Registration Dossier

Administrative data

Endpoint:
short-term repeated dose 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:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
To assess toxicological profile of test chemical to determine target organ of toxicity, its reversibility in the rat after 28 consecutive days of oral administration.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material : decan-5-olide- Substance type: Organic- Physical state: Liquid
Specific details on test material used for the study:
- Name of test material : decan-5-olide
- Substance type: Organic
- Physical state: Liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:National Institute of Biosciences
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation:6 to 8 weeks old
- Weight at study initiation: Male: 145.6-186.4 g; Female: 124.7-154.0 g
- Fasting period before study:
- Housing: The rats were housed in polycarbonate cages with paddy as bedding.
After allocation to respective dose groups rats were housed 2/sex/cage.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences was provided ad libitum from individual feeders on cage top.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. Water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period:5 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19.8 °C to 23.1 °C
- Humidity (%):48.5% to 60.4%
- Air changes (per hr): at least ten air changes per hour of 100% fresh air that has been passed through the HEPA filters.
- Photoperiod (hrs dark / hrs light):An artificial light and dark cycle of 12 hours each was provided to the room.

IN-LIFE DATES:
From: 25-10-2017
To:16 February 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:The test item was diluted with corn oil for preparation of solution(s).
The solution(s) oftest chemical were made at volumes suitable for daily use for 28 days. The solution(s) were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500 and 1000 mg/kg body weight, respectively, were administered.

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
- Concentration in vehicle: 0, 250, 500 and 1000 mg/kg body weight
- Amount of vehicle (if gavage): 10 ml/kg body weight
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
concentration and stability were analyzed
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle (Reversal)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Test Item (Reversal)
No. of animals per sex per dose:
Total: 72
0 mg/kg bw/day: 6 male ,6 female
0 mg/kg bw/day (Reversal): 6 male ,6 female
250 mg/kg bw/day: 6 male ,6 female
500 mg/kg bw/day: 6 male ,6 female
1000 mg/kg bw/day: 6 male ,6 female
1000 mg/kg bw/day (Reversal):: 6 male ,6 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): The animals of uniform body weight were selected.
- Rationale for selecting satellite groups: 6 animals /sex /group for reversal group.
- Post-exposure recovery period in satellite groups: 2 week recovery period
- Section schedule rationale (if not random):

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:twice daily
- Cage side observations checked in table [No.?] were included. Viability were observed

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:daily

BODY WEIGHT: Yes
- Time schedule for examinations:Body weights were recorded on the day of randomization, day of first dosing, weekly thereafter and a fasting body weight at scheduled sacrifice on day 29 and day 43.

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

FOOD EFFICIENCY:Not specified
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the initiation of the dosing and at scheduled sacrifice.
- Dose groups that were examined: All dose groups were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:At the end of dosing period on day 29 and at termination of recovery period on day 43.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, overnight
- How many animals: All 72 animals were examined.
- Parameters checked in table [No.?] were examined:Hemoglobin, Red Blood Corpuscles, Hematocri, Mean Corpuscular Volume, Mean Corpuscular Hemoglobin, Mean Corpuscular Hemoglobin Concentration, Platelets, White Blood Corpuscles, Reticulocytes, Neutrophils, Lymphocytes, Eosinophils, Monocytes, Basophil and Prothrombin time were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:At the end of dosing period on day 29 and at termination of recovery period on day 43.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, overnight
- How many animals: All 72 animals were examined.
- Parameters checked in table [No.?] were examined.:Total Protein, Blood Urea Nitrogen, Urea Nitrogen, Calculated, Alanine Aminotransferase, Aspartate Aminotransferase, Alkaline Phosphatase, Gamma Glutamyl Transferase, Glucose (mg/dL), Calcium , Phosphorous, Albumin, Total Bilirubin, Creatinine, Total Cholesterol, Triglycerides, Globulin, Calculated, Sodium Potassium, Chloride and Bile acid were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: during the last week of dosing period and on reversal group rats at termination of recovery period on day 43.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined.: Volume, Appearance, Colour, pH, Specific Gravity, Proteins, Glucose, Ketones, Bilirubin, Urobililogen, Occult Blood and Nitrite were examined.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the exposure period of 28 days and towards the end of the recovery period on day 42
- Dose groups that were examined:All dose group were examined.
- Battery of functions tested: sensory activity, grip strength, motor activity, Visual Placing Response were examined.

