Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 November 2016 to 21 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
OECD Guideline No. 407, “Repeated Dose 28-Day Oral Toxicity Study in Rodents” adopted on 03 October 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
k.A.
IUPAC Name:
k.A.
Test material form:
solid: flakes
Details on test material:
- Name of test material (as cited in study report): LICOCARE RBW 106 FL TP


Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Clariant Produkte (Deutschland) GmbH and RAK-KRS-00022

- Expiration date of the lot/batch: 01.04.2018
- Purity test date: 30.09.2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: Stable at 6 hours at room temperature with a concentration 100 mg/mL
- Solubility and stability of the test substance in the solvent/vehicle: Clearly miscible (at a concentration of 140 mg/mL) with Distilled water evidenced by the in-house miscibility test results and Stable at 6 hours at room temperature with a concentration 100 mg/mL

FORM AS APPLIED IN THE TEST: Liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is one of the recommended species by regulatory agencies for conducting preclinical toxicological studies among rodent species
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 100 to 160 g
- Housing:Maximum of three animals of same sex and group were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and water bottle. Clean sterilized paddy husk was provided as bedding material.
- Diet :ad libitum
- Water: ad libitum
- Acclimation period: 05 Days

DETAILS OF FOOD AND WATER QUALITY: Teklad Certified (2014SC) Global 14 % Protein Rodent Maintenance Diet - Pellet (Manufactured by Envigo); Water: eep bore-well water passed through an activated charcoal filter and exposed to ultraviolet rays in an aquaguard water filter with purifier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1°C to 22.8°C
- Humidity (%): 46% to 67%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 2 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 26 November 2016 To: 12 January 2017

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered through oral route using stainless steel gavage cannula attached to disposable syringe
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared by mixing to the required quantity with the vehicle to get desired concentrations of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Distilled water was used as vehicle for formulation preparation as the test item was clearly miscible (at a concentration of 140 mg/mL) with Distilled water evidenced by the in-house miscibility test results.
- Concentration in vehicle: G2: 10 mg/mL; G3: 30 mg/mL; G4: 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For active ingredient (a.i.) concentration analysis, the prepared formulations were sampled in duplicate sets (5 mL for each set from middle layer) from each dose level during week 1 and week 4 of the treatment period.
The results were considered acceptable, as the mean results are within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is equal to or less than 10.0%.
Duration of treatment / exposure:
28 Days
Frequency of treatment:
Daily once
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses of 100, 300 and 1000 mg/kg body weight were selected as low (G2),mid (G3) and high dose (G4/G4R) levels based on the NOAEL)of Genamin DMG 75 (N, N-Dimethyl-D-glucamine) was 1000 mg/kg/day when administered for a period of 14 consecutive days by oral (gavage) to Sprague Dawley rats obtained from the dose range finding study.
- Rationale for animal assignment: Randamization and grouping based on the body weight
- Rationale for selecting satellite groups: based on the OECD 407 guidelines
- Post-exposure recovery period in satellite groups: 14 days

The dose applied refers to the amount of N, N-Dimethyl-D-glucamine.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily once
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly once.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly once

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Week 4 for main group animals and week 6 for recovry group animals
- Dose groups that were examined: Vehicle control and high dose group animals of both sex.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination (Day 29 for amin group animals and Day 43 for recovery group animals)
- Anaesthetic used for blood collection: Yes (identity): isolflurane mild
- Animals fasted: Yes
- How many animals: all nimals one day prior to sacrifice
- Parameters checked in table [No.10] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination (Day 29 for amin group animals and Day 43 for recovery group animals)
- Animals fasted: Yes
- How many animals: all nimals one day prior to sacrifice
- Parameters checked in table [No.11] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at termination (Day 29 for amin group animals and Day 43 for recovery group animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.12] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 4 for main group animals and week 6 for recovery group animals
- Dose groups that were examined: Vehicle control and high dose group animals of both sex.
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 15)

HISTOPATHOLOGY: Yes (see table 16)
Other examinations:
Not applicable
Statistics:
SPSS software version 22. One way ANOVA followed by Dunnett’s post-test for main group parameters and independant sample t-test for recovery groups comparing with the control group data at the 95% level of confidence (P<0.05).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity and mortality/morbidity observed at any of the doses tested in both the sex.
Mortality:
no mortality observed
Description (incidence):
There were no mortality/morbidity observed at any of the doses tested in both the sex.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
statistical significant increase in mean body weight on day 22 with respect to day 1 in G2 females; statistical significant decrease in percent change in body weight during week 1 in G3 and G4 males; statistical significant increase in percent change in body weight during week 1 and 3 in G4R females was observed when compared with respective vehicle control groups. These changes can be considered as incidental, but not treatment related as the mean body weights and feed consumption during this period was unaffected and there were no clinical signs were observed.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment related changes noted in feed consumption at any of the tested doses in both the sex.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular changes observed during the ophthalmological examination carried out during week 4 for vehicle control and high dose group animals and during week 6 for vehicle control recovery and high dose recovery group animals.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Main group: Statistical significant increase in mean platelet volume in G2 and G4 males; statistical significant decrease in hemoglobin in G4 females; statistical significant decrease in mean corpuscular volume in G4 females; statistical significant decrease in mean corpuscular hemoglobin in G3 and G4 females; statistical significant decrease in mean corpuscular haemoglobin concentration in G4 females; statistical significant increase in percent eosinophills in G2 females; statistical significant increase in absolute eosinophills in G4 females when compared with vehicle control group animals were noted.

Recovery group: Statistical significant decrease in percent monocytes in G4R males, statistical significant decrease in absolute reticulocytes in G4R females when compared with vehicle control recovery group animals were noted.

All the observed changes are considered incidental and not treatment related because of the lack of dose dependency and/or due to random biological variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decrease in total cholesterol and statistically significant increase in sodium levels in G4R males; statistically significant decrease in glucose in G4R females; statistically significant increase in total bilurubin in G4R females were noted when compared with vehicle control recovery group.

These observed changes are considered as incidental and not treatment related as there were no changes were noted in main group animals of the same group and these changes can be due to random biological variation.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment related changes observed in the urinalysis parameters at any of the doses tested in either sex
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment related changes observed in neurological/functional examination battery carried out during week 4 for vehicle control and high dose group animals and during week 6 for vehicle control recovery and high dose recovery group animals
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increase in absolute spleen weight and decrease in relative thymus weight in G2 females; statistically significant increase in relative kidneys weight in G4R males; statistically significant decrease in absolute kidneys weight in G4R females; statistically significant decrease in relative thymus, kidneys and liver weight in G4R females were noted.
All the observed changes are considered incidental and not treatment related because of the lack of dose dependency and/or there were no associated macroscopic and microscopic abnormalities noted at the high dose main groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed at any of the tested doses of either sex during gross pathological examination.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related changes observed on histopathological examination of high dose group animals.
All observed lesions were considered as spontaneous and incidental to Sprague Dawley rats.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Details on results:
The active ingredient (a.i.) concentration analysis of the prepared dose formulations of Genamin DMG 75 (N, N-Dimethyl-D-Glucamine) was performed on week 1 and week 4 of the experiment as per the validated method (BIO-ANM 374). The results of the analysed formulations were within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is equal to or less than 10.0%.

All animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight and feed consumption. Ophthalmological examination and neurological/functional examination was carried out during week 4 for main groups (G1 and G4 group animals) and during week 6 for recovery groups (G1R and G4R group animals). Haematology, clinical chemistry analysis, urinalysis, gross pathology and organ weighing were performed on day 29 for main groups and on day 43 for recovery groups. Histopathological examination was conducted on the tissues from the vehicle control (G1) and high dose (G4) group animals.

The animals did not reveal any clinical signs of toxicity throughout the treatment and recovery period. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, ophthalmoscopic examination, neurological/functional examination, haematology, clinical chemistry, urinalysis, organ weights (both absolute and relative) were observed.

There were no gross pathological changes observed at any of the doses tested in either sex. No treatment related histopathological findings were noticed.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
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
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

TABLE 2. SUMMARY OF BODY WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight)

Body Weight on Days

1

8

15

22

28

G1, M & 0

Mean

180.39

218.05

262.18

285.29

314.09

±SD

14.38

14.68

17.88

16.52

20.15

n

5

5

5

5

5

G2, M & 100

Mean

181.26

218.46

268.26

296.23

330.72

±SD

9.54

10.58

9.66

10.36

13.09

n

5

5

5

5

5

G3, M & 300

Mean

182.53

213.85

259.27

292.11

322.75

±SD

13.00

14.32

20.46

21.16

22.16

n

5

5

5

5

5

G4, M & 1000

Mean

178.28

207.94

255.99

284.52

307.84

±SD

9.94

8.40

16.36

14.83

24.04

n

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Body Weight on Days

1

8

15

22

28

G1, F & 0

Mean

154.65

171.95

195.12

211.85

227.03

±SD

9.72

11.49

13.22

14.98

17.61

n

5

5

5

5

5

G2, F & 100

Mean

152.53

172.77

198.01

220.45

232.59

±SD

8.28

8.25

8.54

6.66

9.39

n

5

5

5

5

5

G3, F & 300

Mean

152.87

169.42

193.68

217.50

230.59

±SD

7.20

7.03

11.25

9.30

7.68

n

5

5

5

5

5

G4, F & 1000

Mean

155.74

172.60

199.21

219.72

232.71

±SD

9.05

12.29

11.15

14.71

15.13

n

5

5

5

5

5

Group, Sex &

Dose

(mg/kg body weight)

Days

1

8

15

22

28

36

42

G1R, M & 0

Mean

178.54

211.73

250.07

276.32

302.69

329.01

353.91

±SD

9.08

8.43

21.25

19.61

18.03

18.41

18.96

n

5

5

5

5

5

5

5

G4R, M & 1000

Mean

179.48

212.43

255.13

282.25

310.70

335.70

360.55

±SD

9.01

9.99

11.41

10.56

12.26

14.76

15.51

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

154.90

174.67

199.31

214.97

232.80

247.30

264.98

±SD

6.48

8.93

10.63

10.58

13.11

15.16

14.22

n

5

5

5

5

5

5

5

G4R, F & 1000

Mean

156.04

182.14

204.64

227.30

241.57

254.50

271.34

±SD

6.62

11.49

14.82

15.80

16.74

15.94

16.42

n

5

5

5

5

5

5

5

TABLE 3. SUMMARY OF PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose (mg/kg body weight)

Days

1 to 8

1 to 15

1 to 22

1 to 28

G1, M & 0

Mean

20.98

45.46

58.43

74.30

±SD

1.73

2.91

5.88

3.27

n

5

5

5

5

G2, M & 100

Mean

20.54

48.16

63.65

82.70

±SD

0.77

5.63

7.15

8.70

n

5

5

5

5

G3, M & 300

Mean

17.19

42.00

60.02

76.87

±SD

1.35

3.24

1.93

3.83

n

5

5

5

5

G4, M & 1000

Mean

16.76

43.64

59.69

72.62

±SD

3.79

6.06

5.37

7.97

n

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Days

1 to 8

1 to 15

1 to 22

1 to 28

G1, F & 0

Mean

11.17

26.14

36.93

46.71

±SD

1.41

1.57

1.65

3.78

n

5

5

5

5

G2, F & 100

Mean

13.31

30.04

44.76

52.69

±SD

1.05

7.64

6.57

6.75

n

5

5

5

5

G3, F & 300

Mean

10.85

26.67

42.35

50.96

±SD

1.44

3.44

4.43

4.46

n

5

5

5

5

G4, F & 1000

Mean

10.76

27.96

41.04

49.39

±SD

1.64

3.59

3.05

3.07

n

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Days

1 to 8

1 to 15

1 to 22

1 to 28

1 to 36

1 to 42

G1R, M & 0

Mean

18.67

39.96

54.72

69.62

84.35

98.31

±SD

2.99

7.15

6.34

7.62

6.70

7.17

n

5

5

5

5

5

5

G4R, M & 1000

Mean

18.37

42.24

57.41

73.26

87.16

101.01

±SD

0.87

4.78

5.59

6.05

5.70

5.89

n

5

5

5

5

5

5

G1R, F & 0

Mean

12.74

28.68

38.79

50.30

59.61

71.04

±SD

2.27

4.52

4.09

6.01

5.83

4.60

n

5

5

5

5

5

5

G4R, F & 1000

Mean

16.65

31.02

45.54

54.68

63.00

73.79

±SD

2.89

4.39

4.13

4.38

3.65

3.77

n

5

5

5

5

5

5

TABLE 3. SUMMARY OF AVERAGE FEED CONSUMPTION (g/rat/day) RECORD

Group, Sex & Dose (mg/kg body weight)

Week

Week 1

Week 2

Week 3

Week 4

G1, M & 0

Mean

21.1

23.0

24.4

26.8

±SD

1.2

0.9

2.1

1.2

n

5

5

5

5

G2, M & 100

Mean

21.7

23.3

23.9

27.4

±SD

1.2

2.1

2.6

1.7

n

5

5

5

5

G3, M & 300

Mean

20.9

22.6

23.6

26.9

±SD

0.5

0.7

1.1

1.8

n

5

5

5

5

G4, M & 1000

Mean

20.6

21.3

23.8

26.2

±SD

1.7

2.1

2.7

2.0

n

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Week

Week 1

Week 2

Week 3

Week 4

G1, F & 0

Mean

18.4

19.4

20.7

22.8

±SD

0.1

1.0

2.2

3.1

n

5

5

5

5

G2, F & 100

Mean

18.5

19.6

20.8

22.8

±SD

0.1

0.5

0.9

0.8

n

5

5

5

5

G3, F & 300

Mean

18.8

20.1

21.0

22.8

±SD

0.3

1.1

1.4

1.5

n

5

5

5

5

G4, F & 1000

Mean

18.6

19.6

20.9

22.2

±SD

0.3

0.1

0.6

0.3

n

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Week

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

G1R, M & 0

Mean

20.4

21.9

24.0

25.7

24.6

27.0

±SD

1.3

2.6

2.6

1.6

2.0

0.3

n

5

5

5

5

5

5

G4R, M & 1000

Mean

20.2

21.5

23.6

25.8

25.2

25.7

±SD

1.4

2.1

2.3

1.7

2.4

0.7

n

5

5

5

5

5

5

G1R, F & 0

Mean

18.6

19.7

20.3

21.8

21.6

25.5

±SD

0.1

0.3

0.6

0.1

0.6

0.4

n

5

5

5

5

5

5

G4R, F & 1000

Mean

18.3

19.0

20.3

21.2

21.9

24.2

±SD

0.7

0.5

0.9

0.8

0.3

0.8

n

5

5

5

5

5

5

TABLE 5. SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

Week 4

Week 6

Parameters↓

Group & Sex

G1 & M

G4 & M

G1R & M

G4R & M

Dose (mg/kg body weight)

