, fasted overnight- How many animals: five males and five females, randomly selected from each group- Parameters ] were examined. Total Erythrocyte Count (RBC), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Hemoglobin (HGB), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (PLT), Total Leukocyte count (WBC), Prothombin Time (PT) and Activated Partial Thromboplastin time (aPTT) were examined.CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: Just prior to necropsy.- Animals fasted: Yes, fasted overnight- How many animals: five males and five females, randomly selected from each group- Parameters were examined. : Glucose (Glu), Cholesterol (Chol), Triglycerides (TRIG), Alanine amino transferase (ALT), Aspartate amino transferase (AST), Calcium, Albumin (Alb), Total Protein (TP), Creatinine (Crea), Phosphorus, Urea, Sodium (Na), Potassium (K), Blood urea nitrogen (BUN), Globulin (Glob), Alb/ Glb (A:G) and Bile acids were examined.URINALYSIS: No data- Time schedule for collection of urine: No data- Metabolism cages used for collection of urine: No data- Animals fasted: No data- Parameters checked in table [No.?] were examined. No data
NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for exam

Study 4: Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: yes
DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: daily
BODY WEIGHT: Yes
Time schedule for examinations: Body weights were determined on days 0, 4, 7, and weekly thereafter
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):YesFeedconsumption was determined on days 4, 7, and twice weekly thereafter.
Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data available
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Time schedule for examinations: No Data Available
Oestrous cyclicity (parental animals):
Study 2: Estrous cyclicity was monitored for two weeks from begining of the treatment period till cohabitation and thereafter until the evidence of mating.
Study 3: Estrous cycle were monitored daily from beginning of the treatment period until evidence of mating. When taking vaginal smear care was taken to avoid disturbance to vaginal mucosa.
Sperm parameters (parental animals):
Study 2: spermatogenesis were examined.
Litter observations:
Study 2: Number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities.
Study 3: Number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), body weight and ano-genital distance (AGD) were examined.
Postmortem examinations (parental animals):
Study 2: Organ Weight, Gross Pathology and Histopathology were examined.
Study 3: GROSS PATHOLOGY: Yes, At scheduled sacrifice date, all rats of main and recovery groups were euthanized by over dose of carbon dioxide followed by exsanguination. The animals were examined externally in unopened condition. This was followed by opening of the carcasses and topographic examination of different organs. This included careful examination of the external surface of the body, all orifices, cranial, thoracic and visceral cavities and their contents.Similarly, necropsy of terminally sacrificed and found dead pups during study period were conducted andgross pathological observations were recorded.HISTOPATHOLOGY: Yes,Full histopathology was carried out on the preserved organs (ovaries, uterus, cervix with vagina, testes, epididymides, prostate, seminal vesicle with coagulating glands) of all animals and all tissues of five males and females, randomly selected from each group animals in the control and high dose groups.
Study 4: Postmortem examinations (Parent Animal)
SACRIFICE : on day 90
GROSS NECROPSY: yes
HISTOPATHOLOGY / ORGAN WEIGHTS: yes
Postmortem examinations (offspring):
Study 2: Organ Weight, Gross Pathology and Histopathology were examined.
Study 3: Presence of any gross abnormalities were examined.
Statistics:
Study 2: Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks.

Study 3: Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks.

Study 4: All numerical data were evaluated using the following computer generatedstatistical tests: one-way analysis of variance (ANOVA). Bartlett's test,and Duncan's multiple range test where appropriate. A significance level of p<0.05 was chosen to indicate a statistically significant difference.
Reproductive indices:
Study 2: Survival Index of pups, Pregnancy index and Fertility index were examined.
Study 3: Pregnancy Index (%), Post-natal Loss (%), Gestation Length were examined.
Offspring viability indices:
Study 2: yes, viability on day 0 and 4 were examined.
Study 3: Fetal Survival Index at Post-natal Day 4 (%) were examined.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Study 2: No apparent treatment related clinical signs were observed in any of the animals throughout the treatment and recovery period.
Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period.
Statistically significant decrease was observed in number of rears of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male on pre-treatment as compared to control G1 (0 mg/kg body weight). The statistically significant increase was observed in number of urine pools of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of fecal bolus of G3 (556 mg/kg body weight) male at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of rears of G4 (1000 mg/kg body weight) male at week 4 as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of urine pools of G3 (556 mg/kg body weight) male at week 6 as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of rears of G2 (308 mg/kg body weight), G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) female at pre-treatment as compared to control G1 (0 mg/kg body weight). Statistically significant increase was observed in number of fecal bolus of G4 (1000 mg/kg body weight) female at week 5 as compared to control G1 (0 mg/kg body weight).
The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence considered as incidental and not attributed to the effect of test item administration.

Study 3: No apparent treatment related clinical signs were observed in any of the animals throughout the treatment. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period. Statistically significant increase in female was observed in urine pool at Week 5 in 750 mg/kg bw as compared to control. The above changes observed were inconsistent/ biologically insignificant and not dose dependent.

Study 4: Red and blue discoloration of the urine under the cages of all male and female rats fed the 0.32% diets. The abnormality was seen as distinct patches of red or blue urine stained paper under the cages.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
Study 2: No mortality or morbidity was observed in any animal of the control and treatment groups throughout the study period.

Study 3: No mortality or morbidity was observed in any animals of the control and treatment groups throughout the study period.

Study 4: There were no premature deaths during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Study 2: A statistically significant decrease was observed in body weight of G4 (1000 mg/kg body weight) male on day 30 as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in body weight of G4 (1000 mg/kg body weight) female on day 20 of gestation as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in body weight of G4-R (1000 mg/kg body weight) male on day 29, 36, 41 as compared to control G1-R (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change of G3 (556 mg/kg body weight) and G4 (1000 mg/kg body weight) male on day 1-8, 1-14 whereas statistically significant decrease was observed in percent body weight change of G4 (1000 mg/kg body weight) male on day 1-21, 1-28, 1-30, 1-37, 1-44, 1-46 as compared to control G1-R (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change during gestation period of G4 (1000 mg/kg body weight) female on day 0-14, 0-20 as compared to control G1 (0 mg/kg body weight). Statistically significant decrease was observed in percent body weight change of G4-R (1000 mg/kg body weight) male on day 1-8, 1-15, 1-22, 1-29 as compared to control G1-R (0 mg/kg body weight).
Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group.
These changes observed were inconsistent, hence not considered as effect of the test item administration.

Study 3: When treated with 500 mg/kg bw, statistically significant decrease was observed in percent body weight change on day 1-15 and day 1-28 as compared to control group.Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. These changes observed were inconsistent, hence not considered as effect of the test item administration.

Study 4: All groups of animals gained weight although the 0.08% and 0.32% diets clearly retarded weight gain for both males and females. The 0.02% diet had no effect on body weight gain
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Study 2: Statistically significant decrease in feed consumption was observed in G4 (1000 mg/kg body weight) female on gestation day 14-20 as compared to the control group G1. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group.
Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration.

Study 3: No treatment related changes were observed in treated male and female rats as compared to control.

