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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2021 to 29 November 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
28-day (Sub-acute) inhalation toxicity study” adopted on 25 June 2018.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
28-day (Sub-acute) inhalation toxicity study” adopted on 25 June 2018.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(4-chloro-2-nitrophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide
EC Number:
235-462-4
EC Name:
2-[(4-chloro-2-nitrophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide
Cas Number:
12236-62-3
Molecular formula:
C17H13ClN6O5
IUPAC Name:
2-[(4-chloro-2-nitrophenyl)diazenyl]-3-oxo-N-(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)butanamide
Test material form:
solid: nanoform

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is the preferred laboratory rodent species for inhalation toxicity assessment and is also recommended by various regulatory guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-House
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 - 8 weeks on the day of randomization
- Weight at study initiation: Males: 140.14 g to 153.11 g
Females: 120.06 g to 137.46 g
- Fasting period before study: No
- Housing: Maximum of three animals
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum):yes
- Acclimation period:7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0°C to 22.7°C
- Humidity (%): 46% to 65%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: To: 17 May 2021 to 16 August 2021

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 1 - < 2 µm
Geometric standard deviation (GSD):
3
Remarks on MMAD:
1.82 to 1.97
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was conducted using a flow-past, nose-only dynamic inhalation exposure system (Refer Annexure 5) supplied by CH Technologies, USA, with a provision of at least 12 air changes per hour.
- Method of holding animals in test chamber: restraining tubes
- Source and rate of air: Compressor air and 20 L/min
- Method of conditioning air: Filtered air
- System of generating particulates/aerosols: The Rotating Brush Generator (Palas RBG 1000 - supplied by Palas GmbH) was used to generate the dust particles (aerosols) into an airborne state.
- Temperature, humidity, pressure in air chamber: 23.2, 57.6, 60psi
- Air flow rate: 20 L/min
- Air change rate: 12 air cahnges per hour
- Method of particle size determination: gravimetric
- Treatment of exhaust air: NaoH

TEST ATMOSPHERE
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 Hours
Frequency of treatment:
Weekly 5 days
Doses / concentrationsopen allclose all
Dose / conc.:
0.003 mg/L air
Remarks:
Low dose
Dose / conc.:
0.01 mg/L air
Remarks:
Mid dose
Dose / conc.:
0.03 mg/L air
Remarks:
High dose
No. of animals per sex per dose:
5 males + 5 females
Details on study design:
- Dose selection rationale: Inhalation is one of the most possible routes of exposure to human. Primarily used to derive regulatory concentrations for assessing worker risk in occupational settings, also used to identify and assess human residential, consumer, transportation, and environmental risk.
- Rationale for selecting satellite groups: persistence of toxic effects.
- Post-exposure recovery period in satellite groups: Following the 28 days exposure period, the animals in the recovery groups (G1R and G4R) were not be given any treatment and was maintained for 56 days post treatment period and observed for reversibility or persistence of toxic effects.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes / No / Not specified
- Time schedule: twice daily
- Cage side observations checked in table [yes] were included.
Table 1

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly once

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before exposure and week 4 and week 12
- Dose groups that were examined: control and high dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 27 and 85
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in table [yes] were examined.
table 10

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 27 and 85
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in table [yes] were examined.
Table 12

URINALYSIS: Yes
- Time schedule for collection of urine: 27
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [yes] were examined.
Table 13

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Yes
- Time schedule for examinations: week 4 and 12
- How many animals: 40
- Dose groups that were examined: control and high dose
- Parameters checked in table [yes] were examined.
Tables 5, 6, 7 & 8

OTHER:
BALF Fluid
Table 11
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The raw data were subjected to statistical analysis. The computer printout of the data (in the form of the appendix) was verified with the original raw data. After verification, the data were subjected to statistical analysis using SPSS Software version 22. Body weight, percent change in body weight with respect to Day 1, feed consumption, FOB, organ weight ratios, hematology, and clinical chemistry estimations, urine volume, pH, and specific gravity were subjected to statistical analysis. One-way ANOVA followed by Dunnett’s post-test was done for different treatment groups comparing with the control group data. All analyses and comparisons were evaluated at the 95% level of confidence (P<0.05). Statistically significant changes obtained from the aforementioned tests were designated by the superscripts in the summary table throughout the study report, as stated below:
*: Statistically significant (p<0.05).

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All animals survived to scheduled sacrifice, and there were no clinical signs attributable to the test item. Post-exposure, all animals were normal throughout the experimental period. No clinical signs were noted during the recovery period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No statistically significant treatment-related variations were observed in all the tested dose groups when compared with control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No statistically significant treatment-related variations were observed in all the tested dose groups when compared with control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ocular abnormalities were observed during ophthalmoscopic examination
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant treatment related changes in haematology parameters were noted. However, the following statistically significant variations were noted.
In main group males, increase in RBC, haematocrit, MPV, PT in G4 and decrease in MCHC in G3 was noted. In females increase in APTT in G4 was noted.
In recovery males, decrease absolute and relative reticulocyte count (G4R) was noted. In recovery females decrease in percent lymphocytes, absolute and relative reticulocyte count and increase in percent neutrophils (G4R) was noted.
All the noted variations in main group are considered incidental in the absence of dose responsiveness. Further, in the absence of any microscopic variations in the marrow (sternum with marrow), the noted changes are not considered to be adverse. Variations noted at the end of recovery period are considered incidental as similar changes were not noted at the end of treatment period.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant changes were observed in clinical chemistry parameters when compared to the control group. However, the following statistically significant variations were noted.
In main group males, increase in AST and calcium in G3 group and sodium in G4 was noted; in females increase in glucose, urea and BUN in G4 group was noted.
In recovery group females, increase in potassium (G4R) was noted.
The variations noted are considered incidental due to lack of dose responsiveness and/or could be due to random biological variation.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes were observed in main group and recovery when compared with control group.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse effects during neurological/functional examinations in main group (G1 and G4) and recovery group (G1R and G4R). However statistically significant decrease in rearing in G4 males, increase in movement counts in G4R females was noted. The noted variations are considered incidental in the absence of any changes in other neurological parameters. Also, all the animals were normal during the experimental period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant variations were noted in organ weights and its ratios.
Absolute and relative to fasting body weight
In main group females, decrease in absolute and relative weight of liver (G4), thymus (G2 and G4) was noted.
In recovery group females, decrease in absolute weight of spleen, uterus and relative weight of spleen, uterus (G4R) was noted.

Organ weight changes (relative to brain weight)
In main group females, decrease in relative weight of liver (G4) and thymus (G2 and G4) was noted; in males increase in relative weight of testis (G4M) was noted.
In recovery group females, decrease in relative weight of spleen and uterus (G4R) was noted.
All the observed changes are considered incidental in the absence of any associated gross and histopathological changes in the high dose main group animals.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no gross pathological findings in the study.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopically, test item-related changes were observed in lungs and are summarized as below.
Sex Males
Group G1 G2 G3 G4 G1R G4R
Number of Animals Examined 5 5 5 5 5 5
Lungs
Pigment, golden brown - 5 5 5 - 5

Sex Females
Group G1 G2 G3 G4 G1R G4R
Number of Animals Examined 5 5 5 5 5 5
Lungs
Pigment, golden brown - 5 5 5 - 5
In lungs, minimal to moderate, multifocal, variably sized, pigmented (golden brown) granular material was observed throughout the parenchyma of lung at low, mid, high and high dose recovery groups of rats. This pigment was distributed both in alveolar and bronchiolar spaces with no accompanying inflammation or tissue destruction to the lung parenchyma. This pigment was suggestive of inhaled test item particles. In the absence of cellular changes to lung parenchyma, mere presence of pigment in lungs could be considered as non-adverse effect up to the nominal target dose level of 0.03 mg/L. (Nikula KJ et.al., 2014).
Few microscopic findings observed in this study such as cyst/s in kidneys, dilatation of uterus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
13.6.2 BALF Fluid
The bronchio-alveolar lavage fluid measured at termination revealed the following statistically significant changes:
In main group females, decrease in percent lymphocytes and increase in percent neutrophils in G4 group was noted; increase in WBC and absolute lymphocytes in G3 group was noted.
In recovery group, males increase in LDH and decrease in percent basophils (G4R) was noted. The basophils (%) in recovery males appear decreased because of higher values in the control recovery group and hence the significance noted is of no toxicological relevance. Increase in LDH in recovery males is because of higher value in one animal, and hence considered incidental.
Few variations in differential counts could be secondary to accumulation of test item in the lungs. All other changes are considered incidental as they lack dose responsiveness and/or were noted in only one sex.
Details on results:
Based on the observed results, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item was found to be 0.03 mg/L when exposed for 6 hours/day, 5 days/week, for 4 weeks by flow-past nose-only inhalation route to Sprague Dawley rats. The nominal dose of 0.03 mg/L (6 hrs/day, 5 days/week) is the actual exposure concentration in males and females.

Effect levels

Key result
Dose descriptor:
NOAEC
Effect level:
> 30 mg/m³ air
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
serum/plasma biochemistry
urinalysis

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

TABLE 1.   SUMMARY OF CLINICAL SIGNS AND MORTALITY RECORD

                                                                                                                        Refer Appendix 1

Group, Sex & Dose

(mg/L)

No. of

Animals

Clinical Signs of Toxicity

Mortality                               (No. of Incidences/

No. of Animals)

G1, M & 0

5

N

0/5

G2, M & 0.003

5

N

0/5

G3, M & 0.01

5

N

0/5

G4, M & 0.03

5

N

0/5

G1R, M & 0

5

N

0/5

G4R, M & 0.03

5

N

0/5

M: Male; N: None; R: Recovery.


TABLE 1 (Contd…). SUMMARY OF CLINICAL SIGNS AND MORTALITY RECORD

                                                                                                                              Refer Appendix 1

Group, Sex & Dose

(mg/L)

No. of

Animals

Clinical Signs of Toxicity

Mortality                               (No. of Incidence/

No. of Animals)

G1, F & 0

5

N

0/5

G2, F & 0.003

5

N

0/5

G3, F & 0.01

5

N

0/5

G4, F & 0.03

5

N

0/5

G1R, F & 0

5

N

0/5

G4R, F & 0.03

5

N

0/5

F: Female; N: None; R: Recovery.