0

1000

300

1000

Number of Animals

5

5

5

5

Home cage observations

 

a. Convulsions

1

1

1

1

b. Tremors

1

1

1

1

c. Palpebral closure

1

1

1

1

Handling observation

 

 

 

 

a. Ease of removal from the cage

2

2

2

2

b. Ease of handling animal in hand

2

2

2

2

c. Lacrimation

1

1

1

1

d. Red deposits around

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

e. Crusty deposits around

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

f. Salivation

1

1

1

1

g. Fur appearance

1

1

1

1

h. Piloerection

1

1

1

1

i. Palpebral closure

1

1

1

1

j. Respiratory character

1

1

1

1

k. Eye prominence

1

1

1

1

l. Muscle tone

1

1

1

1

Home cage observations: a. Convulsions - 1=Absent, 2=Present, b. Tremors- 1=Absent, 2=Present, c. Palpebral closure - 1=Normal, Handling observation : a. Ease of removal from the cage - 2=Normal, b. Ease of handling animal in hand - 2=Normal, c. Lacrimation - 1=None, 2=Slight, 3=Severe, d. Red deposits around - 1=Absent, 2=Present, e. Crusty deposits around - 1=Absent, 2=Present , f. Salivation- 1=Normal , g. Fur Appearance - 1=Normal, h. Piloerection - 1=None, 2=Slight, 3=Severe, i. Palpebral Closure - 1=Normal, j. Respiratory character 1=Normal, k. Eye Prominence - 1=Normal, l. Muscle tone - 1=Normal

 

Week 4

Week 6

Parameters↓

Group & Sex

G1 & M

G4 & M

G1R & M

G4R & M

Dose (mg/kg body weight)

0

1000

300

1000

Number of Animals

5

5

5

5

Open field Observation

 

a. Mobility

1

1

1

1

b. Gait

1

1

1

1

c. Arousal

3

3

3

3

d. Rearing

Mean

3.2

3.6

2.0

2.8

±SD

0.8

1.1

0.7

0.8

e. Urination

Mean

1.8

1.6

2.6

2.2

Numbers of pools of urine

±SD

0.8

1.3

0.5

0.8

f. Defecation

Mean

1.4

2.0

2.0

1.6

Number of fecal pellets

±SD

0.5

0.7

0.7

0.5

g. Stereotypies- repetitive circling

1

1

1

1

h. Excessive grooming

Mean

2.6

3.0

2.8

2.8

±SD

0.9

0.0

0.8

0.8

Sensory observations

 

a. Startle response

2

2

2

2

b. Touch response

2

2

2

2

c. Pupil response

2

2

2

2

d. Response to nociceptive stimuli

2

2

2

2

e. Righting reflex

1

1

1

1

Physiological observation

Mean

98.2

98.9

98.8

98.4

Body temperature (°F)

±SD

0.9

0.6

0.4

0.6

Open field Observation: a. Mobility - 1=Normal, b. Gait - 1=Normal, c. Arousal - 3=Normal, g. Stereotypies - repetitive circling- 1= Absent, 2=Present,Sensory Observations: a.Startle Response -2=Normal,b. Touch Response -2=Normal,c. Pupil Response 2=Normal,d. Response to Nociceptive stimuli - 2=Normal,e. Righting Reflex -1=Present, 2=Slow, 3=Absent

 

Week 4

Week 6

Parameters↓

Group & Sex

G1 & F

G4 & F

G1R & F

G4R & F

Dose (mg/kg body weight)

0

1000

300

1000

Number of Animals

5

5

5

5

Home cage observations

 

a. Convulsions

1

1

1

1

b. Tremors

1

1

1

1

c. Palpebral closure

1

1

1

1

Handling observation

a. Ease of removal from the cage

2

2

2

2

b. Ease of handling animal in hand

2

2

2

2

c. Lacrimation

1

1

1

1

d. Red deposits around

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

e. Crusty deposits around

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

f. Salivation

1

1

1

1

g. Fur appearance

1

1

1

1

h. Piloerection

1

1

1

1

i. Palpebral closure

1

1

1

1

j. Respiratory character

1

1

1

1

k. Eye prominence

1

1

1

1

l. Muscle tone

1

1

1

1

 

Week 4

Week 6

Parameters↓

Group & Sex

G1 & F

G4 & F

G1R & F

G4R & F

Dose (mg/kg body weight)

0

1000

300

1000

Number of Animals

5

5

5

5

Open field Observation

 

a. Mobility

1

1

1

1

b. Gait

1

1

1

1

c. Arousal

3

3

3

3

d. Rearing

Mean

2.4

3.0

2.8

2.8

±SD

1.1

1.0

0.8

0.8

e. Urination

Mean

1.8

2.4

2.0

2.2

Numbers of pools of urine

±SD

0.8

1.1

1.0

0.8

f. Defecation

Mean

1.8

1.8

1.8

1.2

Number of fecal pellets

±SD

0.8

0.8

0.8

0.5

g. Stereotypies- repetitive circling

1

1

1

1

h. Excessive grooming

Mean

3.2

2.4

2.6

2.8

±SD

1.3

1.1

0.5

0.8

Sensory observations

 

a. Startle response

2

2

2

2

b. Touch response

2

2

2

2

c. Pupil response

2

2

2

2

d. Response to nociceptive stimuli

2

2

2

2

e. Righting reflex

1

1

1

1

Physiological observation

Mean

98.8

98.6

98.5

98.7

Body temperature (°F)

±SD

1.2

0.6

0.5

0.8

Open field Observation: a. Mobility - 1=Normal, b. Gait - 1=Normal, c. Arousal - 3=Normal, g. Stereotypies - repetitive circling- 1= Absent, 2=Present,Sensory Observations: a.Startle Response -2=Normal,b. Touch Response -2=Normal,c. Pupil Response 2=Normal,d. Response to Nociceptive stimuli - 2=Normal,e. Righting Reflex -1=Present, 2=Slow, 3=Absent

Home cage observations: a. Convulsions - 1=Absent, 2=Present, b. Tremors- 1=Absent, 2=Present, c. Palpebral closure - 1=Normal, Handling observation : a. Ease of removal from the cage - 2=Normal, b. Ease of handling animal in hand - 2=Normal, c. Lacrimation - 1=None, 2=Slight, 3=Severe, d. Red deposits around - 1=Absent, 2=Present, e. Crusty deposits around - 1=Absent, 2=Present , f. Salivation- 1=Normal , g. Fur Appearance - 1=Normal, h. Piloerection - 1=None, 2=Slight, 3=Severe, i. Palpebral Closure - 1=Normal, j. Respiratory character 1=Normal, k. Eye Prominence - 1=Normal, l. Muscle tone - 1=Normal

TABLE 6. LANDING FOOT SPLAY (cm) RECORD

Group, Sex & Dose                            (mg/kg body weight)

Landing Foot Splay (cm)

G1, M & 0

Mean

8.0

±SD

1.0

n

5

G4, M & 1000

Mean

8.6

±SD

1.5

n

5

G1R, M & 0

Mean

8.5

±SD

1.2

n

5

G4R, M &1000

Mean

8.7

±SD

1.2

n

5

Group, Sex & Dose                            (mg/kg body weight)

Landing Foot Splay (cm)

G1, F & 0

Mean

7.9

±SD

0.7

n

5

G4, F & 1000

Mean

8.2

±SD

1.1

n

5

G1R, F & 0

Mean

7.9

±SD

0.7

n

5

G4R, F & 1000

Mean

7.3

±SD

1.1

n

5

TABLE 7. SUMMARY OF PHOTOACTOMETER READINGS (No. OF MOVEMENTS) RECORD

Group, Sex & Dose                                    (mg/kg body weight)

Movement Counts (no.)