Study 4: The 0.08% and 0.32% diets reduced (p <0.05) feed consumption for both male and female rats, although not for every time period. Feed consumption was reduced the most at the introduction of the testdiets as reflected in the data for day 4. Reduction of feed consumption wasalso greater for males than for females. Diets of 0.02% dose group generally did not alter feed consumption with fewexceptions. These exceptions (p<0.05) included decreased feed consumptionmeasured on day 73 for males and increased feed consumption for females ondays 35, 39 (90-day group), 59, 63, and 87.
Food efficiency:
not specified
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):
Study 2: All hematological parameters in animals of different treated groups of both the sexes were comparable to their respective control groups, except statistically significant decrease observed for MCHC, WBC in males of G4 (1000 mg/kg body weight) as compared to G1, statistically significant increase observed for aPTT in males of G4 (1000 mg/kg body weight) and G3 (556 mg/kg body weight) as compared to G1. Statistically significant decrease observed for RBC, HCT, HGB, WBC in males of G4-R (1000 mg/kg body weight) as compared to G1-R. Statistically significant decrease observed for PT in females of G3 (556 mg/kg body weight) as compared to G1. Statistically significant decrease observed for MCHC and statistically significant increase observed for RBC, HCT, HGB in females of G4-R (1000 mg/kg body weight) as compared to G1-R.
The above changes were inconsistent, not related to the test item and may be due to the preanalytical and analytical variables.

Study 3: At the end of treatment period revealed statistically significant decreased was noted in WBC of females (R) at 750 mg/kg body weight. The observed variation in WBC was considered to be of no toxicological significance, of a minimal in nature and occurred in the absence of clear dose related response.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Study 2: All clinical chemistry parameters in animals of different treated groups of both the sexes were comparable to their respective control groups, except statistically significant increase observed for ALT and statistically significant decrease observed for Sodium (Na) in males of G4 (1000 mg/kg Body weight) as compared to G1. Statistically significant increase observed for Creatinine in males of G2 (308 mg/kg Body weight) as compared to G1. Statistically significant decrease observed for Total Protein and statistically significant increase observed for A/G ratio in females of G3 (556 mg/kg Body weight) as compared to G1.
The above changes were inconsistent, not dose dependent hence considered as incidental in nature.
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Study 2: The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes.
Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups except a statistically significant decrease was observed in hindlimb foot splay in G4-R (1000 mg/kg body weight) male as compared to the repective control group G1-R.
The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence, considered as incidental and not attributed to the effect of test item administration.
Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group except statistically significant decrease was observed in ST=Stereotypic time in G2, G3 and G4 male as compared to control group G1 and G4-R in female as compared to G1-R.
The above changes observed were inconsistent, hence considered as incidental and not attributed to the effect of test item administration.

Study 3: The functional observation battery/neurobehavioral observation were comparable and no changes were revealed i any of the animals of all the treated groups in both the sexes.The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes.Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups. However in recovery male,statistically significant increase was noted in grip strength (hindlimb) in 750 mg/kg bw (R) when compare to control recovery group.Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group. Howerver, statistically significant increase in female were noted in Distance travelled (DT),Ambulatory time (AT), and in Horizontal counts (HC) at 750 mg/kg body weight when compare to control group.The above changes observed were not dose dependent, hence considered as incidental and not attributed to the effect of test item administration.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Study 2: Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality.
Microscopic examination of control group and rats treated at 308, 556 and 1000 mg/kg revealed varying degree of pathological changes in different organs. This includes Liver: focal to multifocal minimal lymphocytic infiltration (Male: G1:1/5, G4:2/5; Female: G1: 1/5; G4: 2/5); focal minimal necrosis (Male: G1:1/5; Female: G1: 1/5); Kidneys: focal minimal lymphocytic infiltration (Male: G1:2/5; Female: G4:1/5); focal mild mineralization (Female: G1:1/5); Lungs: multifocal minimal lymphocytic infiltration (Male:G1:1/5, G4:1/5; Female: G1: 2/5, G4: 3/5); focal minimal histiocyte infiltration (Female: G1: 1/5, G4: 1/5); Heart: focal minimal lymphocytic infiltration (Male: G1:1/5, G4:1/5); Aorta: focal minimal aneurysm (Male:G1:1/5, G4:1/5); Mandibular Lymph Node: focal moderate cystic dilation of cortex (Female: G4:1/5); Stomach: focal mild squamous epithelium hyperplasia (Female: G1: 1/5); Mesenteric lymph node: focal moderate cystic dilation of cortex (Female:G1:1/5); Spleen: focal to diffuse minimal to mild extramedullary hematopoesis (Female: G1: 2/5, G4: 3/5); Thymus: mild to moderate atrophy (Female: G1:3/5, G4:4/5); focal mild cystic epithelial dilation (Male: G4:1/5; Female: G1: 1/5, G4:1/5); Trachea: focal to multifocal minimal to moderate Neutrophilic/lymphocytic infiltration (Male: G1:3/5, G4:3/5; Female: G1: 2/5, G4:1/5); Adrenals: unilateral accessory adrenocortical tissue (Male: G1:1/5, G4:1/5); Testes: focal to multifocal minimal to mild retention of mature sperm (Male: G1:4/13, G2:8/13, G3:8/13, G4:8/13); focal minimal to mild degeneration of seminiferous tubules (Male: G1:2/13, G2:1/13, G3:1/13, G4:1/13); focal to multifocal minimal sloughing of Pachytene Spermatocyte (Male: G1:2/13, G2:2/13, G3:2/13, G4:2/13); focal minimal sloughing of round spermatid (Male: G1:1/13, G2:1/13, G3:1/13, G4:1/13); focal mild infiltration of multinucleated giant cells (Male: G1:1/13); Seminal Vesicles: multifocal mild neutrophilic/lymphocytic infiltration (Male: G1:1/13); Prostate: focal moderate necrotic debris in lumen (Male: G2:1/13); Uterus: multifocal to diffuse mild reduction of stromal cells (Female: G1:1/13; G4:2/13); focal moderate necrosis (Female: G3:1/13); multifocal mild to moderate nodular hyperplasia (Female: G1:1/13; G2:1/13; G4:1/13); Cervix: focal minimal lymphocytic infiltration (Female: G2:1/13).

Study 3: Microscopic examination of control group and rats treated at 250, 500 and 750 mg/kg revealed varying degree of pathological changes in different organs. This includes in Liver: focal to multifocal minimal to mild lymphocyte infiltration (Male: G1:2/5; Female: G1/5, G4:1/5), focal to multifocal mild degeneration (Male: G1:1/5, Female: G4/5), in Kidneys: focal mild tubular degeneration (Male: G1:2/5), focal mild lymphocyte infiltration (Female: G1/5); in Lungs: multifocal mild lymphocyte infiltration (Male: G1:1/5, G4:1/5; Adrenals: accessory adrenocortical tissue (Bilateral: Female: G4:1/5), in Testes: Male: multifocal mild cytoplasmic vacuolation at sertoli cell (Male: G1: 1/13), focal mild multinucleated giant cell infiltration (Male: G1: 1/13) focal mild seminiferous tubule degeneration (Male: G4: 2/13); focal minimal retention of mature sperm (Male: G1: 1/13, G2:1/13, G3: 1/13, G4: 1/13, G1-R: 1/5); focal minimal to mild sloughing of round spermatid (Male: G1: 1/13, G2:1/13). in Epididymis: Male: multifocal mild reduced sperm count (Male: G1: 1/13). Lesions observed in liver, kidneys, lungs, adrenals and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item.