 

                            


TABLE 2.  SUMMARY OF BODY WEIGHTS (g)RECORD

 Refer Appendix 2

Group, Sex & Dose (mg/L)

Body Weight (g) on Days

1

5

8

12

15

22

26

G1, M & 0

Mean

166.51

171.75

178.32

185.81

197.59

214.19

225.14

±SD

9.48

10.36

10.11

10.38

12.00

12.39

13.10

n

5

5

5

5

5

5

5

G2, M & 0.003

Mean

170.65

174.98

180.83

188.27

204.29

219.31

229.19

±SD

14.03

13.78

13.91

13.41

13.53

13.31

11.58

n

5

5

5

5

5

5

5

G3, M & 0.01

Mean

163.66

168.12

174.43

181.48

198.20

213.03

224.77

±SD

9.42

9.17

9.25

9.10

10.10

9.87

9.39

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

163.99

168.87

175.43

182.93

198.64

214.21

225.74

±SD

9.27

9.25

8.92

8.36

8.87

8.00

8.84

n

5

5

5

5

5

5

5

G1, F & 0

Mean

148.51

152.03

155.66

160.28

172.22

183.16

191.20

±SD

11.18

11.98

11.73

11.04

11.11

10.71

11.54

n

5

5

5

5

5

5

5

G2, F & 0.003

Mean

150.58

154.23

158.35

163.93

177.05

187.21

195.08

±SD

9.04

8.92

8.43

8.83

8.94

8.94

9.12

n

5

5

5

5

5

5

5

G3, F & 0.01

Mean

147.71

151.43

155.06

160.47

173.49

182.83

191.88

±SD

9.33

9.83

9.54

9.60

9.58

9.25

9.64

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

147.30

150.33

154.63

159.60

172.10

182.59

191.08

±SD

8.92

9.02

9.36

8.45

6.81

6.49

6.69

n

5

5

5

5

5

5

5

 M: Male; F: Female; SD: Standard Deviation; n: Number of animals


TABLE 2 (Contd...). SUMMARY OF BODY WEIGHTS (g) RECORD

 Refer Appendix 2

Group, Sex & Dose (mg/L)

Body Weight (g) on Days

1

5

8

12

15

22

29

36

43

50

57

64

71

78

84

G1R, M & 0

Mean

166.20

171.02

177.66

185.62

200.07

214.49

232.63

253.26

274.79

299.32

325.27

349.03

372.79

397.94

413.98

±SD

11.24

11.18

10.45

10.60

12.29

11.09

10.88

10.72

10.05

10.33

9.74

8.98

8.83

7.95

5.30

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

164.61

169.11

175.81

183.14

199.36

215.18

234.98

256.10

278.55

303.45

328.88

353.30

374.05

398.71

415.74

±SD

5.82

6.41

6.91

6.52

8.72

7.25

7.24

8.04

7.24

7.94

8.28

7.95

4.25

4.86

3.34

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

148.78

151.72

155.52

160.51

173.51

184.31

198.22

210.64

225.19

239.40

255.46

270.87

285.04

301.89

317.03

±SD

6.32

5.93

5.77

5.65

6.78

6.41

6.86

8.13

7.78

7.89

7.61

8.09

9.30

8.70

8.48

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

146.83

150.42

154.40

158.83

170.78

181.28

196.23

210.43

224.38

239.40

255.44

270.86

283.41

300.91

314.61

±SD

5.66

5.28

5.62

5.57

5.71

4.93

4.84

5.04

5.63

4.95

4.82

4.24

7.37

7.36

5.82

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

 M: Male; F: Female; R: Recovery; -: Not applicable; SD: Standard Deviation; n: Number of animals


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

Refer Appendix 3

Group, Sex & Dose (mg/L)

Percent Change in Body Weight (%) during Days

1 to 5

1 to 8

1 to 12

1 to 15

1 to 22

1 to 26

G1, M & 0

Mean

3.13

7.09

11.59

18.65

28.64

35.21

±SD

0.42

0.29

0.62

1.61

1.60

1.89

n

5

5

5

5

5

5

G2, M & 0.003

Mean

2.56

6.00

10.41

19.85

28.72

34.60

±SD

0.57

0.62

1.30

2.90

3.74

4.82

n

5

5

5

5

5

5

G3, M & 0.01

Mean

2.74

6.61

10.93

21.15

30.24

37.45

±SD

0.74

0.86

1.34

1.71

2.10

2.63

n

5

5

5

5

5

5

G4, M & 0.03

Mean

2.98

7.00

11.60

21.19

30.73

37.77

±SD

0.25

0.90

1.19

1.65

2.41

3.00

n

5

5

5

5

5

5

G1, F & 0

Mean

2.35

4.81

7.96

16.04

23.45

28.86

±SD

0.73

0.44

0.97

1.64

2.43

2.48

n

5

5

5

5

5

5

G2, F & 0.003

Mean

2.44

5.19

8.90

17.64

24.40

29.64

±SD

0.57

0.96

0.74

1.44

1.75

1.82

n

5

5

5

5

5

5

G3, F & 0.01

Mean

2.51

4.99

8.66

17.51

23.86

29.99

±SD

0.84

0.80

0.54

1.62

1.82

1.86

n

5

5

5

5

5

5

G4, F & 0.03

Mean

2.06

4.98

8.39

16.96

24.11

29.88

±SD

0.24

0.39

1.16

2.98

3.66

3.41

n

5

5

5

5

5

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals

 

 

 

 

 

 

 

 


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

Refer Appendix 3

Group, Sex & Dose (mg/L)

Percent Change in Body Weight (%) during Days

1 to 5

1 to 8

1 to 12

1 to 15

1 to 22

1 to 29

1 to 36

1 to 43

1 to 50

1 to 57

1 to 64

1 to 71

1 to 78

1 to 84

G1R, M & 0

Mean

2.91

6.95

11.75

20.43

29.17

40.12

52.59

65.61

80.42

96.11

110.48

124.83

140.04

149.84

±SD

0.67

1.58

1.96

1.83

2.75

3.14

4.02

5.24

6.27

7.78

8.81

10.07

11.39

14.09

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

2.72

6.79

11.26

21.08

30.74

42.77

55.60

69.26

84.40

99.85

114.71

127.39

142.38

152.75

±SD

0.52

0.60

0.54

1.10

1.63

1.00

1.14

1.90

2.29

2.19

2.89

5.59

5.82

6.84

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

1.99

4.55

7.91

16.63

23.91

33.27

41.61

51.41

60.97

71.80

82.16

91.70

103.06

113.25

±SD

0.66

0.67

1.07

0.62

1.05

1.18

1.88

2.74

2.57

3.42

3.53

5.57

6.21

6.31

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

2.45

5.16

8.18

16.34

23.52

33.72

43.39

52.91

63.15

74.09

84.61

93.13

105.07

114.44

±SD

0.52

0.95

0.96

2.29

2.66

3.11

3.00

3.85

3.92

4.45

4.61

5.07

5.26

6.20

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of animal


TABLE 4.  SUMMARY OF AVERAGE FEED CONSUMPTION (g/rat/day)RECORD

 Refer Appendix 4

Group, Sex & Dose (mg/L)

Week 1

Week 2

Week 3

Week 4

G1, M & 0

Mean

18.67

18.63

18.49

28.45

±SD

2.73

2.83

2.81

3.70

n

2

2

2

2

G2, M & 0.003

Mean

18.61

18.66

18.53

28.03

±SD

3.03

2.77

2.62

3.35

n

2

2

2

2

G3, M & 0.01

Mean

18.78

18.78

18.54

28.01

±SD

3.04

2.87

2.77

3.54

n

2

2

2

2

G4, M & 0.03

Mean

18.53

18.76

18.56

28.05

±SD

2.85

3.06

3.00

3.38

n

2

2

2

2

G1, F & 0

Mean

14.63

14.73

14.61

21.69

±SD

1.59

1.62

1.48

1.31

n

2

2

2

2

G2, F & 0.003

Mean

14.65

14.80

14.64

21.44

±SD

1.41

1.46

1.40

1.43

n

2

2

2

2

G3, F & 0.01

Mean

14.55

14.80

14.68

20.95

±SD

1.40

1.41

1.48

1.22

n

2

2

2

2

G4, F & 0.03

Mean

14.49

14.66

14.53

20.60

±SD

1.37

1.38

1.43

1.13

n

2

2

2

2

 M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: number of cages.


TABLE 4 (Contd...). SUMMARY OF AVERAGE FEED CONSUMPTION (g/rat/day) RECORD

 Refer Appendix 4

Group, Sex & Dose (mg/L)

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

G1R, M & 0

Mean

18.68

18.77

18.50

18.66

18.72

18.78

18.92

19.23

19.43

19.62

19.84

23.11

±SD

3.04

3.04

2.90

2.93

3.02

3.02

3.00

3.27

3.32

3.32

3.44

4.06

n

2

2

2

2

2

2

2

2

2

2

2

2

G4R, M & 0.03

Mean

18.69

18.76

18.59

18.72

18.73

18.80

18.96

19.35

19.41

19.59

19.76

23.06

±SD

2.60

2.87

2.78

2.91

3.06

3.03

2.98

3.21

3.25

3.37

3.40

4.08

n

2

2

2

2

2

2

2

2

2

2

2

2

G1R, F & 0

Mean

14.53

14.76

14.64

14.85

14.99

14.99

15.18

15.56

15.67

15.83

16.03

18.64

±SD

1.39

1.47

1.34

1.74

1.77

1.68

1.78

1.90

1.91

1.91

2.12

2.44

n

2

2

2

2

2

2

2

2

2

2

2

2

G4R, F & 0.03

Mean

14.38

14.61

14.59

14.85

14.94

14.96

15.21

15.41

15.59

15.74

15.87

18.39

±SD

1.31

1.38

1.25

1.52

1.58

1.52

1.60

1.59

1.65

1.76

1.71

2.03

n

2

2

2

2

2

2

2

2

2

2

2

2

 M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: number of cages.