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

OTHER:Organ Weights were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
after 28 consecutive days of oral administration, all surviving study rats were sacrificed on day 29 (Group I, III, IV, V). In addition all rats from reversal groups were sacrificed on day 43 (Group II and VI).

HISTOPATHOLOGY: Yes
From each rat, samples or the whole of the tissues listed below were preserved. All tissues were fixed in 10% neutral buffered formalin except, eyes and testes of all animals were preserved in Davidson’s solution for 24 hours and transferred to 10% neutral buffered formalin.
Following tissue samples of organs were subjected to histopathological examination: Adrenals, Aorta, Brain (cerebrum, cerebellum and pons), Caecum, Cervix, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mesenteric Lymphnodes, Muscles - Skeletal muscle, Oesophagus, Ovaries, Pancreas, Pharyngeal Lymphnodes, Pituitary, Prostate, Rectum, Sciatic Nerve, Seminal Vesicles with coagulation gland, Skin with Mammary Gland, Spleen, Spinal Cord (Cervical, mid thoracic and lumbar), Sternum with bone marrow, Stomach, Testes, Thymus, Trachea, Thyroid / Parathyroid, Urinary Bladder, Uterus, Vagina.
Statistics:
Raw data was processed and analyzed for reporting group means and standard deviations with significance between the controls and treated groups, using SYSTAT 13 validated statistical software supplied by Starcom Information Technology Limited, Bangalore developed by Systat Software, Inc. USA. All the parameters characterized by continuous data such as body weight, feed consumption (calculated as gram per animal), organ weight, relative organ weight, haematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. Where the data was not meet the homogeneity of variance, Student’s t-test were performed to calculate significance.

Significance was calculated at 5% level and indicated in the summary tables as follows:

* = Significant than control at 95% level of confidence (p<0.05).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days and and during the post-dosing recovery period.
Mortality:
no mortality observed
Description (incidence):
All animals from control and different dose groups survived throughout the dosing period of 28 days and the post-dosing recovery period of 14 days.

Body weight and weight changes:
no effects observed
Description (incidence and severity):
Male -
Animals from control and different dose groups exhibited normal body weight gain throughout the dosing period of 28 days.
During the post-dosing recovery period, animals from 1000 mg/kg reversal group exhibited normal body weight gain when compared with that of control animals.

Female -
Animals from control and different dose groups exhibited normal body weight gain throughout the dosing period of 28 days.
Body weight gain of 8.45% above that of control was observed in female animals from 1000 mg/kg reversal group. This effect was not considered to be of any toxicological importance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Animals from control and different dose groups exhibited normal feed consumption at the end of the dosing period of 28 days.
Animals from control reversal and high reversal dose groups exhibited normal feed consumption at the end of the recovery period of 14 days.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ocular abnormalities were observed on ophthalmological examination in the animals during pre-exposure and at the end of the respective termination.
Haematological findings:
no effects observed
Description (incidence and severity):
Male :
MCHC : Increased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).
Female :
MCV and MCH : Increased values were obtained for animals from 500 mg/kg dose group, sacrificed on day 29 (p<0.05) and
Total RBC and HCT : Decreased values were obtained for animals from 500 mg/kg dose group, sacrificed on day 29 (p<0.05).

However, the increase/decrease in the values obtained was within normal biological and laboratory limits or the effect was not dose dependent.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Male :
Aspartate Aminotransferase, Calcium and Gamma Glutamyl Transferase : Decreased levels were observed in animals from 250 mg/kg dose group, sacrificed on day 29 (p<0.05),
Total Protein, Alanine Aminotransferase and Aspartate Aminotransferase : Decreased levels were observed in animals from 1000 mg/kg dose group, sacrificed on day 29 (p<0.05) and
Calcium : Decreased levels were observed in animals from 500 mg/kg dose group, sacrificed on day 29 (p<0.05).

