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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
28-Day Inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 March 2021 to 12 October 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
adopted on 25 June 2018
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethyl 2-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]terephthalate
EC Number:
252-650-1
EC Name:
Dimethyl 2-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]terephthalate
Cas Number:
35636-63-6
Molecular formula:
C21H19N5O7
IUPAC Name:
dimethyl 2-({2-oxo-1-[(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)carbamoyl]propyl}diazenyl)terephthalate
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 bred animals
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 140.06 g to 159.97 g; Females: 120.63 g to 139.41 g
- Fasting period before study: No
- Housing: Maximum of three animals per cage 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 drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized corn cob was provided as bedding material.
- Diet (e.g. ad libitum): Altromin Maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG).
- Water (e.g. ad libitum): Deep bore-well water passed through a reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 7 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6°C to 22.9°C
- Humidity (%): 46% to 65%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 15 March 2021 To: 10 August 2021

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2 - ca. 2 µm
Geometric standard deviation (GSD):
3
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past, nose-only dynamic inhalation exposure system supplied by CH Technologies, USA.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes which were positioned radially around the flow-past, nose-only dynamic inhalation exposure chamber.
- Source and rate of air: Air flow from the generation system into the chamber was controlled and monitored through control panel (supplied by CH Technologies, USA) using calibrated flow-meters (rotameters) and outlet of the chamber air flow was controlled by rotameter. The actual flow rate of chamber inlet was 20 L/min with 60 psi pressure and outlet air from chamber was 15 L/min maintained throughout the exposure period. The air inlet flow rate was recorded hourly once during exposure (i.e. ± 10 minutes) after equilibration period (T95) 0.11min.
- Method of conditioning air: Compressed 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: 22 ± 3°C, 30 to 70%, 60psi
- Air flow rate: 20L/min
- Air change rate: 12 to 15 air changes per cycle
- Method of particle size determination: Cascade impactor
- Treatment of exhaust air: NaOH

TEST ATMOSPHERE
- Brief description of analytical method used: No
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
Animals were exposed to aerosolized test item continuously for 6 hours per day, 5 days per week, for a total of 20 actual exposures, after equilibration period of the chamber concentration.
Following the 28 days exposure period, the animals in the recovery groups (G1R and G4R) were not given any treatment and was maintained for 56 days post treatment period and observed for reversibility or persistence of toxic effects.
Frequency of treatment:
6 hours per day, 5 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/L air (nominal)
Remarks:
Air Only
Dose / conc.:
0.001 mg/L air (nominal)
Remarks:
Low Dose
Dose / conc.:
0.003 mg/L air (nominal)
Remarks:
Mid Dose
Dose / conc.:
0.03 mg/L air (nominal)
Remarks:
High Dose
No. of animals per sex per dose:
5 Males + 5 Females
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
- Rationale for selecting satellite groups: Following the 28 days exposure period, the animals in the recovery groups (G1R and G4R) were not given any treatment and was maintained for 56 days post treatment period and observed for reversibility or persistence of toxic effects.
- Post-exposure recovery period in satellite groups: 56 Days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the animals were observed for clinical signs and pre-terminal deaths at 1.5 hrs (±10 mins), 3 hrs (±10 mins), 4.5 hrs (±10 mins) and 6 hrs (±10 mins) during exposure period. Post-exposure clinical signs and 30 to 40 minutes and 1 hour (±10 mins) following exposure to the test chemical, thereafter once a day for clinical signs and twice daily for mortality.
- Cage side observations checked in table No.1

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once in a week

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weights were recorded at receipt, prior to exposure (Day 1) and twice weekly thereafter for first two weeks. Since there was no significant body weight effects in the first two weeks, body weights were recorded weekly thereafter (post exposure period) for remaining period of the study. Fasting body weight of all the animals of all the groups were recorded at their scheduled terminal sacrifice.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination was carried out once during acclimatization (Pre-treatment) and during week 4 in control and high dose group animals. No, treatment related ocular changes are noted in high dose during week 4, the examination was not extended to lower dose groups (G2, G3) of main study animals and during week 12 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1/G1R and G4/G4R