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

Refer Appendix 5

Week 4

Parameters↓

Group & Sex

G1 & M

G4 & M

G1 & F

G4 & F

Dose (mg/L)

0

0.03

0

0.03

Number of Animals

5

5

5

5

Home cage observations

Home Cage posture

1

1

1

1

Respiratory pattern

1

1

1

1

Clonic involuntary movements

1

1

1

1

Tonic involuntary movements

1

1

1

1

Vocalization

1

1

1

1

Palpebral Closure

1

1

1

1

Handling observation

 

 

Ease of removal from the cage

2

2

2

2

Ease of handling animal in hand

2

2

2

2

Red or crusty deposits

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

Lacrimation

1

1

1

1

Salivation

1

1

1

1

Fur Appearance

1

1

1

1

Piloerection

1

1

1

1

Eye Prominence

1

1

1

1

Muscle Tone

1

1

1

1

Home cage observations: Home cage posture- 1=Normal,Respiratory Pattern -1=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Vocalization -1=Absent (Normal),Palpebral closure -1=Eyelids wide open (Normal),Handling observation : Ease of removal from the cage -2=Normal,Ease of handling animal in hand -2=Normal,Red/Crusty deposits -1=Absent,Lacrimation- 1=None,Salivation-1=Absent (Normal),Fur Appearance -1=Normal,Piloerection -1=None, Eye Prominence -1=Normal,Muscle tone -1=Normal

M: Male; F: Female

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

Refer Appendix 5

Week 4

Parameters↓

Group & Sex

G1 & M

G4 & M

G1 & F

G4 & F

Dose (mg/L)

0

0.03

0

0.03

Number of Animals

5

5

5

5

Open field Observation

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of Rearing

Mean

8.0

5.4*

8.8

8.6

±SD

1.0

1.7

1.1

1.1

Numbers of Urination

Mean

1.2

1.2

1.4

1.4

±SD

0.4

0.4

0.5

0.9

Number of Defecation

Mean

0.8

0.8

0.8

1.2

±SD

0.4

0.4

0.4

0.8

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype Behaviour

1

1

1

1

Excessive Grooming

Mean

4.8

5.4

5.2

7.2

±SD

0.8

1.1

0.8

1.8

Sensory Observations

 

Approach Response

2

2

2

2

Auditory Response

2

2

2

2

Touch Response

2

2

2

2

Pupil Reflex

2

2

2

2

Tail Pinch Response

2

2

2

2

Righting Reflex

1

1

1

1

Physiological observation

 

Body temperature (°F)

Mean

96.7

97.1

98.5

98.5

±SD

0.8

1.1

0.1

0.1

Open field Observation: Mobility -1=Normal,Gait -1=Normal,Arousal -3=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Stereotype Behaviour -1=Absent, Sensory Observations: Approach response -2=Normal, Auditory Response -2=Normal,Touch Response -2=Normal,Pupil Reflex -2=Normal,Tail Pinch Response -2=Normal,Righting Reflex -1=Present

*: Statistically significant (p<0.05)


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

Refer Appendix 5

Week 12

Parameters↓

Group & Sex

G1R & M

G4R & M

G1R & F

G4R & F

Dose (mg/L)

0

0.03

0

0.03

Number of Animals

5

5

5

5

Home cage observations

Home Cage posture

1

1

1

1

Respiratory pattern

1

1

1

1

Clonic involuntary movements

1

1

1

1

Tonic involuntary movements

1

1

1

1

Vocalization

1

1

1

1

Palpebral Closure

1

1

1

1

Handling observation

 

 

Ease of removal from the cage

2

2

2

2

Ease of handling animal in hand

2

2

2

2

Red or crusty deposits

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

Lacrimation

1

1

1

1

Salivation

1

1

1

1

Fur Appearance

1

1

1

1

Piloerection

1

1

1

1

Eye Prominence

1

1

1

1

Muscle Tone

1

1

1

1

Home cage observations: Home cage posture- 1=Normal,Respiratory Pattern -1=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Vocalization -1=Absent (Normal),Palpebral closure -1=Eyelids wide open (Normal),Handling observation : Ease of removal from the cage -2=Normal,Ease of handling animal in hand -2=Normal,Red/Crusty deposits -1=Absent,Lacrimation- 1=None,Salivation-1=Absent (Normal),Fur Appearance -1=Normal,Piloerection -1=None, Eye Prominence -1=Normal,Muscle tone -1=Normal

M: Male; F: Female; R: Recovery

 


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

                                                                                                     Refer Appendix 5

Week 12

Parameters↓

Group & Sex

G1R & M

G4R & M

G1R & F

G4R & F

Dose (mg/L)

0

0.03

0

0.03

Number of Animals

5

5

5

5

Open field Observation

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of Rearing

Mean

6.8

5.6

8.8

8.0

±SD

1.5

1.3

0.8

2.0

Numbers of Urination

Mean

1.0

1.2

1.2

1.2

±SD

0.0

0.4

0.4

0.4

Number of Defecation

Mean

1.0

1.2

1.0

1.2

±SD

0.7

0.8

0.7

0.8

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype Behaviour

1

1

1

1

Excessive Grooming

Mean

5.0

5.0

5.6

5.8

±SD

0.7

0.7

1.3

0.8

Sensory Observations

 

Approach Response

2

2

2

2

Auditory Response

2

2

2

2

Touch Response

2

2

2

2

Pupil Reflex

2

2

2

2

Tail Pinch Response

2

2

2

2

Righting Reflex

1

1

1

1

Physiological observation

 

Body temperature (°F)

Mean

96.9

96.6

98.4

98.6

±SD

0.2

0.4

0.2

0.2

Open field Observation: Mobility -1=Normal,Gait -1=Normal,Arousal -3=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Stereotype Behaviour -1=Absent, Sensory Observations: Approach response -2=Normal, Auditory Response -2=Normal,Touch Response -2=Normal,Pupil Reflex -2=Normal,Tail Pinch Response -2=Normal,Righting Reflex -1=Present

M: Male; F: Female; R: Recovery


TABLE 6.  SUMMARY OF HIND LIMB FOOT SPLAY (cm)RECORD

                                                                                                                                                       Refer Appendix 6

# Week 4 & 12

Group, Sex & Dose (mg/L)

Hind Limb Foot Splay (cm)

G1, M & 0

Mean

6.2

±SD

0.5

n

5

G4, M & 0.03

Mean

6.1

±SD

0.4

n

5

G1, F & 0

Mean

5.7

±SD

0.5

n

5

G4, F & 0.03

Mean

5.6

±SD

0.4

n

5

G1R, M & 0

Mean

8.7

±SD

0.5

n

5

G4R, M & 0.03

Mean

8.1

±SD

0.5

n

5

G1R, F & 0

Mean

5.5

±SD

0.2

n

5

G4R, F & 0.03

Mean

5.5

±SD

0.1

n

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: number of animals

# Week 4 for main group animals and week 12 for recovery group animals.

 


TABLE 7.SUMMARY OF ACTIMETER READING RECORD

                                                                                                                                                       Refer Appendix 7

# Week 4 & 12

Group, Sex & Dose (mg/L)

Movement Counts (no.)

G1, M & 0

Mean

2171.8

±SD

70.6

n

5

G4, M & 0.03

Mean

2202.0

±SD

53.8

n

5

G1, F & 0

Mean

2407.8

±SD

28.4

n

5

G4, F & 0.03

Mean

2395.8

±SD

65.2

n

5

G1R, M & 0

Mean

2239.6

±SD

33.0

n

5

G4R, M & 0.03

Mean

2260.8

±SD

57.3

n

5

G1R, F & 0

Mean

2341.8

±SD

39.5

n

5

G4R, F & 0.03

Mean

2420.6*

±SD

35.9

n

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: number of animals;

# Week 4 for main group animals and week 12 for recovery group animals; *: Statistically significant (p<0.05).

 

 


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

Refer Appendix 8

# Week 4 & 12

Group, Sex & Dose (mg/L)

Fore limb

Hind limb

G1, M & 0

Mean

1.505

0.534

±SD

0.051

0.087

n

5

5

G4, M & 0.03

Mean

1.486

0.472

±SD

0.049

0.022

n

5

5

G1, F & 0

Mean

1.432

0.407

±SD

0.039

0.048

n

5

5

G4, F & 0.03

Mean

1.410

0.375

±SD

0.022

0.058

n

5

5

G1R, M & 0

Mean

1.507

0.533

±SD

0.052

0.085

n

5

5

G4R, M & 0.03

Mean

1.489

0.478

±SD

0.056

0.021

n

5

5

G1R, F & 0

Mean

1.432

0.407

±SD

0.041

0.048

n

5

5

G4R, F & 0.03

Mean

1.416

0.381

±SD

0.024

0.062

n

5

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: number of animals

# Week 4 for main group animals and week 12 for recovery group animals.

 

 

 

 

 

 

 

 

 

 

 

 


TABLE 9.     SUMMARY OF OPHTHALMOSCOPIC EXAMINATION RECORD

Week 4 and 12                                                                                                                               Refer Appendix 9

Week 4

Week 12

Group, Sex & Dose

(mg/L)

G1, M & 0

G4, M & 0.03

G1R, M & 0

G4R, M & 0.03

Number of Animals

05

05

05

05

Observations

Eye Parameters ↓

Eye

L

R

L

R

L

R

L

R

Eye Lids

N

N

N

N

N

N

N

N

Cornea

N

N

N

N

N

N

N

N

Iris

N

N

N

N

N

N

N

N

Aqueous Humour

N

N

N

N

N

N

N

N

Lens

N

N

N

N

N

N

N

N

Vitreous Humour

N

N

N

N

N

N

N

N

Retina/Optic disc

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N: No Abnormality Detected; L: Left; R: Right; M: Male; F: Female.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


TABLE 9 (Contd…).  SUMMARY OF OPHTHALMOSCOPIC EXAMINATION RECORD

Week 4 and 12                                                                                                                              Refer Appendix 9 

Week 4

Week 12

Group, Sex & Dose

(mg/L)

G1, F & 0

G4, F & 0.03

G1R, F & 0

G4R, F & 0.03

Number of Animals

05

05

05

05

Observations

Eye Parameters ↓

Eye

L

R

L

R

L

R

L

R

Eye Lids

N

N

N

N

N

N

N

N

Cornea

N

N

N

N

N

N

N

N

Iris

N

N

N

N

N

N

N

N

Aqueous Humour

N

N

N

N

N

N

N

N

Lens

N

N

N

N

N

N

N

N

Vitreous Humour

N

N

N

N

N

N

N

N

Retina/Optic disc

N

N

N

N

N

N

N

N

N: No Abnormality Detected; L: Left; R: Right; F: Female.