Female :
Total Bilirubin : Elevated levels were observed in animals from 250 mg/kg dose group, sacrificed on day 29 (p<0.05),
Gamma Glutamyl Transferase : Decreased levels were observed in animals from 250 mg/kg dose group, sacrificed on day 29 (p<0.05),
Calcium and Triglycerides : Decreased levels were observed in animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05),
Sodium and Chloride : Elevated levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05) and
Total Protein, Globulin and Potassium : Decreased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).
However the increase/decreased in the values obtained was within normal biological and laboratory limits or the effect was not dose dependent.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No statistically significant variation was observed in the urine analyses conducted at the end of the dosing period in week 4 and 6 (on day 23, 24, 25 and 43) in male and female animals of different dose groups as compared to control group animals, except for higher volume of urine was observed in female animals from 1000 mg/kg reversal dose group (p<0.05). This higher volume of urine analyses were considered to be incidental and of no toxicological importance.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Before commencement of treatment:
In home cage observation, rat from different dose groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closer.
During handling observation, handling of rats did not reveal any abnormality from different dose groups and control group.
In the open field observation, rat did not reveal any abnormality from different dose groups and control group.

During treatment:
In home cage observation, rat from different dose groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closer.
During handling observation, handling of rats did not reveal any abnormality from different dose groups and control group.
In the open field observation, rat did not reveal any abnormality from different dose groups and control group.
Detailed clinical observation did not reveal any abnormality in all groups during the dosing period of 28 days and during the post-dosing recovery period.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Male -
In comparison with controls at the end of dosing on day 29, male animals from 500 mg/kg dose group revealed increased relative weights of liver. In addition, decreased relative weights of spleen were observed in male animals from 250 mg/kg and 500 mg/kg dose groups when compared with that of controls (p<0.05).
In comparison with controls at the end of post-dosing recovery period on day 43, organ weight data of animals from 1000 mg/kg reversal group was found to be comparable.

Female -
In comparison with controls at the end of dosing on day 29, organ weight data of animals from 250 mg/kg dose group revealed increased relative weights of liver, kidneys and heart (p<0.05). Increased relative weights of liver, kidneys and uterus (p<0.05) were observed in animals from 500 mg/kg dose group. In addition, decreased relative weights of ovaries (p<0.05) were observed in animals from 500 mg/kg and 1000 mg/kg dose groups. Decreased relative weights of adrenals (p<0.05) were observed in animals from 1000 mg/kg dose group.
In comparison with controls at the end of post-dosing recovery period on day 43, organ weight data of animals from 1000 mg/kg reversal group revealed increased relative weights of ovaries (p<0.05).

Although significant changes in the values of organ weight were observed in male and female animals from different dose groups, no related gross pathological and histopathological findings were seen, hence these findings were considered to be of no toxicological importance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross pathological examination on male and female animals from control and different dose groups did not reveal any abnormality.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related histopathological changes were evident in male and female animals from control and high dose groups.
Incidental and physiological histopathological changes which were covered in the background historical data of the pathology from control and high dose groups includes minimal, focal to multifocal periportal mononuclear cells infiltration in liver; minimal, focal mononuclear cells infiltration and/or tubular dilatation in the kidneys; minimal, focal to multifocal increase brown pigmentation in spleen; minimal, diffuse dilatation of zona reticularis and/or minimal, multifocal vacuolation in zona fasciculata in the adrenals; minimal, luminal seminal coagulum in urinary bladder; minimal, diffuse luminal dilatation in the uterus in male and female animals from control and high dose group.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

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:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
neuropathology
histopathology: non-neoplastic
Remarks on result:
other: No effect 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

 

Dose (mg/kg)

Mortality

Group

Males

Females

Number

Male

Female

Absolute

Relative %

Absolute

Relative %

I

0

0

0/6

0

0/6

0

II

0 (Reversal)