G1, M & 0

Mean

670.0

±SD

56.6

n

5

G4, M & 1000

Mean

629.6

±SD

56.1

n

5

G1R, M & 0

Mean

692.2

±SD

108.1

n

5

G4R, M & 1000

Mean

741.0

±SD

46.7

n

5

Group, Sex & Dose                                    (mg/kg body weight)

Movement Counts (no.)

G1, F & 0

Mean

651.4

±SD

78.4

n

5

G4, F & 1000

Mean

620.0

±SD

47.8

n

5

G1R, F & 0

Mean

754.4

±SD

44.3

n

5

G4R, F & 1000

Mean

760.4

±SD

52.9

n

5

TABLE 8. SUMMARY OF GRIP STRENGTH (kgf) MEASUREMENT RECORD

Group, Sex & Dose (mg/kg body weight)

Fore limb

Hind limb

G1, M & 0

Mean

1.541

0.590

±SD

0.077

0.043

n

5

5

G4, M & 1000

Mean

1.430

0.502

±SD

0.187

0.073

n

5

5

G1R, M & 0

Mean

1.579

0.568

±SD

0.038

0.020

n

5

5

G4R, M & 1000

Mean

1.558

0.550

±SD

0.023

0.034

n

5

5

Group, Sex & Dose (mg/kg body weight)

Fore limb

Hind limb

G1, F & 0

Mean

1.567

0.577

±SD

0.025

0.038

n

5

5

G4, F & 1000

Mean

1.564

0.557

±SD

0.011

0.031

n

5

5

G1R, F & 0

Mean

1.565

0.556

±SD

0.022

0.038

n

5

5

G4R, F & 1000

Mean

1.555

0.562

±SD

0.026

0.020

n

5

5

TABLE 10. SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose

(mg/kg body weight)

Total Leucocyte Count(WBC)              (103cells/µL)

Total Erythrocyte Count             (RBC)               (106cells/µL)

Hemoglobin (HGB)  (g/dL)

Haematocrit (HCT)                   (%)

Mean Corpuscular Volume (MCV)                  (fL)

Mean Corpuscular Hemoglobin (MCH)                      (pg)

Mean Corpuscular Haemoglobin Concentration (MCHC)                (g/dL)

Platelet Count               (PLT)                (103cells/µL)

Mean Platelet Volume            (MPV)                 (fL)

G1, M & 0

Mean

7.99

7.17

13.50

41.82

58.28

18.82

32.32

1060.40

6.78

±SD

0.81

0.20

0.40

1.26

0.64

0.46

0.58

111.01

0.23

n

5

5

5

5

5

5

5

5

5

G2, M & 100

Mean

9.40

7.36

13.96

43.28

58.86

19.02

32.28

871.20

7.34

±SD

2.60

0.31

0.57

1.72

1.43

0.61

0.38

260.67

0.34

n

5

5

5

5

5

5

5

5

5

G3, M & 300

Mean

8.48

7.50

13.92

42.98

57.34

18.58

32.36

1073.20

7.18

±SD

2.77

0.32

0.51

1.31

1.25

0.59

0.40

50.57

0.13

n

5

5

5

5

5

5

5

5

5

G4, M & 1000

Mean

7.31

7.36

13.92

42.20

57.34

18.90

32.98

953.00

7.52

±SD

1.70

0.45

0.59

2.29

1.09

0.56

0.53

138.38

0.33

n

5

5

5

5

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Reticulocyte   Count                       (%)

Neutrophils             (%)

Lymphocytes             (%)

Monocytes                                 (%)

Eosinophils                         (%)

Basophils                          (%)

G1, M & 0

Mean

2.74

19.30

75.76

2.36

0.68

0.18

±SD

0.16

4.45

5.28

0.42

0.20

0.08

n

5

5

5

5

5

5

G2, M & 100

Mean

2.55

20.10

75.26

2.12

0.86

0.26

±SD

0.22

2.87

3.96

0.62

0.47

0.15

n

5

5

5

5

5

5

G3, M & 300

Mean

2.56

20.46

74.46

2.78

0.70

0.22

±SD

0.46

5.12

4.98

0.48

0.32

0.08

n

5

5

5

5

5

5

G4, M & 1000

Mean

2.36

24.84

69.80

3.22

0.56

0.18

±SD

0.45

3.19

3.20

0.83

0.18

0.04

n

5

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Absolute Reticulocyte Count                  (109cells/L)

Absolute Neutrophils              (103cells/µL)

Absolute Lymphocytes              (103cells/µL)

Absolute Monocytes              (103cells/µL)

Absolute Eosinophils              (103cells/µL)

Absolute Basophils              (103cells/µL)

Prothrombin Time                      (Seconds)

Activated Prothrombin Time                       (Seconds)

G1, M & 0

Mean

196.42

1.53

6.07

0.19

0.06

0.01

14.74

33.06

±SD

6.51

0.30

0.91

0.04

0.02

0.01

0.69

32.13

n

5

5

5

5

5

5

5

5

G2, M & 100

Mean

187.66

1.86

7.13

0.20

0.07

0.03

15.26

46.06

±SD

17.80

0.42

2.21

0.07

0.03

0.02

1.56

6.21

n

5

5

5

5

5

5

5

5

G3, M & 300

Mean

190.70

1.64

6.41

0.24

0.06

0.02

14.36

49.52

±SD

27.20

0.30

2.42

0.09

0.04

0.01

0.46

2.20

n

5

5

5

5

5

5

5

5

G4, M & 1000

Mean

175.56

1.81

5.11

0.25

0.04

0.01

14.34

41.60

±SD

41.83

0.45

1.20

0.11

0.02

0.00

0.79

16.11

n

5

5

5

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Total Leucocyte Count              (WBC)              (103cells/µL)

Total Erythrocyte Count             (RBC)               (106cells/µL)

Hemoglobin (HGB)  (g/dL)

Haematocrit (HCT)                   (%)

Mean Corpuscular Volume (MCV)                  (fL)

Mean Corpuscular Hemoglobin (MCH)                      (pg)

Mean Corpuscular Haemoglobin Concentration (MCHC)                (g/dL)

Platelet Count               (PLT)                (103cells/µL)

Mean Platelet Volume            (MPV)                 (fL)