TABLE 10. SUMMARY OF HAEMATOLOGY RECORD

                                        Refer Appendix 10

Group, Sex & Dose (mg/L)

Total Leucocyte Count

Total Erythrocyte Count

Hemoglobin

Haematocrit

Mean Corpuscular Volume

Mean Corpuscular Hemoglobin

Mean Corpuscular Hemoglobin Concentration

Platelet Count

Mean Platelet Volume

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(PLT)

(MPV)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

G1, M & 0

Mean

7.63

7.81

15.52

44.20

56.60

19.94

35.26

759.00

6.18

±SD

2.12

0.60

0.98

3.50

0.52

1.32

2.33

344.07

0.48

n

5

5

5

5

5

5

5

5

5

G2, M & 0.003

Mean

9.19

8.42

15.68

46.98

55.86

18.66

33.40

1063.80

6.14

±SD

2.75

0.57

0.69

1.92

1.81

0.85

0.61

178.32

0.23

n

5

5

5

5

5

5

5

5

5

G3, M & 0.01

Mean

9.38

8.17

15.58

47.12

57.68

19.06

33.06*

1081.40

6.44

±SD

3.42

0.34

1.16

3.13

1.71

0.78

0.48

214.16

0.36

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

8.11

8.92*

16.50

49.08*

55.04

18.52

33.64

1039.80

6.80*

±SD

0.95

0.66

1.00

2.92

1.46

0.64

0.39

149.35

0.40

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

9.48

9.58

16.80

51.10

53.34

17.54

32.86

783.60

6.42

±SD

1.23

0.14

0.52

1.08

0.70

0.44

0.43

122.98

0.19

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

10.11

9.07

16.88

48.48

53.58

18.76

34.96

930.60

6.38

±SD

1.65

0.86

0.15

3.15

1.88

1.94

2.50

172.00

0.33

n

5

5

5

5

5

5

5

5

5

      M: Male; R: Recovery; SD: Standard Deviation; n: number of animals; *: Statistically significant (p<0.05).

 

 

 

 

 

 

 

 


TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

                                                                                      Refer Appendix 10

Group, Sex & Dose (mg/L)

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Reticulocyte Count

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Retic)

(%)

(%)

(%)

(%)

(%)

(%)

(109cells/L)

G1, M & 0

Mean

2.94

23.88

70.18

3.50

1.16

0.32

229.14

±SD

0.85

6.93

6.12

0.89

0.33

0.08

68.43

n

5

5

5

5

5

5

5

G2, M & 0.003

Mean

2.63

18.86

76.02

3.10

0.82

0.34

221.26

±SD

0.96

2.70

2.61

0.92

0.11

0.09

76.02

n

5

5

5

5

5

5

5

G3, M & 0.01

Mean

2.77

23.30

71.54

2.48

1.30

0.38

225.82

±SD

0.70

3.45

2.77

0.82

0.81

0.11

54.77

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

2.49

23.78

71.38

2.70

1.00

0.36

217.74

±SD

0.95

7.63

8.14

0.83

0.34

0.11

67.75

n

5

5

5

5

5

5

5

G1R, M & 0

Mean

1.81

25.66

69.22

2.68

1.32

0.54

173.32

±SD

0.22

7.18

7.42

0.83

0.40

0.24

21.10

n

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

1.43*

26.90

68.00

2.80

1.08

0.46

129.64*

±SD

0.11

2.90

3.09

0.88

0.29

0.11

18.15

n

5

5

5

5

5

5

5

 M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

                                       

 

 

 

 

TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

   Refer Appendix 10

Group, Sex & Dose (mg/L)

Absolute Neutrophils

Absolute Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated Prothrombin Time

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, M & 0

Mean

1.82

5.35

0.27

0.09

0.02

14.42

16.94

±SD

0.78

1.55

0.11

0.04

0.01

0.68

3.96

n

5

5

5

5

5

5

5

G2, M & 0.003

Mean

1.78

6.94

0.28

0.07

0.03

15.82

19.02

±SD

0.72

1.90

0.11

0.02

0.02

1.30

5.09

n

5

5

5

5

5

5

5

G3, M & 0.01

Mean

2.10

6.77

0.24

0.13

0.04

15.34

23.22

±SD

0.53

2.67

0.15

0.12

0.02

1.20

9.17

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

1.90

5.82

0.22

0.08

0.03

17.42*

24.40

±SD

0.53

1.17

0.06

0.02

0.01

1.59

2.02

n

5

5

5

5

5

5

5

G1R, M & 0

Mean

2.48

6.51

0.26

0.12

0.05

16.14

28.36

±SD

0.97

0.54

0.11

0.04

0.02

1.85

11.80

n

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

2.70

6.88

0.29

0.12

0.05

15.66

27.48

±SD

0.45

1.21

0.12

0.05

0.01

1.19

4.01

n

5

5

5

5

5

5

5

       M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals

 

                                                                                                                                  


TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

       Refer Appendix 10

Group, Sex & Dose (mg/L)

Total Leucocyte Count

Total Erythrocyte Count

Hemoglobin

Haematocrit

Mean Corpuscular Volume

Mean Corpuscular Hemoglobin

Mean Corpuscular Hemoglobin Concentration

Platelet Count

Mean Platelet Volume

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(PLT)

(MPV)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

G1, F & 0

Mean

9.14

8.51

16.12

48.02

56.32

18.92

33.66

906.20

6.96

±SD

2.24

0.30

0.64

2.65

1.48

0.71

1.88

313.91

0.23

n

5

5

5

5

5

5

5

5

5

G2, F & 0.003

Mean

8.33

8.39

16.02

48.42

57.76

19.08

33.08

782.40

6.90

±SD

1.71

0.45

0.82

2.72

1.55

0.48

0.60

256.78

0.22

n

5

5

5

5

5

5

5

5

5

G3, F & 0.01

Mean

8.87

9.00

16.52

51.16

56.86

18.36

32.28

1092.80

6.92

±SD

1.95

0.49

0.67

2.35

2.08

0.73

0.25

89.68

0.22

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

8.54

8.95

16.42

50.80

56.78

18.34

32.32

977.40

6.90

±SD

1.49

0.28

0.52

1.16

1.14

0.40

0.41

117.58

0.19

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

9.67

8.51

16.04

47.36

55.70

18.82

33.80

918.40

6.62

±SD

2.17

0.44

0.68

1.43

2.09

0.65

0.66

130.17

0.33

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

7.58

9.06

16.56

48.78

53.90

18.32

33.94

597.80

6.98

±SD

2.19

0.33

0.72

2.11

1.12

0.53

0.40

308.99

1.65

n

5

5

5

5

5

5

5

5

5

      F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals

  

 

 


TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

       Refer Appendix 10

Group, Sex & Dose (mg/L)

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Reticulocyte Count

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Retic)

(%)

(%)

(%)

(%)

(%)

(%)

(109cells/L)

G1, F & 0

Mean

2.55

24.28

69.98

3.34

1.34

0.26

217.08

±SD

0.59

5.92

6.34

0.91

0.82

0.09

48.26

n

5

5

5

5

5

5

5

G2, F & 0.003

Mean

2.36

24.52

70.00

3.38

0.78

0.32

198.08

±SD

0.37

5.33

5.42

1.13

0.36

0.08

31.99

n

5

5

5

5

5

5

5

G3, F & 0.01

Mean

2.64

25.72

68.92

3.36

0.82

0.36

236.94

±SD

0.48

7.95

7.60

0.68

0.42

0.11

38.20

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

2.35

22.20

72.14

3.44

0.80

0.34

209.38

±SD

0.69

5.59

6.30

0.80

0.33

0.05

55.93

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

2.43

24.74

69.50

3.04

1.16

0.54

205.22

±SD

0.83

1.48

1.73

0.70

0.42

0.25

62.92

n

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

1.31*

30.22*

64.16*

2.96

1.50

0.56

118.78*

±SD

0.16

3.47

3.51

0.94

0.72

0.30

18.19

n

5

5

5

5

5

5

5

      F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

      

 

 

 


TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

     Refer Appendix 10

Group, Sex & Dose (mg/L)

Absolute Neutrophils

Absolute Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated Prothrombin Time

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, F & 0

Mean

2.16

6.46

0.31

0.11

0.03

16.36

16.84

±SD

0.45

1.93

0.12

0.05

0.02

1.87

2.96

n

5

5

5

5

5

5

5

G2, F & 0.003

Mean

2.08

5.80

0.28

0.07

0.02

17.10

21.68

±SD

0.83

1.02

0.11

0.04

0.01

2.14

4.63

n

5

5

5

5

5

5

5

G3, F & 0.01

Mean

2.32

6.09

0.29

0.07

0.04

13.74

19.86

±SD

0.98

1.38

0.05

0.03

0.02

1.90

2.10

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

1.91

6.13

0.30

0.07

0.03

16.46

27.30*

±SD

0.71

0.97

0.12

0.04

0.00

1.62

3.79

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

2.38

6.74

0.30

0.11

0.05

16.92

33.88

±SD

0.46

1.64

0.10

0.03

0.03

3.47

3.09

n

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

2.25

4.88

0.22

0.12

0.05

15.44

33.02

±SD

0.58

1.45

0.12

0.08

0.03

2.09

7.57

n

5

5

5

5

5

5

5

      F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

      

 

      


TABLE 11.SUMMARY OF BALF RECORD

                                                                                                                                                                                            Refer Appendix 11

Group, Sex & Dose (mg/L)

Total Leucocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Neutrophils

(WBC)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Neut)

(103cells/µL)

(%)

(%)

(%)

(%)

(%)

(103cells/µL)

G1, M & 0

 

Mean

1.19

29.60

54.24

3.00

0.86

1.44

0.58

±SD

1.45

20.44

18.64

2.65

1.06

0.52

1.05

n

5

5

5

5

5

5

5

G2, M & 0.003

Mean

0.77

32.70

56.88

2.08

0.80

2.60

0.23

±SD

0.51

9.76

10.60

1.55

0.63

1.89

0.17

n

5

5

5

5

5

5

5

G3, M & 0.01

Mean

0.32

30.42

59.32

1.92

0.88

1.84

0.10

±SD

0.16

5.85

5.34

1.31

0.73

0.90

0.05

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

0.72

27.22

66.56

2.18

1.58

2.18

0.16

±SD

0.85

10.58

10.94

0.64

1.06

0.39

0.13

n

5

5

5

5

5

5

5

G1R, M & 0

Mean

0.12

16.92

68.74

1.64

12.34

10.54

0.01

±SD

0.12

13.35

32.50

2.05

20.46

6.17

0.01

n

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

0.03

27.54

63.42

3.88

4.22

2.94*

0.01

±SD

0.01

24.10

29.51

5.23

3.22

3.20

0.01

n

5

5

5

5

5

5

5

      M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

       


TABLE 11 (Contd…). SUMMARY OF BALF RECORD

       Refer Appendix 11

Group, Sex & Dose (mg/L)

Absolute Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Lactate Dehydrogenase

Total Protein

(Lymph)

(Mono)

(Eos)

(Baso)

(LDH)

(TPR)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(u/L)

(g/dL)