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from all the surviving rats of each group on day 27 (within 24 hours after the last given dose) and day 85 of recovery groups respectively.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in table [No.10] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from all the surviving rats of each group on day 27 (within 24 hours after the last given dose) and day 85 of recovery groups respectively.
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in table [No.12] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected and analyzed from all rats on day 27 (within 24 hours after the last given dose). The animals were kept in urine collection cages overnight for urine collection. Animal was not given access to feed but water was provided ad libitum during their stay in urine collection cages.
The recovery group animal’s urine was collected on day 85.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.13] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Neurological/Functional examination was carried out during Week 4 and 12 for vehicle control and high dose animals. Since no apparent treatment related effects persisted in high dose animals, the examination was not carried out for low and mid dose groups (G2 and G3) during Week 4 of treatment period.
- Dose groups that were examined: G1/G1R and G4/G4R
- Battery of functions tested: sensory activity / grip strength / motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No. 19)
HISTOPATHOLOGY: Yes (see table No. 20)
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:
no effects observed
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
Description (incidence):
All animals survived to scheduled sacrifice, and there were no mortality attributable to the test item. Post-exposure, all animals were normal throughout the experimental period. No mortalities were noted during the recovery period.
Body weight and weight changes:
no effects observed
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:
no effects observed
Description (incidence and severity):
No statistically significant treatment-related variations were observed in all the tested dose groups when compared with control group.
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, decrease in HCT (G2) was noted. In females decrease in APTT (G2, G3 and G4), MCV and MCH (G2 and G4) was noted.
In recovery males decrease in Percent monocytes (G4R) was noted. However, in female no significance observed.
All the noted variations in main group are considered incidental in the absence of dose responsiveness. Variations noted at the end of recovery period are considered incidental as no such variations were noted in recovery females.
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 vehicle control group. However, the following statistically significant variations were noted.
In main group males, increase in chloride (G4) was noted; In females no significance was noted.
In recovery group no significance was observed.
The observed 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 adverse treatment-related effects were observed in urinalysis parameters when compared to the control group. However, a single incidence of a statistically significant decrease in specific gravity was observed in G4R females. This effect, which was minimal in nature, lacked dose-dependency and was therefore not attributed to the test item.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse effects during neurological/functional examinations were observed in main group (G1 and G4) and recovery group (G1R and G4R), however in females (G4R) excessive grooming was noted, this is considered as incidental due to lack of dose response.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In main group males, decrease in absolute weight of testis in G2; increase in absolute and relative weight of lungs in G2 and G4 was noted. In females decrease in absolute and relative weight of spleen in G3 was noted.
In recovery group, increase in absolute and relative weight of thymus (G4R); decrease in absolute and relative weight in spleen (G4R) was noted. In females, increase in absolute and relative weight of ovaries (G4R) were noted.
In main group males, statistically significant increase in relative weight of lungs in G2 and G4 was noted. In females, decrease in relative weight of spleen (G3) and liver (G2); increase in relative weight of lungs (G4) were noted.
In recovery group males, decrease in relative weight of spleen (G4R); increase in relative weight of thymus (G4R) was noted. In females increase in relative weight of ovaries (G4R) was noted.
All the observed changes are considered incidental in the absence of gross and histopathological changes in the high dose group animals and also lack dose dependency.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related gross pathological lesions were observed in main group and recovery group animals.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related, microscopic findings in animals evaluated after terminal and recovery period in the study.
Few microscopic findings observed in this study such as ultimobranchial cyst in thyroid gland, epithelial cysts in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected in laboratory rats.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
BALF Fluid: The bronchio-alveolar lavage fluid measured at termination revealed the following statistically significant changes: In main group males, increase in percent eosinophils and basophils (G3) was noted; In females, increase in percent monocytes (G4) was noted.
In recovery group, females increase in LDH (G4R) was noted; however, in males no significance was noted.
There were no test item-related microscopic findings in animals evaluated after terminal and recovery period in the study and the noted variations are considered non adverse effect.