Study 4: No histopathology lesions in the spleen, liver, kidneys. Testes, epididymides and adipose tissue.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Study 2: In control group G1 and treatment group G2 all the females showed regular cyclicity i.e. 3-5 days estrous cycle; while in group G3, 3 females and in group G4, 4 females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period. In cohabitation or mating period, all females from control and treated groups showed evidence of copulation i.e. sperm positive vaginal smear. Precoital Interval was calculated, all females showed precoital interval less than 5 days, except 1, 1 and 4 females from G1, G3 and G4, respectively which showed precoital interval more than 5 days.

Study 3: In control group G1 and treatment group G2 , G3 and G4 all the females showed regular cyclicity i.e. 3-5 days estrous cycle. However in group G1 two female, group G2 one female and group G4 one female showed prolong diestrus and found to be non-pregnant. In cohabitation or mating period, all females from control and treated groups showed evidence of copulation i.e. sperm positive vaginal smear. All females showed precoital interval less than 5 days, except 1, 2 and 1 females from G2, G3 and G4, respectively which showed precoital interval more than 5 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Study 2: no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Study 2: No statistically significant difference between the control (G1) and treatment groups (G2, G3 and G4) in the maternal parameters i.e. Gestational length, Litter size, No. of live births, Post-implantation loss and Post-natal loss were observed.

Study 3: There was no difference between the control (G1) and treatment groups (G2, G3 and G4) in the maternal and fetal parameters. Gestational length, Litter size, No. of live births, Post-implantation loss, pups weight at birth and PND14, Post-natal loss, Survival Index and weight gain for pups at PND13. Pregnancy index was found to be 84.62, 92.31, 100.00 and 92.31 in G1, G2, G3 and G4 respectively. Pups sex ratio (Male/Female) was found to be 61/57, 72/79, 80/61 and 71/56 at birth in G1, G2, G3 and G4 respectively and 56/53, 48/51, 64/54 and 69/56 at Day 4 in G1, G2, G3 and G4 respectively.

Details on results (P0)

Study 4: The growth of the testes was depressed .Males given the 0.32% diets for 90 days had lower absolute, testes/body weight ratio, and testes/brain weight ratio. Males given the 0.32% diets for 42 days had lower absolute and testes/brain weight ratio but comparable testes/body weight ratio. Degeneration of epididymal spermatozoa indicates a higher incidence of spermatogenic effects in the 0.32% dose group (8 of 10 rats after 42 days and 13 of 20 rats after 90 days). The mid dose group (1 of 10 ratsafter 42 days) and the control group (1 of 20 rats after 90 days) had single rats with testicular atrophy and degenerative epididymal spermatozoa. The 0.02% dose group had no animals with spermatogenic lesions.
Ovarian growth was not affected by exposure to any of the test diets.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
556 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
haematology
clinical biochemistry
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:
other: Not Specified

Target system / organ toxicity (P0)

Critical effects observed:
not specified
System:
other: Not Specified

Results: F1 generation

General toxicity (F1)

Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
Study 2: No statistically significant effect were observed on No. of live births and on PND 4 of pups.

Study 3: No effect on No. of live births were observed in treated pups as compared to control.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Study 2: no statistically significant effect were observed on pups weight at birth and PND4 as compared to control.

Study 3: No effect on pups weight at birth and PND14 were observed in treated pups as compared to control.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Study 2: At the end of treatment and recovery period, absolute and relative weight of organs of treated rats of either sex did not differ significantly except a significant increase in relative wieght of Adrenal of G4-R male group when compared to the respective control group rats.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Study 2: Terminally sacrificed pups of all treated groups did not reveal any lesion of pathological significance in any of the group when compared with control group. Pups died during course of study revealed various lesions among the control and treated groups viz., external examination emaciated carcass (Male: G1:2/55, G2:1/44, G3:5/35; Female: G1:3/56, G2:1/30, G3:6/54); Cannibalism (Male: G1:3/55, G3:2/35; Female: G1:2/56, G3:3/54); Tearing of Neck Muscle (Female: G3:1/54; G4:1/18) and internal examination absence of milk in stomach (Male: G1: 6/55, G2: 6/44, G3: 12/35, G4: 3/16; Female: G1: 8/56, G3: 14/54, G4: 2/18); blood clot in thoracic cavity (Male: G1: 2/55, G2: 3/44, G3: 1/35; Female: G1: 1/56, G3: 1/54, G4: 1/18); reddish discoloration of brain (Male: G1: 1/55, G2: 1/44, G3: 1/35; Female: G1: 1/56, G3: 3/54, G4: 1/18); reddish discoloration of lungs (Male: G1: 5/55, G2: 5/44, G3: 7/35, G4: 1/16; Female: G2: 1/30, G3: 10/54, G4: 2/18); paleness of liver (Male: G1: 1/55, G2: 2/44, G3: 1/35; Female: G3: 4/54, G4: 2/18); congested intestine (Female: G1: 1/56, G3: 1/54); autolytic changes (Female: G2: 1/30, G3: 2/54, G4: 1/18).

Study 3: Terminally sacrificed pups of all treated groups did not reveal any lesion of pathological significance in any of the group when compared with control group. Pups died during course of study revealed various lesions among the control and treated groups viz., external examination Cannibalism (Male: G1: 1/59, G2:13/60, G3: 7/81, Female:G1 :1/59, G2:14/81, G3:1/60, G4: 1/55 ; Tail absent (Anury) G3: 1/60 and internal examination Right skin swelling G1: 1/57.
Histopathological findings:
no effects observed
Description (incidence and severity):
Study 2: Microscopic examination of thyroid of male and female pups of control group and treated group did not revealed any lesion of pathological significance.
Other effects:
no effects observed
Description (incidence and severity):
Study 2: No effect on Litter size and Pups sex ratio were observed as compared to control.
Study 3: No effect on Litter size and Pups sex ratio were observed as compared to control.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
556 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Not Specified

Target system / organ toxicity (F1)

Critical effects observed:
not specified
System:
other: Not Specified

Overall reproductive toxicity

Reproductive effects observed:
not specified
Treatment related:
not specified

Any other information on results incl. tables

Study 2:

Mortality and Morbidity

Sex: Male

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Observation During Study Period

G1

Control

0

13

NMM

G2

Low

308

13

NMM

G3

Mid

556

13

NMM

G4

High

1000

13

NMM

G1-R

Control- Recovery

0

5

NMM

G4-R

High- Recovery

1000

5

NMM

 Sex: Female

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Observation During Study Period

G1

Control

0

13

NMM

G2

Low

308

13

NMM

G3

Mid

556

13

NMM

G4

High

1000

13

NMM

G1-R

Control -Recovery

0

5

NMM

G4-R

High- Recovery

1000

5

NMM

Keys:NMM = No mortality and morbidity observed, No.= Number

Clinical Signs and Symptoms

Sex: Male

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Clinical Sign

Incidences During Study period

G1

Control

0

13

Normal

13/13

G2

Low

308

13

Normal

13/13

G3

Mid

556

13

Normal

13/13

G4

High

1000

13

Normal

13/13

G1-R

Control -Recovery

0

5

Normal

5/5

G4-R

High- Recovery

1000

5

Normal

5/5

 Sex: Female

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

 

Clinical Sign

Incidences During Study period

G1

Control

0

13

Normal

13/13

G2

Low

308

13

Normal

13/13

G3

Mid

556

13

Normal

13/13

G4

High

1000

13

Normal

13/13

G1-R

Control -Recovery

0

5

Normal

5/5

G4-R

High- Recovery

1000

5

Normal

5/5

Key:No.= Number

Mean Body Weight (g)

Sex: Male

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose

(mg/kg b. wt.)