0 (Reversal)

0/6

0

0/6

0

III

250

250

0/6

0

0/6

0

IV

500

500

0/6

0

0/6

0

V

1000

1000

0/6

0

0/6

0

VI

1000 (Reversal)

1000 (Reversal)

0/6

0

0/6

0

Body weigth:

Male

Group

Dose (mg/kg)

 

Weeks

Number

 

Day 0

Day 1

1

2

3

I

0

Mean

164.57

170.70

198.15

235.82

262.07

±SD

10.29

9.93

9.96

20.27

21.65

II

0 (Reversal)

Mean

163.35

170.22

195.60

230.38

265.55

±SD

13.81

13.50

14.21

27.24

26.08

III

250

Mean

163.50

169.97

196.42

233.98

261.33

±SD

13.78

13.39

13.36

21.90

30.19

IV

500

Mean

166.10

172.58

200.70

231.82

262.00

±SD

13.17

13.48

13.43

28.81

32.02

V

1000

Mean

166.42

173.48

203.13

237.82

271.97

±SD

13.06

12.93

13.63

30.83

28.75

VI

1000 (Reversal)

Mean

164.53

171.78

200.17

234.32

263.33

±SD

9.87

10.00

9.67

22.08

22.63

Group

Dose (mg/kg)

 

Weeks

Number

 

4

5

6

I

0

Mean

275.78

 

±SD

20.68

II

0 (Reversal)

Mean

289.00

307.97

325.57

±SD

24.34

24.67

25.48

III

250

Mean

267.57

 

±SD

30.52

IV

500

Mean

277.37

±SD

33.53

V

1000

Mean

282.53

±SD

30.43

VI

1000 (Reversal)

Mean

283.92

300.87

317.08

±SD

22.82

21.49

20.62

Female:

Group

Dose (mg/kg)

 

Weeks

Number

 

Day 0

Day 1

1

2

3

I

0

Mean

138.37

141.80

159.03

184.82

200.58

±SD

8.78

9.84

9.12

6.86

6.62

II

0 (Reversal)

Mean

138.45

142.13

162.85

185.73

202.58

±SD

9.14

8.94

8.79

6.34

8.73

III

250

Mean

138.32

144.12

166.50

192.50

201.43

±SD

9.07

9.61

11.94

9.02

5.26

IV

500

Mean

139.13

144.87

165.63

193.93

211.25

±SD

8.88

11.29

10.40

14.92

14.32

V

1000

Mean

138.92

143.68

166.98

190.15

206.65

±SD

8.54

10.60

6.11

7.58

9.52

VI

1000 (Reversal)

Mean

138.53

144.35

166.28

191.80

210.43

±SD

6.62

9.48

15.85

15.88

12.18

Group

Dose (mg/kg)

 

Weeks

Number

 

4

5

6

I

0

Mean

205.22

 

±SD

5.34

II

0 (Reversal)

Mean

214.33

217.98

221.78

±SD

8.88

8.28

9.35

III

250

Mean

212.65

 

±SD

9.68

IV

500

Mean

219.72

±SD

14.07

V

1000

Mean

218.75

±SD

4.28

VI

1000 (Reversal)

Mean

222.15

230.57

240.53*

±SD

11.56

8.29

10.29

HAEMATOLOGY

Sex : Male

Day : 29 and 43

 

Group

Dose

(mg/kg)

 

Hb

Total RBC

Rt

HCT

MCV

MCH

MCHC

Number

 

(g/dL)

(6/µL)

(%)

(%)

(fL)

(pg)

(g/dL)

I

0

Mean

13.52

6.67

4.38

41.93

62.92

20.32

32.32

±SD

0.56

0.43

0.99

2.19

2.58

0.64

1.28

II

0 (Reversal)