Effect levels

Key result
Dose descriptor:
NOAEC
Effect level:
>= 0.03 - ca. 0.03 mg/L air (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 efficiency
gross pathology
haematology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
other: BALF findings

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

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 & 0.001

5

N

0/5

G3, M & 0.003

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.

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

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 & 0.001

5

N

0/5

G3, F & 0.003

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.

TABLE 2.  SUMMARY OF BODY WEIGHTS (g) RECORD

Group, Sex & Dose (mg/L)

Body Weight (g) on Days

1

5

8

12

15

22

26

G1, M & 0

Mean

187.88

191.93

205.03

209.22

220.95

234.65

241.23

±SD

14.19

14.38

13.69

13.43

13.45

13.00

12.55

n

5

5

5

5

5

5

5

G2, M & 0.001

Mean

183.58

187.41

199.18

203.48

214.50

228.10

235.87

±SD

14.65

14.59

13.65

13.86

13.30

14.17

14.78

n

5

5

5

5

5

5

5

G3, M & 0.003

Mean

182.16

185.60

197.45

201.89

214.35

228.81

235.88

±SD

15.60

15.80

14.39

13.60

13.57

14.20

13.89

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

180.15

183.99

196.54

201.16

213.32

227.63

235.65

±SD

18.66

18.08

17.15

16.54

15.36

14.94

14.81

n

5

5

5

5

5

5

5

G1, F & 0

Mean

152.09

155.06

161.34

163.66

172.01

182.69

189.18

±SD

12.49

12.10

10.71

10.09

10.81

11.06

10.92

n

5

5

5

5

5

5

5

G2, F & 0.001

Mean

153.99

157.68

163.82

166.47

175.30

184.89

190.90

±SD

11.36

11.61

11.46

11.06

11.32

11.43

11.75

n

5

5

5

5

5

5

5

G3, F & 0.003

Mean

149.86

153.07

160.44

162.55

170.62

180.12

186.94

±SD

11.55

12.04

11.07

10.79

10.78

10.64

11.15

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

151.90

154.59

160.20

162.81

170.66

180.51

186.39

±SD

8.04

7.91

8.06

7.73

7.95

9.00

9.00

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

Group, Sex & Dose (mg/L)

Body Weight (g) on Days

1

5

8

12

15

22

26

29

36

43

50

57

64

71

78

84

G1R, M & 0

Mean

184.58

188.92

201.70

205.86

217.92

232.09

242.91

250.80

272.02

294.25

317.10

339.64

362.85

386.35

411.61

432.52

±SD

10.63

11.00

10.49

9.66

9.90

9.61

9.14

9.32

9.71

10.66

10.84

10.19

10.54

10.56

10.87

10.69

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

182.85

186.62

199.13

203.34

215.44

230.12

243.82

251.75

272.84

295.09

317.85

340.56

363.70

386.94

412.13

433.06

±SD

12.36

12.69

11.96

13.79

13.22

13.17

12.42

12.49

11.83

12.80

13.47

13.68

13.72

13.78

14.37

14.48

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

151.42

154.17

159.60

162.05

170.53

180.30

187.50

192.72

208.02

223.72

239.22

254.79

271.47

288.34

305.89

321.44

±SD

12.00

11.75

10.93

10.98

10.61

8.85

8.38

8.72

9.15

8.87

9.39

9.33

9.70

9.90

9.79

9.86

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

151.49

154.31

160.21

162.55

173.23

182.00

189.45

194.62

209.88

225.43

241.03

256.53

273.35

290.30

307.74

323.36

±SD

11.10

11.31

10.58

9.78

11.74

11.05

10.57

10.69

10.98

10.90

10.94

10.91

11.02

10.83

10.45

10.36

n

5

5

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 animals.