G1, F & 0

Mean

7.23

7.54

14.22

43.28

57.40

18.90

32.88

1026.80

7.76

±SD

1.65

0.36

0.56

1.54

0.85

0.23

0.55

78.28

0.57

n

5

5

5

5

5

5

5

5

5

G2, F & 100

Mean

5.80

7.48

14.02

42.10

56.32

18.74

33.28

1046.40

8.46

±SD

1.01

0.17

0.52

1.04

1.19

0.51

0.36

76.03

0.34

n

5

5

5

5

5

5

5

5

5

G3, F & 300

Mean

7.66

7.50

13.38

41.66

55.62

17.84*

32.08

991.80

7.66

±SD

1.44

0.12

0.54

1.70

2.65

0.86

0.57

141.77

0.27

n

5

5

5

5

5

5

5

5

5

G4, F & 1000

Mean

8.27

7.76

13.32

41.70

53.78

17.18*

31.98*

978.20

7.12

±SD

2.13

0.27

0.43

0.96

1.55

0.58

0.47

90.57

0.70

n

5

5

5

5

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Reticulocyte Count                       (%)

Neutrophils             (%)

Lymphocytes             (%)

Monocytes                                 (%)

Eosinophils                         (%)

Basophils                          (%)

G1, F & 0

Mean

1.87

27.20

68.14

2.62

0.60

0.22

±SD

0.70

12.07

12.79

1.44

0.25

0.08

n

5

5

5

5

5

5

G2, F & 100

Mean

1.97

35.06

59.40

2.96

1.24

0.20

±SD

0.41

4.87

4.93

0.78

0.42

0.10

n

5

5

5

5

5

5

G3, F & 300

Mean

2.10

21.08

73.06

3.10

0.96

0.34

±SD

0.26

4.43

3.22

0.72

0.33

0.21

n

5

5

5

5

5

5

G4, F & 1000

Mean

2.30

21.32

72.28

3.24

1.10

0.38

±SD

0.20

4.55

4.56

0.74

0.45

0.08

n

5

5

5

5

5

5

Group, Sex & Dose

 (mg/kg body weight)

Absolute Reticulocyte Count                  (109cells/L)

Absolute Neutrophils              (103cells/µL)

Absolute Lymphocytes              (103cells/µL)

Absolute Monocytes              (103cells/µL)

Absolute Eosinophils              (103cells/µL)

Absolute Basophils              (103cells/µL)

Prothrombin Time                      (Seconds)

Activated Prothrombin Time                       (Seconds)

G1, F & 0

Mean

140.98

2.09

4.79

0.20

0.04

0.02

15.98

49.46

±SD

53.86

1.46

0.73

0.16

0.02

0.01

0.15

7.98

n

5

5

5

5

5

5

5

5

G2, F & 100

Mean

147.28

2.02

3.45

0.17

0.07

0.01

14.56

51.50

±SD

29.15

0.40

0.72

0.07

0.03

0.00

0.89

14.74

n

5

5

5

5

5

5

5

5

G3, F & 300

Mean

157.60

1.61

5.60

0.24

0.07

0.03

14.26

60.16

±SD

21.28

0.47

1.09

0.07

0.02

0.02

0.80

20.82

n

5

5

5

5

5

5

5

5

G4, F & 1000

Mean

177.92

1.77

5.98

0.27

0.09

0.03

15.26

50.46

±SD

9.54

0.58

1.63

0.08

0.03

0.01

1.82

10.17

n

5

5

5

5

5

5

5

5

Group, Sex & Dose

 (mg/kg body weight)

Total Leucocyte Count              (WBC)              (103cells/µL)

Total Erythrocyte Count             (RBC)               (106cells/µL)

Hemoglobin (HGB)  (g/dL)

Haematocrit (HCT)                   (%)

Mean Corpuscular Volume (MCV)                  (fL)

Mean Corpuscular Hemoglobin (MCH)                      (pg)

Mean Corpuscular Haemoglobin Concentration (MCHC)                (g/dL)

Platelet Count               (PLT)                (103cells/µL)

Mean Platelet Volume            (MPV)                 (fL)

G1R, M & 0

Mean

7.91

7.71

14.02

43.74

56.78

18.20

32.04

863.80

6.80

±SD

3.10

0.30

0.61

1.38

1.37

0.66

0.52

133.80

0.32

n

5

5

5

5

5

5

5

5

5

G4R, M & 1000

Mean

8.04

8.05

14.26

44.94

55.86

17.74

31.76

962.60

6.94

±SD

2.59

0.28

0.25

0.87

1.30

0.53

0.22

124.15

0.11

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

7.85

7.84

14.14

43.64

55.74

18.06

32.40

892.80

7.22

±SD

1.83

0.58

0.65

2.09

1.52

0.71

0.45

240.59

0.34

n

5

5

5

5

5

5

5

5

5

G4R, F & 1000

Mean

5.78

7.97

13.98

43.38

54.42

17.54

32.30

1025.60

7.26

±SD

1.48

0.28

0.37

1.54

1.38

0.53

0.39

207.18

0.32

n

5

5

5

5

5

5

5

5

5

Group, Sex & Dose

(mg/kg body weight)

Reticulocyte Count                       (%)

Neutrophils             (%)

Lymphocytes             (%)

Monocytes                                 (%)

Eosinophils                         (%)

Basophils                          (%)

G1R, M & 0

Mean

2.27

20.62

74.00

2.56

0.96

0.20

±SD

0.35

6.14

6.01

0.58

0.48

0.07

n

5

5

5

5

5

5

G4R, M & 1000

Mean

2.18

19.76

76.00

1.62

0.80

0.18

±SD

0.36

3.21

2.98

0.47

0.37

0.08

n

5

5

5

5

5

5

G1R, F & 0

Mean

1.60

20.98

74.52

1.78

1.08

0.22

±SD

0.41

6.18

6.25

0.22

0.29

0.04

n

5

5

5

5

5

5

G4R, F & 1000

Mean

1.98

25.24

69.50

2.04

1.46

0.16

±SD

0.17

7.91

7.23

0.64

0.48

0.05

n

5

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Absolute Reticulocyte Count                  (109cells/L)

Absolute Neutrophils              (103cells/µL)

Absolute Lymphocytes              (103cells/µL)

Absolute Monocytes              (103cells/µL)

Absolute Eosinophils              (103cells/µL)

Absolute Basophils              (103cells/µL)

Prothrombin Time                      (Seconds)

Activated Prothrombin Time                       (Seconds)

G1R, M & 0

Mean

174.60

1.50

5.98

0.21

0.07

0.02

16.00

47.98

±SD

24.51

0.34

2.59

0.10

0.02

0.01

1.14

6.34

n

5

5

5

5

5

5

5

5

G4R, M & 1000

Mean

174.66

1.58

6.11

0.14

0.06

0.02

18.50

49.32

±SD

24.53

0.53

2.01

0.08

0.02

0.01

2.96

5.75

n

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

124.82

1.63

5.87

0.14

0.08

0.02

16.20

49.80

±SD

28.31

0.54

1.49

0.05

0.03

0.01

3.12

14.47

n

5

5

5

5

5

5

5

5

G4R, F & 1000

Mean

157.46

1.43

4.04

0.13

0.09

0.01

16.60

61.16

±SD

11.39

0.46

1.24

0.06

0.04

0.01

2.99

12.17

n

5

5

5

5

5

5

5

5

TABLE 11. SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/kg body weight)