G1, M & 0

Mean

0.44

0.07

0.01

0.01

135.52

349.19

±SD

0.25

0.12

0.01

0.02

130.92

127.80

n

5

5

5

5

5

5

G2, M & 0.003

Mean

0.43

0.02

0.00

0.01

82.20

321.13

±SD

0.31

0.02

0.01

0.00

39.77

171.66

n

5

5

5

5

5

5

G3, M & 0.01

Mean

0.20

0.01

0.00

0.01

56.22

325.64

±SD

0.11

0.01

0.00

0.01

19.47

107.69

n

5

5

5

5

5

5

G4, M & 0.03

Mean

0.51

0.01

0.01

0.01

80.04

299.91

±SD

0.66

0.02

0.01

0.02

51.42

111.84

n

5

5

5

5

5

5

G1R, M & 0

Mean

0.10

0.00

0.01

0.01

56.16

296.06

±SD

0.12

0.00

0.01

0.02

24.94

116.35

n

5

5

5

5

5

5

G4R, M & 0.03

Mean

0.02

0.00

0.00

0.00

140.88*

185.63

±SD

0.02

0.00

0.00

0.00

56.13

31.78

n

5

5

5

5

5

5

      M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)


TABLE 11 (Contd…). SUMMARY OF BALF RECORD

       Refer Appendix 11

Group, Sex & Dose (mg/L)

Total Leucocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Neutrophils

(WBC)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Neut)

(103cells/µL)

(%)

(%)

(%)

(%)

(%)

(103cells/µL)

G1, F & 0

Mean

0.32

21.94

65.62

1.72

0.32

5.42

0.07

±SD

0.07

2.42

5.73

0.60

0.18

4.49

0.01

n

5

5

5

5

5

5

5

G2, F & 0.003

Mean

0.43

27.32

52.80

1.66

0.30

1.68

0.12

±SD

0.17

5.99

4.72

0.73

0.22

0.73

0.06

n

5

5

5

5

5

5

5

G3, F & 0.01

Mean

0.87*

27.86

59.40

1.76

3.56

2.12

0.23

±SD

0.51

6.20

7.70

0.55

6.22

1.10

0.10

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

0.46

51.66*

42.10*

1.66

2.00

2.78

0.23

±SD

0.28

17.30

17.59

1.73

1.17

4.35

0.16

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

0.27

19.08

69.36

2.72

4.80

11.52

0.06

±SD

0.17

6.77

8.10

1.29

2.53

6.91

0.05

n

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

1.89

22.94

66.34

4.62

3.28

9.90

1.08

±SD

3.60

24.01

30.37

7.23

2.26

7.57

2.30

n

5

5

5

5

5

5

5

      F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

 


TABLE 11 (Contd…). SUMMARY OF BALF RECORD

       Refer Appendix 11

Group, Sex & Dose (mg/L)

Absolute Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Lactate Dehydrogenase

Total Protein

(Lymph)

(Mono)

(Eos)

(Baso)

(LDH)

(TPR)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(u/L)

(g/dL)

G1, F & 0

Mean

0.21

0.00

0.00

0.02

47.56

322.15

±SD

0.06

0.01

0.00

0.01

6.02

135.05

n

5

5

5

5

5

5

G2, F & 0.003

Mean

0.23

0.01

0.00

0.01

41.26

326.31

±SD

0.09

0.00

0.00

0.00

18.91

87.24

n

5

5

5

5

5

5

G3, F & 0.01

Mean

0.50*

0.02

0.05

0.02

102.52

369.37

±SD

0.26

0.02

0.12

0.01

39.86

120.36

n

5

5

5

5

5

5

G4, F & 0.03

Mean

0.20

0.01

0.01

0.02

146.44

371.40

±SD

0.13

0.02

0.01

0.03

188.18

300.11

n

5

5

5

5

5

5

G1R, F & 0

Mean

0.18

0.00

0.01

0.03

49.50

193.58

±SD

0.12

0.01

0.01

0.02

30.42

55.52

n

5

5

5

5

5

5

G4R, F & 0.03

Mean

0.46

0.29

0.04

0.09

140.56

229.77

±SD

0.59

0.65

0.08

0.15

175.38

44.27

n

5

5

5

5

5

5

      F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)


TABLE 12.  SUMMARY OF CLINICAL CHEMISTRYRECORD

                                                                                                                                                                                                                                                                 Refer Appendix 12

Group, Sex & Dose (mg/L)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

Total Protein

Albumin

Alanine aminotransferase

Aspartate aminotransferase

(GLU)

(CRE)

(CHO)

(TRI)

(TPR)

(ALB)

(ALT)

(AST)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

(U/L)

(U/L)

G1, M & 0

Mean

96.80

39.10

0.57

53.20

39.20

7.22

3.57

41.20

92.00

±SD

4.44

2.85

0.06

2.95

7.85

0.33

0.13

5.07

7.11

n

5

5

5

5

5

5

5

5

5

G2, M & 0.003

Mean

103.80

39.82

0.57

46.80

37.20

7.42

3.44

43.20

103.80

±SD

26.07

6.93

0.05

14.24

15.30

0.33

0.27

6.61

13.01

n

5

5

5

5

5

5

5

5

5

G3, M & 0.01

Mean

90.20

41.28

0.54

54.60

40.80

7.58

3.39

54.00

112.80*

±SD

8.67

4.69

0.03

12.97

15.02

0.22

0.14

15.44

13.05

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

95.60

39.16

0.54

42.80

37.00

7.70

3.52

39.40

101.00

±SD

4.83

6.64

0.04

13.07

3.94

0.67

0.23

7.54

9.67

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

107.00

29.42

0.49

48.80

40.00

7.14

3.66

67.60

129.40

±SD

18.19

6.19

0.02

7.01

12.83

0.30

0.09

37.04

90.48

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

101.00

28.38

0.46

56.60

53.00

7.10

3.78

56.40

88.60

±SD

10.37

3.04

0.02

14.66

6.12

0.48

0.25

5.37

10.74

n

5

5

5

5

5

5

5

5

5

              M: Male; R: Recovery; SD: Standard Deviation; n: number of animals; *: Statistically significant (p<0.05)

 

 

TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Refer Appendix 12

Group, Sex & Dose (mg/L)

Alkaline phosphatase

Total Bilirubin

Calcium

Phosphorous

Globulin

Blood Urea Nitrogen

Sodium

Potassium

Chloride

(ALP)

(BIT)

(CAL)

(PHO)

(GLO)

(BUN)

(Na)

(K)

(CLO)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1, M & 0

Mean

135.20

0.01

9.88

6.22

3.65

18.25

141.42

3.50

110.18

±SD

34.04

0.01

0.26

0.36

0.33

1.33

0.68

0.18

0.86

n

5

5

5

5

5

5

5

5

5

G2, M & 0.003

Mean

116.80

0.02

10.06

6.86

3.98

18.58

142.36

4.02

110.62

±SD

29.58

0.02

0.15

0.47

0.25

3.23

0.53

0.59

1.82

n

5

5

5

5

5

5

5

5

5

G3, M & 0.01

Mean

142.60

0.03

10.26*

6.86

4.19

19.26

143.06

3.92

110.80

±SD

39.84

0.03

0.09

1.00

0.20

2.19

1.21

0.31

2.59

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

105.80

0.02

9.68

6.36

4.18

18.28

143.46*

3.66

110.12

±SD

11.95

0.01

0.20

0.45

0.60

3.10

1.78

0.25

2.37

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

143.00

0.01

9.44

6.46

3.48

13.73

142.42

3.74

106.80

±SD

49.28

0.01

0.30

0.34

0.29

2.89

0.71

0.45

0.70

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

161.60

0.01

9.76

6.66

3.32

13.25

142.08

3.57

105.20

±SD

94.73

0.01

0.23

0.55

0.28

1.42

0.73

0.16

1.51

n

5

5

5

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

 

 

TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY

Refer Appendix 12

Group, Sex & Dose (mg/L)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

Total Protein

Albumin

Alanine aminotransferase

Aspartate aminotransferase

(GLU)

(CRE)

(CHO)

(TRI)

(TPR)

(ALB)

(ALT)

(AST)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

(U/L)

(U/L)

G1, F & 0

Mean

79.80

39.56

0.52

42.80

34.40

7.00

3.41

52.20

108.60

±SD

8.56

4.16

0.07

3.96

4.67

0.31

0.11

7.79

16.70

n

5

5

5

5

5

5

5

5

5

G2, F & 0.003

Mean

87.80

37.04

0.51

48.80

55.00

7.20

3.41

60.40

123.40

±SD

11.56

4.05

0.05

8.58

30.10

0.32

0.15

19.63

32.67

n

5

5

5

5

5

5

5

5

5

G3, F & 0.01

Mean

91.80

35.14

0.51

45.00

34.00

7.28

3.60

57.00

108.60

±SD

8.14

1.95

0.03

6.28

8.54

0.16

0.11

11.11

8.11

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

101.20*

49.52*

0.52

40.60

34.20

7.38

3.50

62.40

113.60

±SD

17.84

7.88

0.03

5.77

15.11

0.37

0.18

6.69

9.02

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

98.20

28.50

0.52

73.80

39.40

7.52

3.97

45.00

94.00

±SD

6.91

1.52

0.02

11.28

6.62

0.36

0.22

3.54

8.60

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

110.60

40.98

0.63

67.00

40.40

7.56

3.92

57.80

97.80

±SD

20.18

11.85

0.23

11.25

8.50

0.13

0.06

13.74

13.59

n

5

5

5

5

5

5

5

5

5

 F: Female; R: Recovery; SD: Standard Deviation; n: number of animals; *: Statistically significant (p<0.05)

 

 

 

TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY

Refer Appendix 12

Group, Sex & Dose (mg/L)

Alkaline phosphatase

Total Bilirubin

Calcium

Phosphorous

Globulin

Blood Urea Nitrogen

Sodium

Potassium

Chloride

(ALP)

(BIT)

(CAL)

(PHO)

(GLO)

(BUN)

(Na)

(K)

(CLO)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1, F & 0

Mean

200.40

0.02

10.00

7.58

3.59

18.46

143.34

4.02

110.70

±SD

35.13

0.02

0.31

0.62

0.25

1.94

0.85

0.26

1.70

n

5

5

5

5

5

5

5

5

5

G2, F & 0.003

Mean

179.00

0.03

9.88

7.22

3.79

17.29

142.58

4.12

109.02

±SD

24.21

0.01

0.37

0.38

0.22

1.89

2.66

0.37

1.99

n

5

5

5

5

5

5

5

5

5

G3, F & 0.01

Mean

201.00

0.02

10.20

7.10

3.68

16.40

142.94

3.79

108.82

±SD

27.65

0.02

0.07

0.32

0.17

0.91

1.16

0.05

1.53

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

186.80

0.03

9.92

7.06

3.88

23.11*

144.54

3.99

112.36

±SD

55.68

0.01

0.34

0.98

0.20

3.68

1.30

0.14

1.50

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

67.00

0.03

10.00

5.94

3.55

13.30

141.60

3.42

106.46

±SD

31.44

0.02

0.30

0.38

0.29

0.71

0.95

0.19

0.94

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

95.00

0.03

9.80

5.72

3.64

19.12

141.66

3.82*

106.10

±SD

29.97

0.02

0.21

1.10

0.17

5.53

1.15

0.22

1.75

n

5

5

5

5

5

5

5

5

5

F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

 