Mean

16.23

8.37

4.47

50.50

60.40

19.45

32.18

±SD

0.71

0.52

0.74

2.25

2.51

0.90

0.55

III

250

Mean

14.13

6.89

4.53

41.77

60.63

20.48

33.82

±SD

0.24

0.12

0.93

0.62

1.29

0.32

0.45

IV

500

Mean

14.05

6.84

4.68

42.08

61.55

20.57

33.40

±SD

0.71

0.42

0.87

2.10

1.56

0.50

0.43

V

1000

Mean

13.97

6.93

4.35

41.97

60.65

20.17

33.27

±SD

0.72

0.52

1.03

1.74

2.57

0.91

1.00

VI

1000 (Reversal)

Mean

16.25

8.32

4.43

49.07

58.98

19.53

33.12*

±SD

1.06

0.35

0.77

2.78

1.63

0.94

0.71

 

Group

Dose

(mg/kg)

 

Platelets

Total WBC

Differential %

Pt.

Number

 

(3/ µL)

(3/µL)

N

L

E

M

B

(Sec.)

I

0

Mean

350.17

14.23

17.83

80.67

0.83

0.67

0.00

19.83

±SD

36.25

1.53

2.79

2.73

0.75

0.82

0.00

4.67

II

0 (Reversal)

Mean

414.00

13.42

19.33

79.50

0.67

0.50

0.00

17.17

±SD

29.89

2.63

3.01

3.08

0.82

0.55

0.00

4.17

III

250

Mean

387.33

11.82

19.00

79.33

1.00

0.67

0.00

19.33

±SD

20.11

1.30

3.46

3.39

0.89

0.82

0.00

4.97

IV

500

Mean

375.00

13.38

19.50

79.17

0.67

0.67

0.00

19.00

±SD

44.52

1.67

3.56

3.82

0.82

0.82

0.00

6.03

V

1000

Mean

402.67

12.02

18.50

80.00

0.83

0.67

0.00

20.50

±SD

51.32

2.86

3.08

2.61

0.75

0.52

0.00

4.51

VI

1000 (Reversal)

Mean

403.67

14.22

19.50

78.83

0.83

0.83

0.00

19.17

±SD

30.33

1.00

2.88

3.60

0.75

0.75

0.00

4.96

Sex : Female

Day : 29 and 43

Group

Dose

(mg/kg)

 

Hb

Total RBC

Rt

HCT

MCV

MCH

MCHC

Number

 

(g/dL)

(6/µL)

(%)

(%)

(fL)

(pg)

(g/dL)

I

0

Mean

14.28

7.03

4.37

41.77

59.40

20.33

34.23

±SD

0.38

0.19

1.05

1.01

0.60

0.24

0.23

II

0 (Reversal)

Mean

15.57

8.24

4.28

48.60

58.93

18.90

32.07

±SD

0.96

0.38

0.69

2.92

1.46

0.49

0.56

III

250

Mean

14.35

7.08

4.37

42.00

59.38

20.28

34.15

±SD

0.37

0.20

0.87

0.74

0.79

0.39

0.57

IV

500

Mean

13.57

6.46*

4.35

39.63*

61.43*

21.07*

34.30

±SD

0.63

0.44

0.56

2.02

1.41

0.50

0.54

V

1000

Mean

13.63

6.57

4.63

39.78

60.72

20.83

34.30

±SD

1.06

0.81

0.78

3.48

2.38

1.04

0.59

VI

1000 (Reversal)

Mean

15.47

7.91

4.20

47.88

60.48

19.55

32.30

±SD

0.89

0.29

0.67

2.76

2.55

0.91

0.62

 

Group

Dose

(mg/kg)

 

Platelets

Total WBC

Differential %

Pt.

Number

 

(3/ µL)

(3/µL)

N

L

E

M

B

(Sec.)