Glucose (GLU) mg/dL

Urea

mg/dL

Creatinine (CRE)           mg/dL

Total Cholesterol (CHO) mg/dL

Trigly-

cerides

(TRI)              mg/dL

Total Protein (TPR)             g/dL

Albumin (ALB)           g/dL

Alanine Amino     Transferase (ALT)               U/L

Aspartate Amino Transferase (AST)           U/L

Alkaline phosphatase (ALP)

U/L

Total Bilirubin (BIT)

mg/dL

G1, M & 0

Mean

103.00

23.72

0.52

67.80

65.60

5.52

3.26

41.60

86.80

158.40

0.01

±SD

12.90

2.32

0.05

6.76

18.51

0.26

0.14

3.51

14.20

35.06

0.02

n

5

5

5

5

5

5

5

5

5

5

5

G2, M & 100

Mean

97.80

23.96

0.51

60.80

67.00

5.46

3.24

42.00

85.40

140.80

0.01

±SD

14.81

4.36

0.04

12.46

22.62

0.25

0.16

6.20

10.95

36.68

0.01

n

5

5

5

5

5

5

5

5

5

5

5

G3, M & 300

Mean

105.80

28.46

0.54

59.00

70.40

5.42

3.30

37.60

81.60

208.20

0.02

±SD

18.29

4.55

0.05

8.37

34.44

0.13

0.08

5.77

13.46

43.03

0.02

n

5

5

5

5

5

5

5

5

5

5

5

G4, M & 1000

Mean

110.00

28.40

0.51

58.40

46.80

5.56

3.18

44.60

93.00

194.60

0.03

±SD

5.61

5.51

0.04

6.54

12.34

0.24

0.13

8.41

8.46

62.62

0.02

n

5

5

5

5

5

5

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Calcium (CAL) mg/dL

Phosphorous (PHO) (mg/dL)

Globulin (g/dL)

Albumin/

Globulin Ratio

BUN (mg/dL)

Choline-

esterase

Sodium

(Na)

mmol/L

Potassium (K)   mmol/L

Chloride (CLO) mmol/L

G1, M & 0

Mean

9.92

6.76

2.26

1.44

11.07

110.20

151.36

3.99

106.24

±SD

0.08

0.58

0.14

0.06

1.08

31.46

0.76

0.51

0.88

n

5

5

5

5

5

5

5

5

5

G2, M & 100

Mean

9.90

6.94

2.22

1.47

11.18

102.00

152.06

4.08

108.04

±SD

0.77

0.46

0.17

0.13

2.04

30.34

1.15

0.40

0.67

n

5

5

5

5

5

5

5

5

5

G3, M & 300

Mean

10.22

6.90

2.12

1.56

13.28

93.60

153.26

3.78

107.04

±SD

0.13

0.52

0.07

0.05

2.13

13.35

2.80

0.26

2.04

n

5

5

5

5

5

5

5

5

5

G4, M & 1000

Mean

9.76

6.32

2.38

1.35

13.25

111.40

152.04

3.86

107.72

±SD

0.34

0.71

0.21

0.13

2.57

19.88

1.18

0.14

0.87

n

5

5

5

5

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Glucose (GLU) mg/dL

 Urea

mg/dL

Creatinine (CRE)           mg/dL

Total Cholesterol (CHO) mg/dL

Tri-

glycerides (TRI)              mg/dL

Total Protein (TPR)             g/dL

Albumin (ALB)           g/dL

Alanine Amino     Transferase (ALT)               U/L

Aspartate Amino Transferase (AST)           U/L

Alkaline Phosphatase (ALP)

U/L

Total Bilirubin (BIT)

mg/dL

G1, F & 0

Mean

117.80

35.64

0.58

70.20

29.20

6.04

3.61

36.20

87.60

88.60

0.02

±SD

10.64

6.91

0.08

17.68

9.31

0.46

0.26

5.26

23.46

11.41

0.02

n

5

5

5

5

5

5

5

5

5

5

5

G2, F & 100

Mean

120.60

40.46

0.57

70.00

32.40

5.70

3.33

33.80

87.40

84.00

0.01

±SD

15.47

6.85

0.03

5.24

6.27

0.22

0.20

3.70

9.50

20.75

0.01

n

5

5

5

5

5

5

5

5

5

5

5

G3, F & 300

Mean

110.60

41.48

0.56

68.20

29.00

6.00

3.72

45.60

113.80

95.00

0.02

±SD

16.20

2.41

0.03

7.50

8.66

0.44

0.34

21.62

65.57

29.72

0.03

n

5

5

5

5

5

5

5

5

5

5

5

G4, F & 1000

Mean

112.00

36.28

0.54

61.80

34.40

6.00

3.01

32.20

89.80

99.40

0.03

±SD

4.74

4.48

0.03

4.38

6.58

0.10

1.09

5.40

16.28

13.99

0.01

n

5

5

5

5

5

5

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Calcium (CAL) mg/dL

Phosphorous (PHO) (mg/dL)

Globulin (g/dL)

Albumin/Globulin Ratio

BUN (mg/dL)

Choline-

esterase

 Sodium (Na) mmol/L

Potassium (K)   mmol/L

Chloride (CLO) mmol/L

G1, F & 0

Mean

9.96

6.66

2.43

1.48

16.63

483.20

152.46

3.64

107.24

±SD

0.42

0.91

0.20

0.05

3.22

127.64

1.24

0.32

1.07

n

5

5

5

5

5

5

5

5

5

G2, F & 100

Mean

10.08

6.16

2.37

1.41

18.88

708.20

153.30

3.81

107.32

±SD

0.45

0.59

0.19

0.15

3.20

168.26

2.00

0.26

0.90

n

5

5

5

5

5

5

5

5

5

G3, F & 300

Mean

10.68

6.96

2.28

1.70

19.36

662.80

154.04

3.80

109.30

±SD

0.90

0.53

0.48

0.49

1.13

264.33

1.49

0.50

1.74

n

5

5

5

5

5

5

5

5

5

G4, F & 1000

Mean

10.38

6.90

2.99

1.16

16.93

584.80

153.56

3.70

108.42

±SD

1.53

0.86

1.09

0.53

2.09

134.92

1.00

0.38

1.58

n

5

5

5

5

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Glucose (GLU) mg/dL

Urea mg/dL

Creatinine (CRE)           mg/dL

Total Cholesterol (CHO) mg/dL

Triglycerides (TRI)              mg/dL

Total Protein (TPR)             g/dL

Albumin (ALB)           g/dL

Alanine Amino     Transferase (ALT)               U/L

Aspartate Amino Transferase (AST)           U/L

Alkaline Phosphatase (ALP)

U/L

Total Bilirubin (BIT) mg/dL

G1R, M & 0

Mean

107.80

28.58

0.57

57.20

39.80

5.84

3.41

45.00

97.40

218.80

0.02

±SD

5.40

1.87

0.03

11.52

12.76

0.29

0.29

6.63

13.01

24.91

0.02

n

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 1000

Mean

104.00

30.88

0.55

43.80

31.40

5.46

3.25

40.00

86.80

175.20

0.02

±SD

6.44

2.99

0.02

5.67

7.89

0.23

0.11

6.04

10.31

38.65

0.02

n

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

127.40

35.30

0.61

69.40

61.40

6.36

3.87

32.60

69.20

72.20

0.01

±SD

5.59

5.43

0.03

13.96

32.85

0.21

0.16

2.88

18.29

9.26

0.03

n

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 1000

Mean

105.00

40.52

0.61

75.00

34.40

6.18

3.83

32.00

70.80

70.80

0.05

±SD

13.44

4.51

0.03

19.10

4.56

0.38

0.22

4.53

5.12

15.09

0.01

n

5

5

5

5

5

5

5

5

5

5

5

Group, Sex & Dose (mg/kg body weight)