 

 


TABLE 13.SUMMARY OF URINALYSIS

         Refer Appendix 13

Examination

Group, Sex & Dose (mg/L)

G1, M & 0

G2, M & 0.003

G3, M & 0.01

G4, M & 0.03

G1R, M & 0

G4R, M & 0.03

Number of Animals

5

5

5

5

5

5

Physical

Colour

Pale Yellow

5

5

5

5

5

4

Yellow

-

-

-

-

-

1

Appearance

Clear

5

5

5

5

4

3

Turbid

-

-

-

-

1

2

Volume (mL)

Mean

4.8

5.9

6.8

5.9

5.3

4.2

±SD

1.2

1.7

1.3

1.0

0.9

0.6

Chemical

pH

Mean

7.8

7.8

7.7

7.6

7.2

7.4

±SD

0.3

0.3

0.3

0.4

0.7

0.8

Specific Gravity

Mean

1.009

1.010

1.011

1.012

1.013

1.010

±SD

0.002

0.000

0.002

0.003

0.004

0.004

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.2

0.2

0.2

0.2

±SD

0.0

0.0

0.0

0.0

0.0

0.0

Bilirubin (mg/dL)

Neg

1

4

4

3

5

4

1

4

1

1

2

-

1

Ketones (mg/dL)

Neg

2

1

3

-

-

2

5

3

4

2

5

5

3

Blood (Ery/µL)

Neg

5

3

5

4

4

5

Ca10

-

1

-

1

1

-

Ca25

-

1

-

-

-

-

Proteins (mg/dL)

Neg

2

4

2

3

3

3

Trace

1

-

2

2

1

-

30

2

1

1

-

1

1

>=300

-

-

-

-

-

1

Nitrite

Neg

1

3

1

2

4

3

Pos

4

2

4

3

1

2

Leucocytes (Leu/µL)

Neg

-

-

-

1

-

1

Ca15

-

2

1

2

2

2

Ca70

5

3

4

2

3

2

Glucose (mg/dL)

Neg

5

5

5

5

5

5

Micro albumin (MALB) (mg/dL)

Neg

-

1

-

1

-

1

>15

3

1

3

2

2

2

15

2

3

2

2

3

2

Microscopic

Epithelial cells

0

4

5

5

5

2

4

0-1

1

-

-

-

2

-

1-2

-

-

-

-

1

1

Casts

Absent

5

5

5

5

5

5

Crystals

Present

5

5

5

5

5

5

       M: Male; Neg: Negative; Ca: Calculated Approximately; Pos: Positive; SD: Standard Deviation

        

 

 

 

 

 

 

 

 

 

TABLE 13 (Contd…). SUMMARY OF URINALYSIS

Refer Appendix 13

Examination

Group, Sex & Dose (mg/L)

G1, F &

0

G2, F & 0.003

G3, F & 0.01

G4, F &

0.03

G1R, F &

0

G4R, F

&

0.03

Number of Animals

5

5

5

5

5

5

Physical

Colour

Pale Yellow

5

5

5

5

4

5

Yellow

-

-

-

-

1

-

Appearance

Clear

5

5

5

5

4

4

Turbid

-

-

-

-

1

1

Volume (mL)

Mean

5.8

5.1

5.2

5.0

4.0

4.6

±SD

1.5

1.3

0.8

0.9

0.4

1.1

Chemical

pH

Mean

7.3

7.1

7.7

7.8

7.3

7.0

±SD

0.8

1.0

0.3

0.3

0.8

0.8

Specific Gravity

Mean

1.012

1.016

1.010

1.009

1.009

1.012

±SD

0.008

0.007

0.000

0.002

0.004

0.003

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.2

0.6

0.2

0.2

±SD

0.0

0.0

0.0

0.8

0.0

0.0

Bilirubin (mg/dL)

Neg

4

3

5

4

4

5

1

1

2

-

1

1

-

Ketones (mg/dL)

Neg

5

5

5

5

5

5

Blood (Ery/µL)

Neg

5

3

4

4

5

4

Ca10

-

1

1

1

-

-

Ca25

-

1

-

-

-

1

Proteins (mg/dL)

Neg

5

5

4

3

2

4

Trace

-

-

1

1

2

-

30

-

-

-

-

1

1

>=300

-

-

-

1

-

-

Nitrite

Neg

3

2

4

4

3

4

Pos

2

3

1

1

2

1

Leucocytes (Leu/µL)

Neg

-

2

-

-

3

1

Ca15

5

3

2

2

1

4

Ca70

-

-

3

2

1

-

Ca125

-

-

-

1

-

-

Glucose (mg/dL)

Neg

5

5

5

5

5

5

Micro albumin (MALB) (mg/dL)

Neg

1

2

1

1

1

1

>15

-

-

1

2

3

1

15

4

3

3

2

1

3

Microscopic

Epithelial cells

0

4

4

4

3

1

3

0-1

1

-

1

1

3

1

1-2

-

1

-

1

1

1

Casts

Absent

5

5

5

5

5

5

Crystals

Present

5

5

5

5

5

5

       F: Female; Neg: Negative; Ca: Calculated Approximately; Pos: Positive; SD: Standard Deviation

      


TABLE 14.    SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g)

                                                                                                                                                                                                                                                                    Refer Appendix 14

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Testes

Heart

Kidneys

Brain

Liver

Lungs

G1, M & 0

Mean

0.0591

0.3086

0.4909

2.8529

0.8080

1.6090

1.8356

6.7114

0.6179

±SD

0.0205

0.0655

0.0487

0.0423

0.1208

0.1735

0.0435

0.5112

0.1409

n

5

5

5

5

5

5

5

5

5

G2, M & 0.003

Mean

0.0491

0.3546

0.4750

3.0797

0.9093

1.8123

1.8330

7.3951

0.5748

±SD

0.0017

0.0335

0.0973

0.1235

0.0870

0.1910

0.0669

0.9794

0.0890

n

5

5

5

5

5

5

5

5

5

G3, M & 0.01

Mean

0.0585

0.2789

0.4677

3.0948

0.8801

1.7441

1.7921

6.8768

0.5045

±SD

0.0101

0.0359

0.0834

0.2170

0.1741

0.1330

0.1249

0.8572

0.1224

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

0.0499

0.2645

0.4638

3.0833

0.8060

1.5553

1.7554

6.7946

0.5538

±SD

0.0059

0.0298

0.1002

0.1584

0.1314

0.1997

0.1222

0.7357

0.0879

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

0.0670

0.3694

0.7428

3.4995

1.3398

2.5617

1.9615

10.6978

0.6410

±SD

0.0091

0.0663

0.1451

0.3605

0.2577

0.5982

0.1373

1.3430

0.0477

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

0.0705

0.4383

0.7226

3.1986

1.4393

2.8197

2.0371

11.1423

0.6347

±SD

0.0132

0.0332

0.0480

0.1484

0.0975

0.3347

0.0799

1.4656

0.0587

n

5

5

5

5

5

5

5

5

5

 M: Male; R: Recovery; SD: Standard Deviation, n: Number of animals

.

 

 

TABLE 14 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g)

Refer Appendix 14

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

0.0688

0.4001

0.5071

0.1062

0.5056

0.7483

1.3784

1.7000

6.4285

0.4590

±SD

0.0084

0.0640

0.0527

0.0343

0.0727

0.1094

0.1386

0.0522

0.5623

0.0574

n

5

5

5

5

5

5

5

5

5

5

G2, F & 0.003

Mean

0.0709

0.3071*

0.4388

0.1335

0.6509

0.7224

1.3771

1.7426

5.7190

0.4886

±SD

0.0109

0.0146

0.0602

0.0338

0.1297

0.0724

0.1948

0.0479

0.6038

0.0660

n

5

5

5

5

5

5

5

5

5

5

G3, F & 0.01

Mean

0.0884

0.3436

0.4720

0.1027

0.6082

0.7089

1.3448

1.7532

6.4444

0.5426

±SD

0.0645

0.0508

0.0590

0.0437

0.1803

0.0492

0.1877

0.0476

0.9024

0.0554

n

5

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

0.0581

0.3067*

0.4379

0.0971

0.4466

0.6583

1.3295

1.7579

5.1555*

0.5497

±SD

0.0085

0.0338

0.1303

0.0283

0.1686

0.0274

0.0703

0.0731

0.8599

0.1192

n

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

0.0774

0.3357

0.8291

0.1343

1.1745

0.9412

1.7869

1.9118

8.5142

0.5585

±SD

0.0140

0.0703

0.1620

0.0056

0.2419

0.0437

0.1660

0.0276

1.1044

0.1485

n

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

0.0777

0.2541

0.5076*

0.1404

0.7023*

0.9485

1.6055

1.8672

7.3391

0.5311

±SD

0.0149

0.0375

0.0487

0.0314

0.1485

0.0838

0.1722

0.0575

0.6635

0.0730

n

5

5

5

5

5

5

5

5

5

5

 F: Female, R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05)

 

 

 


TABLE 15.  SUMMARY OF ORGAN WEIGHT RELATIVE TO BODY WEIGHT(%)RECORD

Refer Appendix 15

Group, Sex & Dose (mg/L)