I

0

Mean

404.50

10.12

20.17

78.17

0.83

0.83

0.00

18.33

±SD

33.67

2.17

2.93

4.07

0.75

0.75

0.00

4.41

II

0 (Reversal)

Mean

424.17

10.50

19.83

79.00

0.83

0.33

0.00

19.67

±SD

28.23

3.82

2.32

2.53

0.75

0.52

0.00

5.72

III

250

Mean

400.50

11.22

19.67

78.50

1.00

0.83

0.00

20.17

±SD

25.65

2.55

2.80

2.81

0.89

0.75

0.00

5.49

IV

500

Mean

347.67

9.97

18.33

80.00

1.17

0.50

0.00

18.33

±SD

101.95

2.35

3.44

3.74

1.17

0.55

0.00

4.89

V

1000

Mean

366.17

12.35

18.00

81.00

0.67

0.33

0.00

19.67

±SD

143.88

4.83

2.28

2.10

0.82

0.52

0.00

5.89

VI

1000 (Reversal)

Mean

472.33

9.67

19.67

79.50

0.50

0.33

0.00

19.50

±SD

82.68

3.09

2.58

3.15

0.55

0.52

0.00

4.18

CLINICAL BIOCHEMISTRY

Sex : Male

Day : 29 and 43

Group Number

Dose (mg/kg)

 

TotalProtein (g/dL)

BUN (mg/dL)

Urea

(mg/dL)

ALT (U/L)

AST (U/L)

ALP (U/L)

Glucose (mg/dL)

I

0

Mean

6.69

15.00

32.70

63.50

113.33

216.50

84.17

±SD

0.10

2.45

5.34

10.65

11.76

26.60

6.11

II

0 (Reversal)

Mean

6.72

18.33

39.97

46.00

94.50

104.00

102.67

±SD

0.48

1.51

3.28

11.22

8.02

41.26

7.34

III

250

Mean

6.65

13.17

28.70

61.17

86.33*

185.17

93.17

±SD

0.23

1.17

2.55

10.30

6.62

15.11

12.46

IV

500

Mean

6.66

16.00

34.88

75.83

99.83

217.83

82.00

±SD

0.28

2.97

6.47

13.47

13.14

34.75

8.58

V

1000

Mean

6.57*

13.83

30.16

51.67*

98.50*

192.17

85.67

±SD

0.08

2.32

5.05

2.50

4.68

44.14

4.68

VI

1000 (Reversal)

Mean

6.48

17.17

37.42

57.00

96.00

140.00

100.00

±SD

0.23

2.79

6.08

11.49

12.07

59.58

9.47

 

Group Number

Dose (mg/kg)

 

Calcium(mmol/L)

Phospho-rous (mg/dL)

GGT

(U/L)

Total

Bilirubin (mg/dL)

Albumin (g/dL)

Globulin (g/dL)

Creatinine (mg/dL)

I

0

Mean

4.09

7.58

5.50

0.20

1.21

5.52

0.36

±SD

0.04

0.55

1.05

0.04

0.07

0.10

0.04

II

0 (Reversal)

Mean

4.05

8.70

4.00

0.17

1.10

5.60

0.40

±SD

0.06

0.88

1.55

0.05

0.11

0.41

0.10

III

250

Mean

3.75*

8.30

4.17*

0.20

1.25

5.40

0.34

±SD

0.07

0.67

0.41

0.02

0.06

0.18

0.03

IV

500

Mean

3.78*

7.63

4.67

0.19

1.15

5.50

0.40

±SD

0.04

0.50

0.82

0.03

0.12

0.26

0.06

V

1000

Mean

4.04

7.70

5.50

0.20

1.14

5.42

0.33

±SD

0.22

0.88

0.55

0.03

0.09

0.08

0.04

VI

1000 (Reversal)

Mean

3.92

8.03

4.83

0.22

1.10

5.38

0.35

±SD

0.23

0.60

1.17

0.04

0.09

0.17

0.12

Group Number

Dose (mg/kg)

 

 

Sodium

(mmol/L)

Potassium

(mmol/L)

Chloride

(mmol/L)

Total Cholesterol

(mg/dL)

Triglycerides

(mg/dL)

Bile Acids (µmol/L)

I

0

Mean

145.01

3.69

106.33

50.67

71.17

33.82

±SD

1.11

0.24

1.98

4.93

24.45

10.98

II

0 (Reversal)