Calcium (CAL) mg/dL

Phosphorous (PHO) (mg/dL)

Globulin (g/dL)

Albumin/

Globulin Ratio

BUN (mg/dL)

Cholinesterase

Sodium

(Na)

mmol/L

Potassium (K)   mmol/L

Chloride (CLO) mmol/L

G1R, M & 0

Mean

10.16

6.92

2.43

1.41

13.34

98.60

143.16

3.94

102.52

±SD

0.69

0.80

0.21

0.19

0.87

22.41

0.22

0.26

0.58

n

5

5

5

5

5

5

5

5

5

G4R, M & 1000

Mean

9.74

7.22

2.21

1.47

14.41

83.00

145.02

3.59

103.22

±SD

0.23

0.59

0.13

0.05

1.40

17.89

0.65

0.34

0.84

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

10.24

6.26

2.49

1.56

16.48

926.80

145.42

3.35

102.90

±SD

0.44

0.88

0.08

0.06

2.53

248.64

1.47

0.33

2.00

n

5

5

5

5

5

5

5

5

5

G4R, F & 1000

Mean

10.56

6.00

2.35

1.63

18.91

872.00

144.38

3.31

102.86

±SD

0.27

0.51

0.20

0.10

2.10

199.89

2.72

0.13

0.88

n

5

5

5

5

5

5

5

5

5

TABLE 12. SUMMARY OF URINALYSIS RECORD

      Examination

Group & Sex

G1 & M

G2 & M

G3 & M

G4 & M

G1R & M

G4R & M

Dose (mg/kg body weight)

0

100

300

1000

0

1000

Number of Animals

5

5

5

5

5

5

Physical

Colour

Pale Yellow

5

5

5

4

4

3

Yellow

-

-

-

1

1

2

Appearance

Clear

4

5

5

4

5

5

Turbidity

1

-

-

1

-

-

Volume (mL)

Mean

4.5

2.6

5.3

4.2

3.9

4.5

±SD

1.7

1.1

2.6

2.1

1.3

1.4

Chemical

pH

Mean

6.7

6.8

6.9

7.1

6.8

6.8

±SD

0.5

0.5

0.2

0.6

0.5

0.3

Specific Gravity

Mean

1.027

1.030

1.021

1.022

1.028

1.030

±SD

0.007

0.000

0.005

0.008

0.004

0.000

Urobilinogen (mg/dL)

Mean

0.4

0.4

0.2

0.4

0.4

0.5

±SD

0.4

0.4

0.0

0.4

0.4

0.4

Bilirubin (mg/dL)

Neg

4

3

4

3

4

4

1

1

2

1

2

1

1

Ketones (mg/dL)

Neg

-

-

-

2

-

-

5

5

5

5

3

5

5

Blood (Ery/µL)

Neg

4

5

5

3

4

4

Ca10

1

-

-

2

1

1

Proteins (mg/dL)

Trace

4

3

5

2

1

2

100

-

1

-

-

1

 

30

1

1

-

2

3

3

Neg

-

-

-

1

-

-

Nitrite

Neg

1

4

3

4

3

3

Pos

4

1

2

1

2

2

Leucocytes (Leu/µL)

Neg

5

5

5

4

5

5

Ca15

-

-

-

1

-

-

Glucose (mg/dL)

Neg

5

5

5

5

5

5

Microscopic

Epithelial cells

0

-

-

1

-

-

-

0-1

1

2

3

 

1

1

1-2

3

3

1

3

4

3

2-3

1

-

-

1

-

1

3-4

-

-

-

1

-

-

Casts

Absent

5

5

5

5

5

5

Crystals

Present

5

5

5

5

5

5

TABLE 12 (Contd..,). SUMMARY OF URINALYSIS RECORD

      Examination

Group & Sex

G1 & F

G2 & F

G3 & F

G4 & F

G1R & F

G4R & F

Dose (mg/kg body weight)

0

100

300

1000

0

1000

Number of Animals

5

5

5

5

5

5

Physical

Colour

Pale yellow

5

5

3

5

4

5

Yellow

-

-

2

-

1

-

Appearance

Clear

5

3

4

5

5

4

Turbidity

-

2

1

-

-

1

Volume (mL)

Mean

4.8

6.2

5.9

4.2

3.5

3.6

±SD

2.1

2.5

1.7

1.6

0.8

1.3

Chemical

pH

Mean

6.8

7.1

7.1

6.7

6.8

6.8

±SD

0.7

0.4

1.1

0.3

0.4

0.4

Specific Gravity

Mean

1.021

1.021

1.020

1.028

1.027

1.026

±SD

0.008

0.007

0.010

0.004

0.004

0.007

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.2

0.2

0.4

0.2

±SD

0.0

0.0

0.0

0.0

0.4

0.0

Bilirubin (mg/dL)

Neg

4

4

4

4

4

5

1

1

1

1

1

1

-

Ketones (mg/dL)

Neg

5

5

5

4

4

5

5

-

-

-

1

1

-

Blood (Ery/µL)

Neg

3

4

4

5

4

5

Ca10

-

-

-

-

1

-

Ca25

-

1

1

-

-

-

Ca200

2

-

-

-

-

-

Proteins (mg/dL)

Trace

2

1

2

4

5

1

30

-

-

-

1

-

-

Neg

3

4

3

-

-

4

Nitrite

Neg

4

3

3

5

4

3

Pos

1

2

2

 

1

2

Leucocytes (Leu/µL)

Neg

5

5

4

5

5

5

Ca15

-

-

1

-

-

-

Glucose (mg/dL)

Neg

5

5

5

5

5

5

Microscopic

Epithelial cells

0-1

1

1

2

-

1

-

1-2

4

2

3

3

3

4

2-3

-

1

-

2

1

1

3-4

-

1

-

-

-

-

Casts

Absent

5

5

5

5

5

5

Crystals

Present

5

5

5

5

5

5

TABLE 13. SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose

(mg/kg body weight)

Adrenals

Thymus

Spleen

Epididymes

Testes

Heart

Kidneys

Brain

Liver

PSC

G1, M & 0

Mean

0.0604

0.7514

0.6342

1.0064

3.1735

1.2125

2.2590

2.0835

10.5344

2.3816

±SD

0.0119

0.1533

0.0738

0.0640

0.1397

0.0614

0.2240

0.1289

1.1741

0.3663

n

5

5

5

5

5

5

5

5

5

5

G2, M & 100

Mean

0.0630

0.6919

0.6735

1.0010

2.9228

1.1877

2.5969

2.1525

10.8162

2.2128

±SD

0.0065

0.1076

0.0667

0.0443

0.1672

0.0647

0.2891

0.1217

0.9876

0.2863

n

5

5

5

5

5

5

5

5

5

5

G3, M & 300

Mean

0.0572

0.6269

0.5922

1.0242

3.1694

1.1864

2.3507

2.0956

10.3840

2.3975

±SD

0.0049

0.0664

0.1458

0.0415

0.2142

0.1443

0.2566

0.1243

1.4737

0.1569

n

5

5

5

5

5

5

5

5

5

5

G4, M & 1000

Mean

0.0605

0.5860

0.5928

1.0397

3.1413

1.2067

2.2968

2.0724

9.4292

2.2865

±SD

0.0077

0.1830

0.1249

0.1030

0.3287

0.1716

0.1797

0.0997

0.7478

0.2833

n

5

5

5

5

5

5

5

5

5

5

TABLE 13 (Contd..,). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose

 (mg/kg body weight)