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Testes

Heart

Kidneys

Brain

Liver

Lungs

G1, M & 0

Mean

219.28

0.0267

0.1414

0.2241

1.3049

0.3676

0.7334

0.8392

3.0624

0.2808

±SD

11.91

0.0082

0.0326

0.0213

0.0893

0.0413

0.0633

0.0519

0.2050

0.0541

n

5

5

5

5

5

5

5

5

5

5

G2, M & 0.003

Mean

221.69

0.0222

0.1606

0.2131

1.3908

0.4104

0.8163

0.8278

3.3281

0.2583

±SD

10.68

0.0011

0.0210

0.0340

0.0630

0.0387

0.0554

0.0382

0.3134

0.0295

n

5

5

5

5

5

5

5

5

5

5

G3, M & 0.01

Mean

217.94

0.0269

0.1279

0.2147

1.4231

0.4042

0.8012

0.8234

3.1585

0.2322

±SD

8.20

0.0050

0.0151

0.0387

0.1334

0.0816

0.0686

0.0685

0.4111

0.0607

n

5

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

218.45

0.0229

0.1213

0.2127

1.4128

0.3682

0.7106

0.8039

3.1065

0.2540

±SD

8.44

0.0029

0.0153

0.0480

0.0842

0.0532

0.0727

0.0529

0.2609

0.0436

n

5

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

406.82

0.0165

0.0908

0.1824

0.8601

0.3293

0.6293

0.4820

2.6288

0.1577

±SD

5.43

0.0024

0.0158

0.0342

0.0859

0.0626

0.1442

0.0298

0.3176

0.0131

n

5

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

408.41

0.0173

0.1073

0.1770

0.7833

0.3525

0.6908

0.4988

2.7301

0.1554

±SD

2.91

0.0033

0.0085

0.0123

0.0383

0.0254

0.0858

0.0190

0.3761

0.0144

n

5

5

5

5

5

5

5

5

5

5

 M: Male; R: Recovery; SD: Standard Deviation, n: Number of animals

 


TABLE 15 (Contd…). SUMMARY OF ORGAN WEIGHT RELATIVE TO BODY WEIGHT (%) RECORD

Refer Appendix 15

Group, Sex & Dose (mg/L)

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

186.50

0.0369

0.2152

0.2733

0.0562

0.2709

0.4001

0.7379

0.9151

3.4526

0.2470

±SD

11.64

0.0036

0.0370

0.0400

0.0154

0.0321

0.0433

0.0331

0.0779

0.3026

0.0345

n

5

5

5

5

5

5

5

5

5

5

5

G2, F & 0.003

Mean

190.05

0.0374

0.1620*

0.2316

0.0702

0.3417

0.3795

0.7244

0.9178

3.0099

0.2568

±SD

8.48

0.0058

0.0136

0.0362

0.0169

0.0597

0.0268

0.0942

0.0305

0.2928

0.0299

n

5

5

5

5

5

5

5

5

5

5

5

G3, F & 0.01

Mean

186.46

0.0465

0.1837

0.2538

0.0548

0.3264

0.3811

0.7206

0.9417

3.4488

0.2909

±SD

9.23

0.0311

0.0193

0.0359

0.0222

0.0989

0.0353

0.0845

0.0436

0.3509

0.0235

n

5

5

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

186.01

0.0312

0.1648*

0.2341

0.0522

0.2401

0.3541

0.7154

0.9454

2.7706*

0.2964

±SD

6.42

0.0038

0.0158

0.0647

0.0154

0.0926

0.0156

0.0443

0.0350

0.4482

0.0677

n

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

311.50

0.0249

0.1078

0.2670

0.0431

0.3782

0.3023

0.5743

0.6141

2.7364

0.1797

±SD

8.78

0.0046

0.0227

0.0568

0.0022

0.0840

0.0165

0.0595

0.0180

0.3825

0.0500

n

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

309.87

0.0250

0.0819

0.1637*

0.0453

0.2268*

0.3061

0.5177

0.6027

2.3667

0.1715

±SD

5.44

0.0045

0.0111

0.0142

0.0098

0.0490

0.0260

0.0500

0.0205

0.1834

0.0247

n

5

5

5

5

5

5

5

5

5

5

5

 F: Female, R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05)

 


TABLE 16.  SUMMARY OF ORGAN WEIGHT RELATIVE TO BRAIN WEIGHT (%) RECORD

                                                                                                                                                                                                                                                        Refer Appendix 16

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Testes

Heart

Kidneys

Liver

Lungs

G1, M & 0

Mean

3.2084

16.8417

26.7271

155.4808

44.0710

87.6913

365.9170

33.6076

±SD

1.0667

3.7434

2.3717

4.1007

7.0256

9.6107

30.7326

7.1803

n

5

5

5

5

5

5

5

5

G2, M & 0.003

Mean

2.6843

19.3593

25.9283

168.0469

49.5460

98.9292

403.3509

31.3037

±SD

0.1704

1.9147

5.3241

4.2249

3.4296

10.3827

50.1715

4.3567

n

5

5

5

5

5

5

5

5

G3, M & 0.01

Mean

3.2578

15.6978

26.2370

172.9034

49.2188

97.6369

384.4100

28.5755

±SD

0.4378

2.9017

4.9883

9.4807

9.2781

9.1659

45.1221

8.7837

n

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

2.8690

15.0658

26.7382

176.6741*

46.0977

88.6284

389.0618

31.6333

±SD

0.5374

1.2650

7.5037

19.5080

8.4852

9.9253

55.4416

5.2430

n

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

3.4347

18.7629

37.7116

179.2759

68.2623

130.2046

544.9017

32.7719

±SD

0.5718

2.3064

5.4990

23.6753

11.8016

25.8373

48.5062

2.9682

n

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

3.4637

21.5828

35.4693

157.2705

70.6704

138.4677

547.5343

31.2402

±SD

0.6484

2.2843

1.8665

10.6981

4.3381

16.2928

75.0901

3.6027

n

5

5

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05)

 

 

 

TABLE 16 (Contd…). SUMMARY OF ORGAN WEIGHT RELATIVE TO BRAIN WEIGHT (%) RECORD

Refer Appendix 16

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Liver

Lungs

G1, F & 0

Mean

4.0588

23.5205

29.8022

6.2720

29.7271

44.1110

81.1947

378.1715

27.0106

±SD

0.5596

3.6232

2.5080

2.0577

4.0718

6.9072

8.8951

31.5449

3.3719

n

5

5

5

5

5

5

5

5

5

G2, F & 0.003

Mean

4.0677

17.6305*

25.2137

7.6464

37.3403

41.4107

78.8936

327.7424

28.0605

±SD

0.5998

0.9366

3.7009

1.8651

7.3467

3.4933

9.7042

27.2146

3.9499

n

5

5

5

5

5

5

5

5

5

G3, F & 0.01

Mean

5.0209

19.6054

26.9066

5.8463

34.9025

40.4713

76.6939

367.4592

30.9520

±SD

3.6063

2.8966

3.1648

2.4545

11.1287

3.1944

10.3619

48.8942

3.0575

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

3.3066

17.4195*

24.7852

5.5106

25.3271

37.4696

75.7367

293.0729*

31.3939

±SD

0.4756

1.4201

7.0121

1.5875

9.5655

1.4874

5.0797

46.3627

7.3171

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

4.0565

17.5985

43.3976

7.0279

61.4346

49.2572

93.5633

445.8532

29.2652

±SD

0.7920

3.9173

8.6843

0.3349

12.5922

2.8374

9.8313

62.6420

8.0534

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

4.1760

13.6221

27.2025*

7.4981

37.7341*

50.7802

85.9015

393.3327

28.4678

±SD

0.8846

2.1080

2.6776

1.5337

8.6957

3.9404

7.7389

37.4725

4.0105

n

5

5

5

5

5

5

5

5

5

 F: Female, R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05)

 

 

 

 

 

 

 

 

TABLE 17.  SUMMARY OF CHAMBER (EXPOSURE) CONDITIONS

 Refer Appendix17


Group & Conc.

(mg/L)

 

Temp.

 (°C)

Rh

(%)

O2

(%)

CO2

 (mg/L)

Air inlet flow rate

(L/min)*

BZC

(mg/L)

G1/G1R & 0

Mean

22.51

55.70

20.45

615.25

20.00

-

±SD

0.22

0.94

0.22

5.03

0.00

-

G2 & 0.003

Mean

22.53

55.51

20.48

615.34

20.00

0.0031

±SD

0.21

1.08

0.21

4.72

0.00

0.0001

G3 & 0.01

Mean

22.52

55.48

20.46

615.85

20.00

0.011

±SD

0.24

1.01

0.22

5.22

0.00

0.001

G4/G4R & 0.03

Mean

22.31

55.21

20.45

614.76

20.00

0.032

±SD

2.07

1.27

0.21

5.21

0.00

0.0007

*: Values were constant throughout the exposure; hence standard deviation is zero, Temp.: Temperature; Rh: Relative Humidity; O2: Oxygen Concentration;

     CO2: Carbon Dioxide Concentration;BZC: Breathing Zone Concentration:SD: Standard Deviation;R: Recovery.

 

 

 

 

 

 


TABLE 18.SUMMARY OF NOMINAL CONCENTRATION

                                                                                                                              Refer Appendix 18

Day

Group

Test item Used (mg) (a)

Air flow rate (L/minute)(b)

Minute

(min)

(c)

Nominal Concentration

(mg/L)

(a)    / (b) × (c)

1

G1/G1R 

(Air Only)

-

20

    360

-

G2, G3 and G4/G4R 

(Test Item)

1690

20

360

0.23

2650

0.37

3440

0.48

2

G1/G1R

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1750

20

360

0.24

2600

0.36

3650

0.51

 3

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1530

20

360

0.21

2570

0.36

3590

0.50

4

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1690

20

360

0.23

2700

0.38

3440

0.48

5

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1490

20

360

0.21

2670

0.37

3500

0.49

6

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1600

20

360

0.22

2780

0.39

3480

0.48

7

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1540

20

360

0.21

2680

0.37

3440

0.48


TABLE 18 (Contd…). SUMMARY OF NOMINAL CONCENTRATION

                                                                                                                                                               Refer Appendix 18

Day

Group

Test item Used (mg) (a)

Air flow rate (L/minute)(b)

Minute

(min)

(c)

Nominal Concentration

(mg/L)

(b)    / (b) × (c)

8

G1/G1R 

(Air Only)

-

20

    360

-

G2, G3 and G4/G4R 

(Test Item)

1530

20

360

0.21

2700

0.37

3510

0.49

9

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1500

20

360

0.21

3040

0.42

3570

0.50

 10

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1730

20

360

0.24

2750

0.38

3650

0.51

11

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1780

20

360

0.25

2490

0.35

3440

0.48

12

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1820

20

360

0.25

2530

0.35

3410

0.47

13

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1720

20

360

0.24

2650

0.37

3680

0.51

14

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1780

20

360

0.25

2520

0.35

4040

0.56

 

 

 

 

 

 

 

 

 

 


TABLE 18 (Contd…). SUMMARY OF NOMINAL CONCENTRATION

Refer Appendix 18

Day

Group

Test item Used (mg) (a)

Air flow rate (L/minute)(b)

Minute

(min)

(c)

Nominal Concentration

(mg/L)

(c)    / (b) × (c)

15

G1/G1R 

(Air Only)