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

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

2.16

9.19

11.44

17.71

25.05

28.59

±SD

0.52

1.38

1.95

2.09

2.93

3.31

n

5

5

5

5

5

5

G2, M & 0.001

Mean

2.10

8.58

10.93

16.97

24.38

28.62

±SD

0.42

1.46

1.86

2.19

2.28

2.25

n

5

5

5

5

5

5

G3, M & 0.003

Mean

1.89

8.50

10.98

17.87

25.84

29.76

±SD

0.53

1.67

2.17

3.16

3.61

4.10

n

5

5

5

5

5

5

G4, M & 0.03

Mean

2.18

9.25

11.86

18.72

26.75

31.25

±SD

0.62

1.77

2.42

3.76

4.74

5.28

n

5

5

5

5

5

5

G1, F & 0

Mean

1.99

6.19

7.75

13.25

20.30

24.59

±SD

0.53

1.70

2.28

2.33

2.66

3.28

n

5

5

5

5

5

5

G2, F & 0.001

Mean

2.40

6.41

8.15

13.90

20.15

24.06

±SD

0.28

0.53

1.01

1.18

2.05

1.85

n

5

5

5

5

5

5

G3, F & 0.003

Mean

2.13

7.12

8.54

13.96

20.34

24.89

±SD

0.72

1.16

1.75

2.21

3.07

2.90

n

5

5

5

5

5

5

G4, F & 0.03

Mean

1.78

5.48

7.22

12.39

18.86

22.74

±SD

0.29

0.59

1.35

1.44

0.90

1.30

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

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

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

2.35

9.31

11.59

18.14

25.84

31.73

36.01

47.54

59.58

71.99

84.25

96.85

109.61

123.32

134.68

±SD

0.30

0.61

1.51

1.92

2.46

3.09

3.31

3.90

3.97

4.48

5.40

5.85

6.55

7.11

7.80

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

2.06

8.94

11.20

17.86

25.92

33.48

37.82

49.42

61.60

74.08

86.54

99.24

111.99

125.80

137.28

±SD

0.45

0.81

0.61

0.99

1.38

3.24

3.40

4.36

4.67

5.39

5.93

6.81

7.54

8.13

8.78

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

1.84

5.48

7.11

12.75

19.33

24.13

27.57

37.71

48.14

58.41

68.75

79.81

90.99

102.65

112.97

±SD

0.41

1.23

1.71

2.42

4.43

4.95

4.92

5.32

6.27

6.72

7.66

8.36

9.11

10.22

11.06

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

1.87

5.81

7.39

14.41

20.25

25.20

28.63

38.75

49.05

59.39

69.67

80.81

92.06

103.64

113.99

±SD

0.23

1.11

1.55

2.31

2.97

3.11

3.53

4.27

4.67

5.22

6.01

6.67

7.64

8.66

9.19

n

5

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

Group, Sex & Dose (mg/L)