0

308

556

1000

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Day 1

247.38

11.20

262.38

20.32

261.62

18.86

261.92

17.03

Day 8

282.31

9.33

294.92

19.40

289.31

20.12

278.69

19.98

Day 14

315.00

13.00

324.38

21.35

316.15

20.45

299.15

21.37

Day 21

333.15

15.35

339.46

25.50

336.23

22.65

316.23

18.79

Day 28

350.08

18.06

362.62

30.37

358.54

27.49

334.15

20.60

Day 30

355.77

18.46

368.00

30.44

364.54

27.22

331.62

19.88

Day 37

370.77

22.36

381.92

30.46

381.77

31.58

351.62

24.13

Day 44

393.31

26.08

407.69

34.24

402.23

34.23

368.23

26.45

Day 46

397.23

24.79

414.77

34.07

405.38

34.10

370.92

26.71

Day 47

(Fasting)

372.00

25.57

391.08

33.83

383.38

33.84

350.15

26.15

 Period: Pre-mating                                                                                                    Sex: Female

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose

(mg/kg b. wt.)

0

308

556

1000

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Day 1

226.62

6.50

231.54

7.34

230.15

6.73

228.77

7.90

Day 8

229.85

8.37

233.46

7.85

233.54

9.00

227.92

7.39

Day 14

232.77

8.32

236.92

8.23

237.00

9.65

232.62

8.01

Keys:N = Number of animals in group, g= gram, SD = Standard deviation,↓= Statistically Significant Decrease (atp<0.05)

Mean Body Weight (g) Continued

Sex: Male

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

1000

Day

Mean

SD

Mean

SD

Day 1

261.40

19.99

258.20

13.33

Day 8

294.00

18.51

270.80

17.95

Day 15

326.80

21.25

297.20

19.99

Day 22

348.20

25.65

315.20

20.20

Day 29

370.60

28.03

 328.60

19.01

Day 36

391.60

26.41

 349.40

20.94

Day 41

398.20

28.67

 351.40

20.85

Day 48

415.40

33.16

372.60

26.82

Day 54

420.40

35.56

380.80

31.00

Day 55 (Fasting)

399.20

33.88

362.80

26.88

Sex: Female

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

1000

Day

Mean

SD

Mean

SD

Day 1

227.20

13.14

228.60

7.20

Day 8

233.80

11.95

227.80

9.78

Day 15

234.00

11.55

232.00

11.34

Day 22

236.00

10.93

234.00

10.37

Day 29

239.20

10.80

237.20

10.62

Day 36

240.80

11.61

238.60

11.84

Day 41

242.40

11.84

240.40

12.38

Day 48

243.40

11.87

243.00

13.62

Day 54

245.00

11.98

245.00

14.63

Day 55 (Fasting)

230.20

11.95

229.80

15.53

Keys:N= Number of animals in group, g= gram, SD= Standard deviation,↓= Statistically Significant Decrease (atp<0.05).

Mean Body Weight Change (%)

Sex:Male      

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose (mg/kg b. wt.)

0

308

556

1000

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Day 1-8

14.20

3.21

12.55

3.76

 10.70

4.73

6.40

 3.12

Day 1-14

27.53

7.02

23.94

7.71

21.13

7.89

14.28

5.24

Day 1-21

34.87

7.85

29.71

9.59

28.91

10.07

20.98

7.42

Day 1-28

41.73

8.95

38.68

12.93

37.52

12.61

27.88

8.78

Day 1-30

44.03

9.19

40.72

12.79

39.81

12.51

26.89

8.20

Day 1-37

50.16

11.39

46.09

13.55

46.47

14.70

34.67

11.44

Day 1-44

59.32

13.26

55.96

15.08

54.42

16.81

41.10

13.16

Day 1-46

60.85

12.16

58.69

15.39

55.61

16.53

42.13

13.26

 Period: Pre-mating                                                                                                Sex: Female

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose (mg/kg b. wt.)

0

308

556

1000

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Day 1-8

1.43

2.42

0.83

0.97

1.45

1.70

-0.36

1.27

Day 1-14

2.72

2.25

2.33

1.88

2.95

2.07

1.69

1.40

 Period: Gestation                                                                                                    Sex: Female

Group (N)

G1 (12)

G2 (11)

G3 (11)

G4 (8)

Dose (mg/kg b. wt.)

0

308

556

1000

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Day 0-7

5.23

3.64

5.76

2.32

5.79

2.87

2.60

2.31

Day 0-14

15.18

6.90

13.68

2.32

15.93

6.40

7.54

3.70

Day 0-20

29.44

11.73

28.10

6.90

30.75

11.02

14.52

5.81

Keys:N = Number of animals in group, g= gram, SD = Standard deviation,↓= Statistically Significant Decrease (atp<0.05)

Mean Body Weight Change (%) Continued

 Period: Post-Partum                                                                                                             Sex: Female

Group (N)

G1 (12)

G2 (11)

G3 (11)

G4 (8)

Dose (mg/kg b. wt.)

0

308

556

1000

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Day 0/1-4

0.82

3.33

-1.21

3.07

1.52

3.21

-1.33

3.03

   Sex: Male

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

1000

Day

Mean

SD

Mean

SD

Day 1-8

12.58

3.07

4.83

2.30

Day 1-15

25.26

7.48

15.04

2.48

Day 1-22

33.51

10.23

22.03

3.05

Day 1-29

42.19

12.51

27.31

5.11

Day 1-36

50.34

13.50

35.37

6.14

Day 1-41

52.96

15.34

36.14

5.92

Day 1-48

59.63

17.61

44.33

8.14

Day 1-54

61.54

18.41

47.46

9.14

Keys:N = Number of animals in group, g= gram, SD = Standard deviation,↓= Statistically Significant Decrease (atp<0.05)

Mean Hematology Data Continued

 

Group (N)

G1-R (5)

G4-R (5)

G1-R (5)

G4-R (5)

Sex

Male

Female

Dose (mg/kg body weight)

0

1000

0

1000

Parameter↓

Mean

SD

Mean

SD

Mean

SD

Mean

SD

RBC x 106(µl)

8.70

0.07

7.81

0.46

7.83

0.34

8.29

0.22

HCT( %)

