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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl phenylacetate
EC Number:
202-940-9
EC Name:
Methyl phenylacetate
Cas Number:
101-41-7
Molecular formula:
C9H10O2
IUPAC Name:
methyl phenylacetate
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): methyl phenylacetate
- Molecular Formula: C9H10O2
- Molecular Weight: 150.176 g/mol
- Substance type: Organic
- Physical state: Liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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: 240 g; Maximum: 315 g
Female: Minimum: 210 g; 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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral 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:
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
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
308 mg/kg bw/day (actual dose received)
Dose / conc.:
556 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Total: 124
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:
- 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

Examinations

Observations and examinations performed and frequency:
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:

OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just prior to necropsy.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight (approximately 16-18 hr) prior to blood collection
- How many animals: five males and five females, randomly selected from each group
- Parameters checked in table [No.?] 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, overnight (approximately 16-18 hr) prior to blood collection
- How many animals:five males and five females, randomly selected from each group
- Parameters checked in table [No.?] 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) – Calculated, Globulin (Glob) - Calculated, Alb/ Glb (A:G) – Calculated and Bile acidswere examined.

URINALYSIS: Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the last week of treatment and that of recovery groups, in the last week of recovery period.
- Dose groups that were examined:All dose groups were examined.
- Battery of functions tested: sensory activity / grip strength / motor activity / other:Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted

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

OTHER:
Organ weight were examined.
Weighing of brain, adrenals, ovaries with oviduct, testes, epididymides, heart, liver, kidneys, thymus and spleen was performed for randomly selected 5 male and 5 female rats. Testes and epididymides of all male rats were weighed.
Sacrifice and pathology:
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.

HISTOPATHOLOGY: Yes, All the preserved organs (Testes, epididymides, prostate and seminal vesicle with coagulating glands, ovaries, uterus and cervix with vagina) of all the rats, all the preserved tissues of randomly selected five male and five female rats of groups G1 and G4 and preserved thyroid of one male and one female pup of each litter were subjected to histopathological examination. All the tissues were trimmed, processed, embedded in paraffin wax. Sections were cut at a thickness of 3-5 micron and stained with hematoxylin and eosin stain.
Processed tissues were subjected to histopathological examination. The prepared slides were examined under microscope by the Pathologist to note histopathological lesions, if any in different organs. Special attention was paid to observe effect of test item on reproductive system and spermatogenesis.
Other examinations:
Number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), body weight and the presence of gross abnormalities.
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,

Results and discussion

Results of examinations

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.
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 (J. Robinson and G.O.Evans, 2005).
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:
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:
effects observed, non-treatment-related
Description (incidence and severity):
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
Neuropathological findings:
not specified
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:
no effects observed
Description (incidence and severity):
No effect were observed on Number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), body weight and gross abnormalities of treated pups as compared to control.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
other: No effect observed
Remarks on result:
other: No toxic potential was observed

Target system / organ toxicity

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified

Any other information on results incl. tables

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

 


Applicant's summary and conclusion

Conclusions:
Based on all the observations and results, itwas concluded that the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg / kg body weight when Wistar male and female rats were orally treated with test chemical.
Executive summary:

In a combined repeated and reproductive developmental 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 treatment related changes were observed inhematological and clinical chemistry parameters of treated male and female rats as compared to control.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.In addition, no significant change in organ weight,External and visceral examination of treated and recovery groups as compared to control.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 milddegeneration of seminiferous tubules,focal to multifocal minimalsloughing of Pachytene Spermatocyte,focal minimalsloughing 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. Lesions observed in liver, kidneys, lungs, heart, aorta, stomach, lymph nodes, spleen, thymus, trachea, adrenal gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship Therefore, No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg / kg body weight when Wistar male and female rats were orally treated with the test chemical.