Week 1

Week 2

Week 3

Week 4

G1, M & 0

Mean

18.74

18.84

18.68

32.63

±SD

2.77

2.75

2.72

4.74

n

2

2

2

2

G2, M & 0.001

Mean

18.60

18.66

18.49

32.58

±SD

2.42

2.74

2.71

4.67

n

2

2

2

2

G3, M & 0.003

Mean

18.63

18.74

18.55

32.63

±SD

2.60

2.99

2.92

4.79

n

2

2

2

2

G4, M & 0.03

Mean

18.67

18.48

18.56

32.65

±SD

2.62

2.67

2.59

4.99

n

2

2

2

2

G1, F & 0

Mean

14.20

14.55

14.39

32.70

±SD

1.66

1.72

1.61

4.72

n

2

2

2

2

G2, F & 0.001

Mean

14.46

14.37

14.33

32.70

±SD

1.72

1.44

1.54

4.86

n

2

2

2

2

G3, F & 0.003

Mean

14.22

14.39

14.35

32.66

±SD

1.37

1.57

1.41

4.71

n

2

2

2

2

G4, F & 0.03

Mean

14.12

14.22

14.36

32.57

±SD

1.47

1.64

1.60

4.74

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

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

18.58

18.64

18.42

18.94

18.81

19.28

19.29

19.31

19.34

19.25

19.39

22.32

±SD

2.54

2.78

2.63

3.14

3.13

3.09

3.30

3.52

3.64

3.55

3.65

4.15

n

2

2

2

2

2

2

2

2

2

2

2

2

G4R, M & 0.03

Mean

18.63

18.68

18.50

18.63

18.66

19.20

19.21

19.32

19.45

19.33

19.46

22.34

±SD

2.65

2.89

2.76

2.78

2.98

3.14

3.30

3.21

3.30

3.24

3.29

3.79

n

2

2

2

2

2

2

2

2

2

2

2

2

G1R, F & 0

Mean

14.24

14.26

14.25

14.15

14.30

15.14

15.26

15.30

15.57

15.58

15.67

18.02

±SD

1.57

1.28

1.39

1.84

1.88

1.82

1.84

2.09

2.21

2.01

1.97

2.33

n

2

2

2

2

2

2

2

2

2

2

2

2

G4R, F & 0.03

Mean

14.07

14.19

14.19

14.15

14.36

14.74

14.93

15.16

15.33

15.27

15.43

17.69

±SD

1.48

1.88

1.67

1.78

1.94

2.25

2.19

2.27

2.43

2.11

2.13

2.41

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)

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

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

7.4

6.6

7.8

7.6

±SD

1.5

0.9

1.9

1.1

Numbers of Urination

Mean

1.4

1.4

1.4

1.6

±SD

0.5

0.5

0.5

0.5

Number of Defecation

Mean

1.0

1.0

1.0

0.8

±SD

0.7

0.7

0.7

0.4

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype Behaviour

1

1

1

1

Excessive Grooming

Mean

3.8

4.0

4.2

4.4

±SD

0.4

1.0

0.8

0.5

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

98.2

98.2

98.5

98.5

±SD

0.2

0.2

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

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

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

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

7.8

7.6

8.8

9.0

±SD

0.8

1.7

0.8

1.0

Numbers of Urination

Mean

1.2

1.2

1.0

1.4

±SD

0.8

0.4

0.7

0.5

Number of Defecation

Mean

1.0

1.0

1.2

1.0

±SD

0.7

0.7

0.4

0.7

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype Behaviour

1

1

1

1

Excessive Grooming

Mean

4.2

3.4

4.0

5.4*

±SD

0.8

0.9

0.7

0.5

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

97.9

97.9

98.7

98.7

±SD

0.2

0.2

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; *: Statistically significant (p<0.05).

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

# Week 4 & 12

Group, Sex & Dose (mg/L)

Hind Limb Foot Splay (cm)

G1, M & 0

Mean

6.0

±SD

0.4

n

5

G4, M & 0.03

Mean

5.7

±SD

0.2

n

5

G1, F & 0

Mean

6.4

±SD

0.5

n

5

G4, F & 0.03

Mean

5.9

±SD

0.6

n

5

G1R, M & 0

Mean

8.3

±SD

0.4

n

5

G4R, M & 0.03

Mean

7.8

±SD

0.5

n

5

G1R, F & 0

Mean

6.5

±SD

0.4

n

5

G4R, F & 0.03

Mean

5.8

±SD

0.8

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

# Week 4 & 12

Group, Sex & Dose (mg/L)

Movement Counts (no.)