39.38

0.38

35.80

2.10

34.90

0.96

37.10

1.09

MCV (µm3 

45.26

0.73

45.80

0.98

44.56

1.21

44.68

0.73

HGB (g/dl)

15.40

0.16

14.10

0.73

13.88

0.36

14.54

0.42

MCH (pg)

17.70

0.25

18.06

0.52

17.70

0.51

17.50

0.19

MCHC (g/dl)

39.14

0.25

39.38

0.40

39.78

0.16

39.24

0.39

Platelet x 103(µl)

560.80

53.19

579.20

40.11

590.20

26.76

644.20

131.73

WBC x 103(µl)

9.20

1.06

7.10

1.61

5.80

1.49

5.50

3.20

Neutrophil (%)

17.60

2.30

14.80

3.27

14.80

4.44

14.60

2.88

Lymphocyte (%)

81.80

1.92

84.40

3.58

84.20

3.49

84.60

2.41

Monocyte (%)

0.00

0.00

0.20

0.45

0.00

0.00

0.00

0.00

Eosinophil (%)

0.60

0.55

0.60

0.55

1.00

1.00

0.80

0.84

Basophil (%)

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

PT (Sec.)

22.51

5.59

22.48

2.01

25.19

13.02

29.11

6.84

aPTT (Sec.)

25.24

12.42

29.00

9.25

20.68

18.28

34.71

4.80

Keys: SD = Standard deviation, N = Number of animals in group and NAD = No Abnormality Detected,µm3=cubic micrometer,g/dl= gram per decilitre, pg= picogram, µl= microlitre, Sec= second,↓ =statisticalsignificant decrease at 95% level of significance,↑= statisticalsignificant increase at 95% level of significance.

Mean Gestational Length

Group(N)

G1(12)

G2(11)

G3(11)

G4(8)

Dose(mg/kg bwt)

0

308

556

1000

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Gestation Length

22.17

0.39

22.55

0.82

22.00

0.45

22.75

0.89

Keys:SD= Standard Deviation, N= number of dams in a group

Mean Litter size

Group(N)

G1(12)

G2(9)

G3(11)

G4(7)

Dose(mg/kg bwt)

0

308

556

1000

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Litter size

9.33

3.37

8.22

3.35

9.18

1.72

6.71

2.36

Keys:SD= Standard Deviation, N= number of dams in a group

Mean Post-Implantation Loss (%), Post-natal Loss (%) and Pups Survival Index (%)

Group(N)

G1(12)

G2(9)

G3(11)

G4(7)

Dose(mg/kg bwt)

0

308

556

1000

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

No. of Live Births

9.33

3.37

8.22

3.35

9.18

1.72

6.71

2.36

Post-Implantation Loss

0.16

0.27

0.04

0.07

0.13

0.19

0.36

0.13

No. of alive pups at Post-natal Day 4

7.5

3.03

6.89

2.93

4.73

3.10

3.71

2.29

Post-natal Loss (%)

16.42

19.90

12.22

22.53

49.07

31.15

42.86

30.50

Fetal Survival Index at Post-natal Day 4 (%)

83.58

19.90

87.78

22.53

50.93

31.15

57.14

30.50

Keys:SD= Standard Deviation, N= number of dams in a grou

Summary of Days of Conception and Pregnancy Index (%)

Group/N

G1(13)

G2(13)

G3(13)

G4(13)

Dose (mg/kg b.wt.)

0

308

556

1000

No of females showing evidence of copulation

13

13

13

13

No of females concieving between Days 1-5 of cohabitation

12

13

12

9

No. of females concieving after Day 5 of cohabitation

1

0

1

4

Females achieving pregnancy

12

11

11

8

Pregnancy Index (%)

92.31

84.62

84.62

61.54

Key:N= number of dams in a group

 

Study 3:

Mortality and Morbidity

Sex: Male

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Observation During Study Period

G1

Control

0

13

NMM

G2

Low

250

13

NMM

G3

Mid

500

13

NMM

G4

High

750

13

NMM

G1-R

Control- Recovery

0

5

NMM

G4-R

High- Recovery

750

5

NMM

 Sex: Female

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Observation During Study Period

G1

Control

0

13

NMM

G2

Low

250

13

NMM

G3

Mid

500

13

NMM

G4

High

750

13

NMM

G1-R

Control -Recovery

0

5

NMM

G4-R

High- Recovery

750

5

NMM

Keys:NMM = No mortality and morbidity observed, No.= Number

Mean Body Weight (g)

Sex: Male

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose

(mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

1

328.62

22.46

330.46

21.52

327.92

20.07

329.08

16.28

8

343.08

26.03

348.77

26.14

350.46

24.55

350.00

20.15

14

351.62

27.09

362.92

32.04

370.00

33.60

367.46

26.28

21

364.31

26.48

380.31

33.72

385.38

39.58

379.23

30.18

28

382.77

28.60

398.77

39.39

402.62

42.57

399.00

32.01

35

397.85

31.58

407.62

37.91

412.23

44.44

407.38

32.29

40

401.15

32.82

394.92

53.26

419.54

48.74

414.69

30.05

41{fasting}

385.46

31.17

389.62

35.03

399.23

47.68

394.23

31.15

 Period: Pre-mating                                                                                                    Sex: Female

Group (N)

G1

G2

G3

G4

Dose

(mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

1

247.54

16.31

13

245.00

11.95

13

248.23

14.22

13

247.92

14.28

13

8

251.62

16.29

13

252.00

14.93

13

254.38

16.09

13

255.77

13.40

13

14

258.15

18.38

13

259.62

17.47

13

263.62

20.39

13

265.38

16.00

13

21

267.50

14.85

2

244.00

18.38

2

274.00

12.73

2

289.00

18.38

2

28

288.00

29.70

2

276.00

./.

1

./.

./.

0

295.00

./.

1

35

309.50

28.99

2

300.00

./.

1

./.

./.

0

309.00

./.

1

42

295.00

22.63

2

319.00

./.

1

./.

./.

0

307.00

./.

1

49

282.50

34.65

2

334.00

./.

1

./.

./.

0

306.00

./.

1

56

286.50

30.41

2

328.00

./.

1

./.

./.

0

303.00

./.

1

58

280.00

29.70

2

329.00

./.

1

./.

./.

0

326.00

./.

1

59(Fasting)

272.00

28.28

2

321.00

./.

1

./.

./.

0

312.00

./.