 

Adrenals

Thymus

Spleen

Uterus

Ovaries

Heart

Kidneys

Brain

Liver

G1, F & 0

Mean

0.0648

0.5093

0.4214

0.5513

0.1470

0.8857

1.9527

2.0259

7.4806

±SD

0.0078

0.1181

0.0275

0.1162

0.0144

0.0824

0.1765

0.0857

0.7090

n

5

5

5

5

5

5

5

5

5

G2, F & 100

Mean

0.0703

0.3810

0.5137

0.6402

0.1526

0.9369

1.7481

1.9522

7.5152

±SD

0.0115

0.0378

0.0586

0.2055

0.0172

0.1019

0.1312

0.0413

0.5203

n

5

5

5

5

5

5

5

5

5

G3, F & 300

Mean

0.0705

0.4218

0.4418

0.4521

0.1531

0.9352

1.9264

1.9350

7.3831

±SD

0.0094

0.0893

0.0454

0.0523

0.0225

0.0600

0.1232

0.0525

0.3699

n

5

5

5

5

5

5

5

5

5

G4, F & 1000

Mean

0.0708

0.4073

0.4270

0.4558

0.1571

0.8627

1.7695

1.9476

7.4482

±SD

0.0061

0.0781

0.0400

0.1060

0.0151

0.1085

0.1630

0.1456

0.6254

n

5

5

5

5

5

5

5

5

5

TABLE 13 (Contd..,). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose

(mg/kg body weight)

Adrenals

Thymus

Spleen

Epididymes

Testes

Heart

Kidneys

Brain

Liver

PSC

G1R, M & 0

Mean

0.0647

0.5413

0.6230

1.2683

3.2340

1.2561

2.3803

2.1835

10.7767

2.4993

±SD

0.0082

0.0443

0.0542

0.0911

0.2266

0.0549

0.1389

0.1071

0.9928

0.2145

n

5

5

5

5

5

5

5

5

5

5

G4R, M & 1000

Mean

0.0624

0.5626

0.6442

1.3105

3.1893

1.2834

2.5415

2.2298

11.2594

2.9348

±SD

0.0039

0.0494

0.0530

0.0328

0.1477

0.0958

0.1937

0.1037

1.8236

0.4075

n

5

5

5

5

5

5

5

5

5

5

TABLE 13 (Contd..,). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose

 (mg/kg body weight)

Adrenals

Thymus

Spleen

Uterus

Ovaries

Heart

Kidneys

Brain

Liver

G1R, F & 0

Mean

0.0709

0.6198

0.5040

0.5658

0.1688

1.0534

1.8789

2.0390

9.0836

±SD

0.0059

0.0445

0.0623

0.0621

0.0205

0.0846

0.0549

0.0810

0.7625

n

5

5

5

5

5

5

5

5

5

G4R, F & 1000

Mean

0.0722

0.5087

0.4635

0.5721

0.1695

1.0817

1.6534

2.0396

8.3174

±SD

0.0036

0.0983

0.0519

0.0801

0.0085

0.1088

0.1034

0.0974

0.5321

n

5

5

5

5

5

5

5

5

5

TABLE 15. SUMMARY OF GROSS PATHOLOGY RECORD

Organs/ Lesions

Group

G1

G1R

G2

G3

G4

G4R

Dose (mg/kg body weight/day)

0

0

100

300

1000

1000

Sex

M

F

M

F

M

F

M

F

M

F

M

F

No. of Animals

5

5

5

5

5

5

5

5

5

5

5

5

No Abnormality Detected

5

5

5

5

5

5

5

5

5

5

5

5

TABLE 16. SUMMARY OF HISTOPATHOLOGY RECORD

Group No.

G1

G4

Dose (mg/kg body weight/day)

0

1000

Sex

M

F

M

F

No. of Animals

5

5

5

5

ORGANS AND OBSERVATIONS

KIDNEYS                 

 

 

 

 

Dilation, renal pelvis, bilateral

1

0

0

0

THYMUS

 

 

 

 

Hemorrhage

1

0

1

0

LUNGS

 

 

 

 

Alveolar macrophages

0

0

1

0

Hemorrhage

0

0

0

1

THYROID

 

 

 

 

Ultimobranchial cyst

0

1

0

0

Ectopic thymus

0

0

0

2

RECTUM

 

 

 

 

Parasite

1

0

0

0

MANDIBULAR LYMPH NODES

 

 

 

 

Sinus dilatation

0

1

0

0

UTERUS

X

 

X

 

Dilated lumen

 

1

 

0

Note: The numeral indicates number of animal/s with lesion; M: Male; F: Female; X: Organs not subjected for histopathological examination due to sex difference.

Applicant's summary and conclusion

Conclusions:
The repeated dose toxicity of the registration substance was investigated according to the Guideline OECD 407. Rats were treated at dose of up to 1000 mg/kg/day. No effect was observed. The NOAEL of 1000 mg/kg/day was obtained.
Executive summary:

The registration substance, N, N-Dimethyl-D-glucamine, was evaluated to determine the toxic potential when administered for a period of 28 consecutive days repeatedly by oral (gavage) route to Sprague Dawley rats. This study provides information on any major toxic effects, target organs, possibility of cumulative effects and also to assess the reversibility of effects after 14 days recovery period and to estimate the No Observed Adverse Effect Level (NOAEL).

A total of 60 (30 males + 30 females) Sprague Dawley rats were distributed to main and recovery groups. Each main group and recovery group consisted of 5 males and 5 females. The animals were administered N, N-Dimethyl-D-Glucamine at the dose levels of 0, 100, 300 and 1000 mg/kg/day. The vehicle and test item formulations were administered at the dose volume of 10 mL/kg body weight.

The concentration analysis of the prepared dose formulations was performed on week 1 and week 4 of the experiment as per the validated method. The results of the analysed formulations were within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is equal to or less than 10.0%.

All animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight and feed consumption. Ophthalmological examination and neurological/functional examination was carried out during week 4 for main groups and during week 6 for recovery groups. Haematology, clinical chemistry analysis, urinalysis, gross pathology and organ weighing were performed on day 29 for main groups and on day 43 for recovery groups. Histopathological examination was conducted on the tissues from the vehicle control and high dose group animals.

The animals did not reveal any clinical signs of toxicity throughout the treatment and recovery period. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, ophthalmoscopic examination, neurological/functional examination, haematology, clinical chemistry, urinalysis, organ weights (both absolute and relative) were observed. There were no gross pathological changes observed at any of the doses tested in either sex. No treatment related histopathological findings were noticed.

The NOAEL of 1000 mg/kg/day was obtained.

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