G1, M & 0

Mean

2114.4

±SD

61.2

n

5

G4, M & 0.03

Mean

2067.8

±SD

75.0

n

5

G1, F & 0

Mean

2179.2

±SD

71.7

n

5

G4, F & 0.03

Mean

2164.4

±SD

26.9

n

5

G1R, M & 0

Mean

2074.0

±SD

54.0

n

5

G4R, M & 0.03

Mean

2117.0

±SD

26.3

n

5

G1R, F & 0

Mean

2213.2

±SD

27.6

n

5

G4R, F & 0.03

Mean

2221.8

±SD

9.4

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 8. SUMMARY OF GRIP STRENGTH (kgf) MEASUREMENT RECORD

# Week 4 & 12

Group, Sex & Dose (mg/L)

Fore limb

Hind limb

G1, M & 0

Mean

1.352

0.330

±SD

0.032

0.021

n

5

5

G4, M & 0.03

Mean

1.373

0.354

±SD

0.034

0.017

n

5

5

G1, F & 0

Mean

1.358

0.316

±SD

0.020

0.018

n

5

5

G4, F & 0.03

Mean

1.364

0.337

±SD

0.009

0.019

n

5

5

G1R, M & 0

Mean

1.563

0.536

±SD

0.036

0.020

n

5

5

G4R, M & 0.03

Mean

1.550

0.531

±SD

0.029

0.025

n

5

5

G1R, F & 0

Mean

1.418

0.371

±SD

0.053

0.023

n

5

5

G4R, F & 0.03

Mean

1.399

0.351

±SD

0.056

0.017

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

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

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

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; F: Female.