1

Period: Gestation                                                                                                  Sex: Female

Group

G1

G2

G3

G4

Dose

(mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

0

260.36

19.82

11

264.08

16.89

12

370.00

21.68

13

268.67

18.81

12

7

279.45

18.84

11

282.00

18.92

12

284.69

22.29

13

284.83

19.40

12

14

311.64

22.08

11

308.92

23.12

12

312.31

27.62

13

311.83

22.23

12

20

373.91

25.68

11

368.58

23.13

12

368.46

36.03

13

363.67

25.82

12

 Period: Post-Partum                                                                                                         Sex: Female

Group (N)

G1 (11)

G2 (12)

G3 (13)

G4 (12)

Dose

(mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

0

298.55

28.22

297.42

17.10

306.54

26.49

294.58

21.90

4

303.09

27.13

291.25

20.91

299.38

19.49

296.58

19.46

13

313.91

33.40

297.58

21.47

307.92

23.06

304.50

21.45

14(Fasting)

285.18

25.27

272.00

16.04

282.08

19.95

272.25

18.11

Sex: Male

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

750

Day

Mean

SD

Mean

SD

1

335.60

26.19

334.00

27.00

8

354.60

29.82

358.60

25.36

14

361.40

33.92

371.80

16.83

21

378.00

32.30

393.40

21.48

28

395.00

37.29

412.00

30.81

35

407.20

38.26

419.60

33.00

42

416.60

45.28

427.00

33.85

49

429.80

46.66

446.40

30.76

56

434.60

43.55

447.40

33.76

63

435.80

45.88

452.60

33.81

66

441.40

46.76

457.20

37.34

67(fasting)

421.20

45.17

436.60

34.41

Sex: Female

Group (N)

G1-R (5)

G4-R (5)

Dose (mg/kg b. wt.)

0

750

Day

Mean

SD

Mean

SD

1

246.60

22.73

248.60

20.13

8

255.20

19.20

257.60

23.44

14

263.20

19.68

269.60

19.63

21

270.40

20.32

276.40

22.50

28

275.60

19.93

280.00

27.96

35

279.80

20.27

278.80

25.94

42

287.00

22.44

283.80

22.70

49

292.60

25.86

287.60

23.80

56

287.80

25.97

288.00

25.56

63

291.20

26.33

287.00

23.79

66

291.00

25.66

287.40

23.04

67(fasting)

275.40

26.02

272.80

25.12

Sex:Male      

Group (N)

G1 (13)

G2 (13)

G3 (13)

G4 (13)

Dose (mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

(1-8)

4.37

2.19

5.50

2.43

6.87

3.52

6.34

2.59

(1-15)

7.01

4.39

9.72

4.29

12.70↑

4.92

11.61

4.42

(1-28)

10.87

3.42

15.02

5.90

17.30↑

6.28

15.13

4.96

(1-29)

16.54

5.36

20.54

6.86

22.54

7.07

21.13

5.48

(1-35)

21.12

6.19

23.28

7.21

25.45

7.17

23.72

6.20

(1-40)

22.11

6.39

19.45

17.33

27.61

8.21

25.98

5.72

(1-41)

17.33

5.86

17.77

7.65

21.41

8.18

19.74

6.12

Period: Pre-mating                                                                                                Sex: Female

Group (N)

G1

G2

G3

G4

Dose (mg/kg        b. wt.)

0

250

500

750

Day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

(1-8)

1.66

1.31

13

2.83

2.41

13

2.48

2.86

13

3.20

1.19

13

(1-15)

4.28

2.24

13

5.90

3.15

13

6.10

2.68

13

7.05

2.45

13

(1-21)

9.59

2.27

2

6.02

5.39

2

9.57

2.61

2

10.53

0.44

2

(1-28)

17.89

8.07

2

17.95

./.

1

./.

./.

1

18.47

./.

1

(1-35)

26.71

7.48

2

28.21

./.

1

./.

./.

1

24.10

./.

1

(1-42)

20.81

5.07

2

36.32

./.

1

./.

./.

1

23.29

./.

1

(1-49)

15.60

10.18

2

42.74

./.

1

./.

./.

1

22.89

./.

1

(1-56)

17.27

8.38

2

40.17

./.

1

./.

./.

1

21.69

./.

1

(1-59)

14.61

8.19

2

40.60

./.

1

./.

./.

1

30.92

./.

1

Period: Gestation                                                                                                  Sex: Female

Group

G1

G2

G3

G4

Dose

(mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

(0-7)

7.43

3.09

11

6.78

2.19

12

5.85

2.40

13

6.08

3.55

12

(0-14)

19.85

5.33

11

16.97

4.42

12

16.04

3.47

13

16.17

5.54

12

(0-20)

43.90

8.24

11

39.63

3.82

12

36.88

6.74

13

35.58

8.39

12

 Period: Post-Partum                                                                                                         Sex: Female

Group (N)

G1 (11)

G2 (12)

G3 (13)

G4 (12)

Dose

(mg/kg b. wt.)

0

250

500

750

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

(0-4)

-4.32

4.81

-8.47

4.07

-7.73

5.39

-7.34

6.33

(0-13)

5.29

7.73

0.04

4.14

0.72

6.21

3.61

7.07

(0-14)

-4.32

4.81

-8.47

4.07

-7.73

5.39

-7.34

6.33

Summary of Days of Conception and Pregnancy Index (%)

Group(N)

G1(13)

G2(13)

G3(13)

G4(13)

Dose (mg/kg b.wt.)

0

250

500

750

No. of females showed evidence of copulation

11

12

13

12

No. of females concieved between Days 1-5 of cohabitation

11

11

11

11

No. of females concieved after Day 5 of cohabitation

0

1

2

1

Females achieved pregnancy

11

12

13

12

Pregnancy Index (%)

84.62

92.31

100.00

92.31

Key:N= number of dams in a group, No. = Number

Post-natal Loss (%) and Pups Survival Index (%)

Group(N)

G1(11)

G2(12)

G3(13)

G4(12)

Dose(mg/kg bwt)

0

250

500

750

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

No. of Live Births

11.80

1.93

12.00

2.95

9.85

3.60

10.58

1.68

No. of alive pups at Post-natal Day 4

10.90

3.00

8.33

5.57

9.08

4.37

10.42

1.51

Post-natal Loss (%)

7.85

19.84

33.02

38.93

18.72

37.26

1.28

4.44

Fetal Survival Index at Post-natal Day 4 (%)

92.15

19.84

66.98

38.93

81.28

37.26

98.72

4.44

Mean Gestational Length and Litter size

Group(N)

G1(11)

G2(12)

G3(13)

G4(12)

Dose(mg/kg bwt)

0

250

500

750

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Gestation Length

22.36

0.50

22.00

0.00

22.00

0.00

22.25

0.45

Litter size

(Total No. of litter size)

10.91

3.48

12.67

2.90

10.85

2.61

10.58

1.68

Mean Pups Body Weight, Sex Ratio and Gross Observation

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean Pups Weight

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Day 0

6.34

0.57

118

6.10

0.76

151

6.24

0.86

141

6.59

0.48

127

Day 4

9.76

1.78

109

8.56

1.12

100

9.11

1.08

118

9.50

1.45

125

Day 13

24.88

3.88

87

20.70

3.00

78

22.74

2.65

86

22.05

2.90

100

Group(n)

G1(11)

G2(12)

G3(13)

G4(13)

Pups Body Weight gain

(%)

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Day 0-Day 4

47.42

20.65

109

36.29

13.99

99

41.58

13.79

118

43.66

15.12

125

Day 0-Day 13

148.89

19.84

87

143.22

25.24

78

144.89

19.45

86

135.74

22.14

100

Group

(Number of Litter size)

G1(118)

G2(151)

G3(141)

G4(127)

Sex Ratio at birth

(Male/Female)