-

20

    360

-

G2, G3 and G4/G4R 

(Test Item)

1910

20

360

0.27

2510

0.35

3560

0.49

16

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1630

20

360

0.23

2880

0.40

3610

0.50

 17

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1660

20

360

0.23

2700

0.37

3750

0.52

18

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1510

20

360

0.21

2790

0.39

3690

0.51

19

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1530

20

360

0.21

2660

0.37

3590

0.50

20

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1720

20

360

0.24

2730

0.38

3630

0.50

 

TABLE 1.      SUMMARY OF GROSS PATHOLOGY FINDINGS

Refer Appendices 1 & 2

Sex

Male

Route of administration

Inhalation

Treatment

Air only

Low Dose

Mid Dose

High Dose

Air only

High Dose Recovery

Group

G1

G2

G3

G4

G1R

G4R

Nominal Target Dose (mg/L)

0

0.003

0.01

0.03

0

0.03

Number of animals

5

5

5

5

5

5

No. of dead rats during treatment

-

-

-

-

-

-

No. of moribund sacrificed rats

-

-

-

-

-

-

No. of terminally sacrificed rats

5

5

5

5

5

5

No. of rats showing gross pathology

-

-

-

-

-

-

 

Sex

Female

Route of administration

Inhalation

Treatment

Air only

Low Dose

Mid Dose

High Dose

Air only

High Dose Recovery

Group

G1

G2

G3

G4

G1R

G4R

Nominal Target Dose (mg/L)

0

0.003

0.01

0.03

0

0.03

Number of animals

5

5

5

5

5

5

No. of dead rats during treatment

-

-

-

-

-

-

No. of moribund sacrificed rats

-

-

-

-

-

-

No. of terminally sacrificed rats

5

5

5

5

5

5

No. of rats showing gross pathology

-

-

-

-

-

-

R: Recovery; - : No incidence.


TABLE 2.      SUMMARY OF HISTOPATHOLOGY FINDINGS– MAIN GROUP

Refer Appendices 3 & 4

Route of administration

Inhalation

Treatment

Air only

High Dose

Nominal Target Dose (mg/L)

0

0.03

Group

G1

G4

Sex

M

F

M

F

Number of Animals

5

5

5

5

Adrenals

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Brain

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Cervical lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Esophagus

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Eyes with optic nerve and eye lids

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Heart

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Hilar lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Kidneys

Number examined

5

5

5

5

Within normal limits

4

5

3

5

Cysts

Present

1

-

2

-

Basophilia, tubules

Minimal

-

-

1

-

Larynx

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Liver

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Lungs

Number examined

5

5

5

5

Within normal limits

5

4

-

-

Pigment, golden brown

Moderate

-

-

5

5

Infiltrate, mononuclear cells

Minimal

-

1

-

-

Mandibular lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Mesenteric lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Nasopharyngeal tissue

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Olfactory bulb

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Ovaries

Number examined

X

5

X

5

Within normal limits

X

5

X

5

Parathyroid

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Seminal vesicles

Number examined

5

X

5

X

Within normal limits

5

X

5

X

M: Male; F: Female; -: no incidence; X: not applicable.


TABLE 2 (Contd…). SUMMARY OF HISTOPATHOLOGY FINDINGS – MAIN GROUP

Refer Appendices 3 & 4

Route of administration

Inhalation

Treatment

Air only

High Dose

Nominal Target Dose (mg/L)

0

0.03

Group

G1

G4

Sex

M

F

M

F

Number of Animals

5

5

5

5

Spinal cord

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Spleen

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Stomach

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Testes

Number examined

5

X

5

X

Within normal limits

4

X

5

X

Degeneration/atrophy, tubule

Moderate

1

X

-

X

Thymus

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Thyroid

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Trachea

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Urinary bladder

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Uterus

Number examined

X

5

X

5

Within normal limits

X

4

X

5

Dilatation, glandular

Minimal

X

1

X

-

M: Male; F: Female; -: no incidence; X: not applicable.


TABLE 3.      SUMMARY OF HISTOPATHOLOGY FINDINGS – RECOVERY GROUP

Refer Appendices 5 & 6

Route of administration

Inhalation

Treatment

Air only

High Dose Recovery

Nominal Target Dose (mg/L)

0

0.03

Group

G1R

G4R

Sex

M

F

M

F

Number of Animals

5

5

5

5

Adrenals

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Brain

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Cervical lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Esophagus

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Eyes with optic nerve and eye lids

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Heart

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Hilar lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Kidneys

Number examined

5

5

5

5

Within normal limits

5

5

3

5

Cysts

Present

-

-

2

-

Basophilia, tubules

Minimal

-

-

1

-

Larynx

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Liver

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Lungs

Number examined

5

5

5

5

Within normal limits

5

5

-

-

Pigment, golden brown

Mild

-

-

-

1

Moderate

-

-

5

4

Mandibular lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Mesenteric lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Nasopharyngeal tissue

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Olfactory bulb

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Ovaries

Number examined

X

5

X

5

Within normal limits

X

5

X

5

Parathyroid

Number examined

5

5

5

5

Within normal limits

4

-

-

-

Hyperplasia

Mild

1

-

-

-

M: Male; F: Female; R: Recovery; -: no incidence; X: not applicable.


TABLE 3 (Contd…). SUMMARY OF HISTOPATHOLOGY FINDINGS – RECOVERY GROUP

Refer Appendices 5 & 6

Route of administration

Inhalation

Treatment

Air only

High Dose Recovery

Nominal Target Dose (mg/L)

0

0.03

Group

G1R

G4R

Sex

M

F

M

F

Number of Animals

5

5

5

5

Seminal vesicles

Number examined

5

X

5

X

Within normal limits

5

X

5

X

Spinal cord

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Spleen

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Stomach

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Testes

Number examined

5

X

5

X

Within normal limits

5

X

5

X

Thymus

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Thyroid

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Trachea

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Urinary bladder

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Uterus

Number examined

X

5

X

5

Within normal limits

X

4

X

4

Dilatation, lumen

Minimal

X

1

X

1

M: Male; F: Female; R: Recovery; -: no incidence; X: not applicable.


TABLE 4.      SUMMARY OF HISTOPATHOLOGY FINDINGS – LOW DOSE AND MID DOSE GROUP

Refer Appendices 7 & 8

Route of administration

Inhalation

Treatment

Low Dose

Mid Dose

Nominal Target Dose (mg/L)

0.003

0.01

Group

G2

G3

Sex

M

F

M

F

Number of Animals

5

5

5

5

Lungs

Number examined

5

5

5

5

Within normal limits

-

-

-

-

Pigment, golden brown

Minimal

3

4

2

3

Mild

2

1

3

2

M: Male; F: Female; -: No incidence.


 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
 Based on the observed results, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item,Novoperm Orange HLwas found to be 0.03 mg/L when exposed for 6 hours/day, 5 days/week, for 4 weeks by flow-past nose-only inhalation route to Sprague Dawley rats. The nominal dose of 0.03 mg/L (6 hrs/day, 5 days/week) is the actual exposure concentration in males and females..
Executive summary:

The objective of this study was to determine the toxic potential of the test item when administered for 6 hours/day, 5 days per week, for                4 consecutive weeks by flow-past nose-only inhalation route to Sprague Dawley rats. This study provides information on toxic effects, target organs, the possibility of cumulative effects, the reversibility of effects (after 56 days recovery period), and an estimate of the No Observed Adverse Effects Concentration (NOAEC).A total of 60 (30 males + 30 females) healthy young Sprague Dawley rats were distributed to four main groups viz., control (G1), low dose (G2), mid dose (G3), high dose (G4) and two recovery groups [(G1R (Air only) and G4R (high dose)]. Each main and recovery group comprised of 10 animals (5 males and 5 females). Animals allocated to groups G2, G3 and G4/G4R were exposed to test item for 6 hours per day, 5 days per week, for 4 consecutive weeks., at a nominal target concentration of 0.003, 0.01 and 0.03 mg/L. Animals of the control group (G1/G1R) received air only inhalation for 6 hours/day for 4 consecutive weeks. The inhalation exposure of test item/air was achieved by a flow-past, nose-only dynamic inhalation exposure system supplied by CH Technologies, USA.All animals were observedonce daily for clinical signs and twice daily for mortality. Body weightwas recorded before exposure (day 1and twice weekly for initial two weeks during treatment period and thereafter once weekly.Feed consumption was recorded weekly.Ophthalmoscopic examinationswereperformed duringtheacclimatizationperiodfor all groups, andduringWeek4for main groups (G1and G4),and duringWeek12for recovery groups (G1Rand G4R).Neurological/Functionaltestswereperformed duringWeek4forG1 and G4groupsand duringWeek12for recovery groupanimals.At the end of treatment and recovery periods, all animals were fasted overnight (water was availablead libitum), and the next day, blood, urine, and Broncho alveolar lavage fluid (BALF) samples were collected and analysed. Subsequently, the animals were sacrificed and subjected to gross pathological examination, and the organs were collected, weighed, and preserved. The tissues/organs in vehicle control and high dose group animals including recovery animals were subjected to histopathological examinations.The data recorded for all exposure days relating to the chamber conditions like particle size, temperature, relative humidity, oxygen, and carbon dioxide concentrations determined during the exposure period were found within the specified range.No treatment-related changes in body weight, percent change in body weight with respect to day 1, feed consumption,or ophthalmology were noted.No adverse effects were observed in the neurological/functional examination tests.No adverse effects were observed in haematology, clinical chemistry, coagulation, BALF analysis,or urinalysis parameters. No toxicologically significant changes were observed infasting body weight, absoluteorgan weight, or relative organ weight.There were no test item-related, microscopic findings in animals evaluated after terminal and recovery period in the study.In lungs, minimal to moderate, multifocal, variably sized, pigmented (golden brown) granular material was observed throughout the parenchyma of lung at low, mid, high and high dose recovery groups of rats. This pigment was distributed both in alveolar and bronchiolar spaces with no accompanying inflammation or tissue destruction to the lung parenchyma. This pigment was suggestive of inhaled test item particles. In the absence of cellular changes to lung parenchyma, mere presence of pigment in lungs could be considered as non-adverse effect up to the nominal target dose level of0.03mg/L. (Nikula KJet.al., 2014).


Conclusion: Based on the observed results, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item,Novoperm Orange HLwas found to be 0.03 mg/L when exposed for 6 hours/day, 5 days/week, for 4 weeks by flow-past nose-only inhalation route to Sprague Dawley rats. The nominal dose of 0.03 mg/L (6 hrs/day, 5 days/week) is the actual exposure concentration in males and females.