Mean

145.30

4.31

107.72

51.33

41.33

14.06

±SD

1.38

0.61

1.34

6.92

20.02

8.26

III

250

Mean

145.84

3.67

107.88

49.00

42.17

34.85

±SD

0.88

0.19

1.57

5.76

11.14

22.18

IV

500

Mean

145.56

3.79

106.70

48.50

41.83

27.66

±SD

1.10

0.25

1.84

10.77

17.10

10.71

V

1000

Mean

145.35

3.73

108.45

47.00

62.83

26.80

±SD

1.43

0.28

1.49

4.20

27.74

18.50

VI

1000 (Reversal)

Mean

145.30

4.15

106.65

54.50

48.83

17.89

±SD

1.19

0.68

1.52

16.63

14.43

6.53

Sex : Female

Day : 29 and 43

Group Number

Dose (mg/kg)

 

TotalProtein (g/dL)

BUN (mg/dL)

Urea

(mg/dL)

ALT (U/L)

AST (U/L)

ALP (U/L)

Glucose (mg/dL)

I

0

Mean

6.34

16.17

35.24

50.83

104.33

157.67

91.50

±SD

0.37

1.17

2.55

10.85

19.26

28.70

10.86

II

0 (Reversal)

Mean

7.07

15.50

33.79

56.00

112.00

145.17

91.50

±SD

0.30

2.35

5.11

10.64

13.89

66.28

9.61

III

250

Mean

6.25

15.00

32.70

53.67

90.50

167.33

95.00

±SD

0.13

1.41

3.08

12.93

20.14

85.70

18.60

IV

500

Mean

6.28

16.67

36.33

57.33

92.00

148.00

95.67

±SD

0.14

2.80

6.11

8.41

18.92

20.91

10.60

V

1000

Mean

6.18

15.50

33.79

49.67

93.50

163.83

86.33

±SD

0.12

1.38

3.00

7.84

12.77

21.26

6.02

VI

1000 (Reversal)

Mean

6.51*

14.83

32.34

44.17

103.17

110.83

110.50

±SD

0.43

2.14

4.66

9.02

21.06

36.44

18.05

 

Group Number

Dose (mg/kg)

 

Calcium(mmol/L)

Phospho-rous (mg/dL)

GGT

(U/L)

Total

Bilirubin (mg/dL)

Albumin (g/dL)

Globulin (g/dL)

Creatinine (mg/dL)

I

0

Mean

4.13

7.18

4.83

0.14

1.11

5.23

0.36

±SD

0.21

0.58

0.98

0.01

0.11

0.31

0.03

II

0 (Reversal)

Mean

3.51

6.90

6.00

0.14

1.13

5.95

0.53

±SD

0.54

0.69

0.89

0.03

0.05

0.27

0.04

III

250

Mean

3.68*

8.17

3.50*

0.16*

1.10

5.13

0.33

±SD

0.09

1.12

0.84

0.01

0.11

0.18

0.03

IV

500

Mean

3.77*

7.70

5.17

0.15

1.07

5.22

0.34

±SD

0.12

0.70

0.75

0.03

0.09

0.12

0.05

V

1000

Mean

3.77*

7.40

4.33

0.16

1.10

5.08

0.35

±SD

0.12

0.58

0.52

0.02

0.04

0.12

0.03

VI

1000 (Reversal)

Mean

3.24

8.15

6.00

0.15

1.08

5.43*

0.49

±SD

0.12

1.20

1.55

0.04

0.08

0.37

0.09

 

Group Number

Dose (mg/kg)

 

 

Sodium

(mmol/L)

Potassium

(mmol/L)

Chloride

(mmol/L)

Total Cholesterol

(mg/dL)

Triglycerides

(mg/dL)

Bile Acids (µmol/L)

I

0

Mean

145.05

4.02

108.55

63.67

70.67

29.41

±SD

0.90

0.42

1.58

6.68

12.52

19.77

II

0 (Reversal)