61/57

72/79

80/61

71/56

Sex Ratio at Day 4

(Male/Female)

56/53

48/51

64/54

69/56

Gross Observations

NAD

NAD

NAD

NAD

Hormonal Analysis Data

Sex: Male (Termination)

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

4.62

0.79

13

4.34

0.96

13

4.63

0.75

13

4.18

0.73

13

TSH (uIU/mL)

3.48

2.05

13

4.44

2.52

13

3.31

1.59

13

3.97

1.62

13

Testosterone (ng/dL)

71.85

77.91

13

115.22

96.43

13

99.50

118.60

13

99.75

112.05

13

                  Sex: Female (Day 4)

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

3.54

0.56

11

3.27

0.63

12

3.60

0.74

13

3.46

0.48

12

TSH (uIU/mL)

3.10

1.31

11

3.38

1.58

12

2.69

1.41

13

3.21

1.67

12

                                                                                        Sex: Female (Day 13)

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

3.48

0.60

11

3.28

0.69

12

3.30

0.50

13

3.04

0.31

12

TSH (uIU/mL)

2.45

2.09

11

3.25

1.84

12

3.23

2.71

13

2.21

1.32

12

E2 (pg/mL)

 

33.13

11.94

11

41.09

18.51

12

29.94

15.08

13

25.25

8.02

12

Sex: Female (Non-Pregnenat)

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

5.6

3.39

2

3.90

./.

1

./.

./.

0

3.90

./.

1

TSH (uIU/mL)

5.45

1.15

2

2.62

./.

1

./.

./.

0

2.50

./.

1

E2 (pg/mL)

 

36.4

2.50

2

56.37

./.

1

./.

./.

0

39.28

./.

1

 

Sex: Male (Termination)

 

Group

G1R

G4R

Dose(mg/kg bwt)

0

750

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

2.98

0.63

5

4.76

2.18

5

TSH (uIU/mL)

5.03

1.73

5

3.91

1.65

5

Testosterone (ng/dL)

54.67

37.03

5

160.34

183.40

5

 

Sex: Female (Termination)

 

Group

G1-R

G4-R

Dose(mg/kg bwt)

0

750

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

2.82

0.11

5

2.72

0.43

5

TSH (uIU/mL)

4.69

4.74

5

2.82

2.74

5

Testosterone (ng/dL)

45.64

33.01

5

24.04

21.81

5

Day: 04 (Pups)

 

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

2.17

0.29

10

2.04

0.42

11

2.37

0.49

11

2.13

0.31

12

TSH (uIU/mL)

1.76

0.32

10

1.84

0.51

11

1.84

0.81

11

1.82

0.56

12

 

Day: 13 (Pups)

 

Group(n)

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

750

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4 (ug/dL)

5.87

0.88

9

5.08

0.60

9

5.60

0.70

10

5.64

0.82

12

TSH (uIU/mL)

2.16

1.13

9

1.88

0.49

9

2.05

0.60

10

2.14

0.69

12


Applicant's summary and conclusion

Conclusions:
Study 2: No Observed Adverse Effect Level (NOAEL) is considered to be 556 mg/kg body weight when Wistar male and female rats were orally treated with the test chemical.

Study 3: NOAEL was considered to be 750 mg/kg bw when Wistar male and female Rats were treated with test chemical orally by gavage for more the 63 days.

Study 4: No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 46 mg/kg/day for male and 51mg/kg/day were considered to be the NOAEL for female. Whenmale and female rats were treated with test material orally.
Executive summary:

Reproductive Toxicity Study:

The summaries of the data of the studies are as follows:

Study 2:

In a reproductive toxicity study, Wistar male and female rats were treated with the test chemical in the concentration of 0, 308, 556 and 1000 mg/kg bw orally by gavage in Corn oil for 63 days. No mortality or morbidity and apparent treatment related clinical signs were observed any of the groups of animals throughout the study period. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period. Number of rear, urine pools, fecal bolus in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. Body weight, percent body weight changes and feed consumption in animals of all the test groups of both the sexes was comparable and did not show any treatment related significant difference as compared to the respective control groups. The sensory reactivity measurements, Foot splay, fore limb and hind limb grip strength parameters and Motor activity were comparable and no treatment related changes were revealed in any of the animals of all treated groups as compare to the repective control groups. Similarly, no test item related changes in estrous cyclicity and precoital interval were observed. There was statistically significant decrease in G3 (556 mg/kg body weight) as compared to control G1 (0 mg/kg body weight). This is not dose dependent hence not considered as treatment related. There was no statistically significant difference between the control and treatment groups in the maternal and pups parameters, except markedly decreased pregnancy index / fertility index in G4 (1000 mg/kg body weight), which was considered to be treatment related. No treatment related changes were observed inhematological and clinical chemistry parameters of treated male and female rats as compared to control. In addition, no significant change in organ weight, External and visceral examination of treated and recovery groups as compared to control. Pups that died among the control and treated groups during the course of study, revealed various lesions when examined externally and internally but the observations were not considered treatment related. Emaciated carcass, Cannibalism; Tearing of Neck Muscle and internal examination Absence of milk in stomach, Thoracic cavity: Blood clot present, Reddish discoloration of brain, Reddish discoloration of lungs, Paleness of liver, Congested intestine and Autolytic changes were observed all the treated groups.Focal to multifocal minimal lymphocytic infiltration of male and female and focal minimal necrosis in male in liver, focal minimal lymphocytic infiltration of male and female and focal mild mineralization in female in kidney, multifocal minimal lymphocytic infiltration in male and female andfocal minimal histiocyte infiltration in female lungs, focal minimal lymphocytic infiltration in heart of male, focal minimal aneurysm in aorta of male, focal moderate cystic dilationof cortex of Mandibular Lymph Node in female,focal mildsquamous epitheliumhyperplasia stomach of female, focal moderate cystic dilation of cortex in Mesenteric lymph node, focalto diffuse minimal to mild extramedullary hematopoesis in spleen and mild to moderate atrophy in Thymus of female, focal to multifocal minimal to moderate Neutrophilic/lymphocytic infiltration of Trachea of male and female, unilateral accessory adrenocortical tissue of Adrenals and focal to multifocal minimal to mildretention of mature sperm, focal minimal to mild degeneration of seminiferous tubules,focal to multifocal minimalsloughing of Pachytene Spermatocyte,focal minimal sloughing of round spermatid andfocal mildinfiltration of multinucleated giant cells of Testes, multifocal mild neutrophilic/ lymphocytic infiltration of Seminal Vesicles and focal moderate necrotic debris in lumen of Prostate observed in male rats, multifocal to diffuse mild reduction of stromal cells, focal moderate necrosis, multifocal mild to moderate nodular hyperplasia of Uterus and focal minimal lymphocytic infiltration of Cervix in female rats were observed. Therefore, No Observed Adverse Effect Level (NOAEL) is considered to be 556 mg/kg body weight when Wistar male and female rats were orally treated with the test chemical.

Study 3:

In a experimental study conducted, the Wistar male and female rat treated with test chemical in the concentration of 0, 250, 500 and 750 mg/ kg bw orally by gavage for more than 63 days. No mortality or morbidity was observed in any animals of the control and treatment groups throughout the study period. No apparent treatment related clinical signs were observed in any of the animals throughout the treatment. Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period. Statistically significant increase in female was observed in urine pool at Week 5 in 750 mg/kg bw as compared to control. The above changes observed were inconsistent/ biologically insignificant and not dose dependent. Statistically significant decrease was observed in percent body weight change on day 1-15 and day 1-28 at 500 mg/kg bw as compared to control group. Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. No treatment related changes in food consumption were observed in treated male and female rats as compared to control. Similarly, at the end of treatment period revealed statistically significant decreased was noted in WBC of females (R) at 750 mg/kg body weight. The observed variation in WBC was considered to be of no toxicological significance. At the end of treatment period revealed, statistically significant decrease were noted in Triglyceride in Male at 500 mg/kg body weight, Calcium in Female at 750 mg/kg body weight. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes. During treatment-free recovery period revealed statistically significant increase was noted in cholesterol in Female at 750 mg/kg body weight, A: G ratio, in Male and Female (R) at 750 mg/kg body weight while statistical significant decrease were noted in ALT and Globulin in Female at (R) 750 mg/kg body weight. The observed variations in Triglyceride, Calcium, Cholesterol, ALT, Globulin and A: G ratio were considered to be of no toxicological significance. The functional observation battery/neurobehavioral observation were comparable and no changes were revealed in any of the animals of all the treated groups in both the sexes. The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups. However in recovery male, statistically significant increase was noted in grip strength (hindlimb) in 750 mg/kg bw (R) when compare to control recovery group. Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group. Howerver, statistically significant increase in female were noted in Distance travelled (DT), Ambulatory time (AT), and in Horizontal counts (HC) at 750 mg/kg body weight when compare to control group. The above changes observed were not dose dependent, hence considered as incidental and not attributed to the effect of test item administration. In addition, At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of either sex did not differ significantly when compared to respective control group except increased splenic absolute and relative to brain weight in female rats treated at 500 mg/kg, decreased splenic weight in male rats treated at 750 mg/kg during recovery and increased weight of adrenal relative to brain weight in male rats treated at 500 mg/kg with respective control group. Observed weight variations in the organs did not showed dose dependency and are minor in nature, so could be considered as spontaneous. External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality.

Microscopic examination of control group and rats treated with 250, 500 and 750 mg/kg revealed varying degree of pathological changes in different organs. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item. No effects on reproductive parameters were observed such as regular cyclicity i.e. 3-5 days estrous cycle. However in group G1 two female, group G2 one female and group G4 one female showed prolong diestrus and found to be non-pregnant. In cohabitation or mating period, all females from control and treated groups showed evidence of copulation i.e. sperm positive vaginal smear. All females showed precoital interval less than 5 days, except 1, 2 and 1 female from G2, G3 and G4, respectively which showed precoital interval more than 5 days. There was no difference between the control (G1) and treatment groups (G2, G3 and G4) in the maternal and fetal parameters. Gestational length, Litter size, No. of live births, Postimplantation loss, pups weight at birth and PND14, Post-natal loss, Survival Index and weight gain for pups at PND13. Pregnancy index and Pups sex ratio (Male/Female) respectively of treated rats as compared to control. No treatment related changes were noted in hormonal analysis (T4, TSH, Testosterone and Estrogen). Therefore, NOAEL was considered to be 750 mg/kg bw when Wistar male and female Rats were treated with test chemical orally by gavage for more the 63 days.

Study 4:

The reproductive toxicity study of test material was performed on male and femaleCRL: COBS 'CD '(SD)BR rats. The test material soluble in corn oil and themixture was combined with laboratory rodent chow. The Corn oil was added to all diets at a concentration of 1.0%.Estimated consumption was0, 0.02%, 0.08%,0.32 %( 9, 46, or 214 mg/kg/day for male , 10,51 or 239 mg/kg/day for female ) for 90 day. The dose levels were selected on the basis of a 12 day feeding study in which rats were fed diets of 1.0, 0.1 or 0.0% test material .Thirty males and thirty females were exposed to each dietary concentration. Ten rats of each sex at each concentration were killed approximately half-way through' the study (42 days interim groups) and twenty rats of each sex were killed after approximately 90 days. Body weights were determined on days 0, 4, 7, and weekly thereafter. Feed consumption was determined on days 4, 7, and twice weekly thereafter. Necropsies were conducted according to pathology SOP TP 180. liver, kidneys, spleen, heart, adrenal glands, ovaries, testes, and brain. Paired organs were weighed together. Organ/body weight and organ/brain weight ratios were calculated. No mortality was observed.Red and blue discoloration of the urine under the cages of all male and female rats fed the 0.32% diets. The abnormality was seen as distinct patches of red or blue urine stained paper under the cages.All groups of animals gained weight although the 0.08% and 0.32% diets clearly retarded weight gain for both males and females. The 0.02% diet had no effect on body weight gain.The 0.08% and 0.32% diets reduced (p <0.05) feed consumption for both male and female rats, although not for every timeperiod. Feed consumption was reduced the most at the introduction of the testdiets as reflected in the data for day 4. Reduction of feed consumption wasalso greater for males than for females. Diets of 0.02%dose group generallydid not alter feed consumption with fewexceptions. These exceptions(p<0.05)included decreased feed consumptionmeasured on day 73 for males and increased feed consumption for females ondays 35, 39 (90-day group), 59, 63, and 87. Ovarian growth was not affected by exposure to any of the test diets.The only target organ effects which are apparent involve enlargement of the spleen (0.08% and 0.32% diets) and atrophy of the testes (0.32% diets). The only statistically significant effects in rats given diets of 0.02% were organ to body weight ratios for the liver (90 day), brain (42 day), and adrenal gland (42 day) for females. None of the differences in rats given the 0.02% diets were of sufficient magnitude or consistency to be considered toxicologically significant.Red blood cell toxicity wascharacterized as a macrocytic, very slightly hyperchromatic anemia with Heinzand Howell-Jolly bodies and secondary lesions in the spleen, liver, and kidneydue to increased hemoglobin catabolism and increased hematopoiesis. Theseverity of red blood cell damage was dose related and was detectable at thelowest dose 9-10 mg/kg/day for 90 days (0.02% diet).Nohistopathologic lesions in the spleen, liver, kidneys. Testes, epididymides and adipose tissue. The growth of the testes was depressed .Males given the 0.32% diets for 90 days had lower absolute, testes/body weight ratio, and testes/brain weight ratio. Degeneration of epididymal spermatozoa indicates a higher incidence of spermatogenic effects in the 0.32% dose group (8 of 10 rats after 42 days and 13 of 20 rats after 90 days). The mid dose group (1 of 10 rats after 42 days) and the control group (1 of 20 rats after 90 days) had single rats with testicular atrophy and degenerative epididymal spermatozoa. The 0.02% dose group had no animals with spermatogenic lesions. Hence, No Observed Adverse Effect Level (NOAEL) for male was considered to be 46 mg/kg/day and for female NOAEL was considered to be the 51 mg/kg/day on the basis of effects observed on reproductive organ, when male and female rats were treated with test material orally.