Mean

149.81

3.77

112.06

62.33

69.17

14.81

±SD

2.13

0.32

6.55

14.56

39.97

6.54

III

250

Mean

145.46

4.33

110.46

62.50

37.00*

13.53

±SD

0.73

0.51

1.94

8.85

6.23

3.41

IV

500

Mean

145.52

3.99

108.37

57.50

40.17*

27.58

±SD

1.45

0.42

1.64

7.50

6.62

25.20

V

1000

Mean

145.98

4.31

110.46

59.17

36.00*

19.12

±SD

1.26

0.51

2.16

10.94

12.60

6.01

VI

1000 (Reversal)

Mean

153.31*

3.27*

118.75*

64.67

42.50

16.45

±SD

2.10

0.22

1.41

9.07

18.13

6.86

Applicant's summary and conclusion

Conclusions:
No Observed Adverse Effect Level (NOAEL) of test chemical in the Sprague Dawley rat via oral route, over a period of 28 days was found to be 1000 mg/kg body weight in male and female animals.
Executive summary:

In a Repeated Dose 28-day Oral Toxicity study, male and female Sprague Dawley rats were treated wtih test chemical in the concnetration of 0, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight orally by gavage in corn oil. Two additional dose levels were added to the study as control 0 mg/kg (Rev.) and test item 1000 mg/kg (Rev.), in order to study the reversibility or delayed occurrence of symptoms, if any.

No effect on survival, clinical sign, body weight, feed intake, Ophthalmoscopic examination and functional observation battery of treated male and female rats were noted at the end of dosing period of 28 days and the recovery period of 14 days. Similarly, at the end of the dosing period in male rat on day 29, revealed no statistically significant changes in the values of various parameters. Statistically significant increase in the values of MCHC at 1000 mg/kg were observed at the end of the recovery period. Statistically significant increase in the values of MCV and MCH and statistically significant decrease in the values of Total RBC and HCT at 500 mg/kg in female rats. No statistically significant changes in the values of various parameters at the end of the recovery period on day 43 in female rats. Statistically significant increase in the values of Total Bilirubin at 250 mg/kg in female rats, Statistically significant decrease was observed in the values of Aspartate Aminotransferase, Calcium and Gamma Glutamyl Transferase at 250 mg/kg in male rats as well as in Total Protein, Alanine Aminotransferase and Aspartate Aminotransferase at 1000 mg/kg in male, Calcium at 500 mg/kg in malerats, Gamma Glutamyl Transferase at 250 mg/kg in female rat, Calcium and Triglycerides at 250, 500 and 1000 mg/kg in female rats. were observed. In male at the end of the recovery period on day 43, no statistically significant changes in the values of various parameters. In female rats, statistically significant increase was observed in the values of Sodium and Chloride at 1000 mg/kg and statistically significant decrease was observed in the values of Total Protein, Globulin and Potassium at 1000 mg/kg. The increase/decrease in the values of various parameters was marginal and within the normal biological and laboratory limits. No abnormality attributable to the treatment except for higher volume of urine was observed in female animals at 1000 mg/kg reversal dose group and considered to be incidental and of no toxicological importance. Increased relative weights of liver at 500 mg/kg bw, decreased relative weights of spleen at 250 mg/kg and 500 mg/kg were observed in male rat as compared to control. Increased relative weights of liver, kidneys and heart at 250 mg/kg and increased relative weights of liver, kidneys and uterus were observed in female animals at 500 mg/kg as compared to control. Decreased relative weights of ovaries at 500 mg/kg and 1000 mg/kg and decreased relative weights of adrenals at 1000 mg/kg were observed in female rats as compared to control. On day 43 at 1000 mg/kg reversal group, increased relative weights of ovaries were observed as compared to controls. Although significant changes in the values of organ weight were observed in male and female animals from different dose groups, no related gross pathological and histopathological findings were seen, Hence these findings were considered to be of no toxicological importance. In addition, no Gross pathological and histopathological abnormality were observed in reproductive organ of treated male and femlae rats at the end of the dosing period on day 29 and recovery period on day 43 as compared to control. Therefore, No Observed Adverse Effect Level (NOAEL) of test chemical in the Sprague Dawley rata an oral route, over a period of 28 days was found to be 1000 mg/kg body weight in male and female animals.