TABLE 10.     SUMMARY OF HAEMATOLOGY RECORD

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

10.44

8.93

17.14

53.50

60.02

19.22

32.06

972.00

5.90

±SD

2.75

0.41

0.82

2.05

3.62

1.14

0.84

131.79

0.25

n

5

5

5

5

5

5

5

5

5

G2, M & 0.001

Mean

9.54

8.57

15.86

50.28*

58.84

18.54

31.54

865.20

6.08

±SD

3.23

0.51

0.51

1.88

4.30

1.15

0.33

28.49

0.41

n

5

5

5

5

5

5

5

5

5

G3, M & 0.003

Mean

10.98

8.30

16.30

52.12

63.28

19.72

31.24

760.80

7.22

±SD

3.97

0.81

0.87

1.58

5.95

1.10

1.25

348.58

1.47

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

12.13

8.88

16.24

52.10

58.76

18.30

31.18

877.80

6.66

±SD

3.91

0.56

0.92

1.78

2.46

0.60

0.76

118.40

1.02

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

9.06

8.85

16.46

50.18

56.66

18.60

32.86

835.00

6.78

±SD

2.95

0.22

0.74

2.10

2.28

0.89

0.48

171.39

0.22

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

8.17

9.08

16.00

48.82

53.80

17.62

32.78

757.20

6.70

±SD

1.98

0.33

0.85

2.43

2.94

1.04

0.55

350.57

0.22

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

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

4.03

20.72

72.38

4.02

0.66

1.02

356.90

±SD

1.89

4.02

3.94

1.89

0.42

0.38

160.41

n

5

5

5

5

5

5

5

G2, M & 0.001

Mean

4.09

27.06

65.18

5.10

0.68

0.80

342.64

±SD

1.83

2.48

3.20

0.62

0.13

0.10

124.07

n

5

5

5

5

5

5

5

G3, M & 0.003

Mean

7.29

21.04

71.66

4.18

0.94

1.06

571.88

±SD

5.52

6.75

7.23

1.17

0.27

0.55

374.97

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

4.66

21.72

71.72

4.16

0.88

0.62

406.30

±SD

2.03

6.20

6.74

0.88

0.58

0.22

156.82

n

5

5

5

5

5

5

5

G1R, M & 0

Mean

2.75

24.76

68.60

3.74

1.36

0.48

243.48

±SD

0.51

6.03

5.00

0.65

0.41

0.16

49.00

n

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

2.24

26.74

68.40

2.26*

1.68

0.28

203.34

±SD

0.23

5.60

4.67

0.61

0.71

0.13

17.59

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

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

2.14

7.57

0.42

0.06

0.10

15.72

20.68

±SD

0.63

2.10

0.20

0.03

0.03

1.40

0.77

n

5

5

5

5

5

5

5

G2, M & 0.001

Mean

2.55

6.25

0.48

0.06

0.08

16.12

20.48

±SD

0.84

2.21

0.13

0.03

0.02

0.56

2.41

n

5

5

5

5

5

5

5

G3, M & 0.003

Mean

2.21

8.01

0.43

0.10

0.11

16.94

26.86

±SD

0.78

3.43

0.12

0.03

0.07

2.33

8.44

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

2.75

8.57

0.52

0.10

0.07

16.32

23.94

±SD

1.41

2.46

0.23

0.07

0.03

1.11

4.54

n

5

5

5

5

5

5

5

G1R, M & 0

Mean

2.12

6.32

0.36

0.13

0.04

15.32

21.20

±SD

0.29

2.46

0.17

0.06

0.02

1.49

1.80

n

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

2.14

5.63

0.19

0.13

0.03

17.96

26.26

±SD

0.43

1.63

0.09

0.05

0.02

5.13

6.31

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

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

9.06

8.85

16.60

49.98

56.50

18.84

33.30

1023.80

6.02

±SD

2.15

0.71

0.75

3.61

1.04

1.19

2.14

93.66

0.13

n

5

5

5

5

5

5

5

5

5

G2, F & 0.001

Mean

9.75

9.77

17.18

52.46

53.74*

17.58*

32.74

1092.80

6.16

±SD

4.30

1.21

2.04

6.09

1.24

0.39

0.21

213.31

0.30

n

5

5

5

5

5

5

5

5

5

G3, F & 0.003

Mean

9.40

9.63

17.08

52.46

54.48

17.76

32.58

980.00

6.38

±SD

3.58

0.96

1.70

5.61

1.70

0.51

0.57

262.16

0.49

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

9.93

9.26

16.10

49.62

53.60*

17.38*

32.44

992.60

6.60

±SD

2.52

0.32

0.60

1.67

1.50

0.72

0.62

290.58

0.52

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

8.38

8.72

16.10

47.82

54.82

18.50

33.70

711.40

6.58

±SD

1.44

0.65

1.11

3.32

0.93

0.51

0.73

276.36

0.29

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

8.96

8.55

15.82

47.44

55.50

18.52

33.36

854.20

6.66

±SD

1.56

0.41

0.91

3.04

1.58

0.56

0.54

202.83

0.23

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 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

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

2.43

21.12

72.62

3.46

1.26

0.64

214.50

±SD

0.43

4.43

4.70

0.96

0.68

0.24

40.96

n

5

5

5

5

5

5

5

G2, F & 0.001

Mean

1.72

27.40

66.90

2.64

1.32

0.84

166.78

±SD

0.56

6.67

7.47

0.66

0.34

0.61

47.63

n

5

5

5

5

5

5

5

G3, F & 0.003

Mean

2.15

24.56

70.40

2.38

1.06

0.76

204.00

±SD

0.54

5.33

5.11

1.22

0.43

0.62

37.56

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

1.91

26.70

67.40

2.78

1.58

0.44

176.24

±SD

0.61

3.96

5.05

0.88

0.64

0.11

51.51

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

1.84

22.58

71.16

2.90

2.10

0.38

161.28

±SD

0.29

5.49

6.51

1.01

1.42

0.04

33.92

n

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

1.95

23.14

71.68

2.38

1.64

0.34

165.02

±SD

0.54

5.94

6.51

0.31

0.32

0.09

40.29

n

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

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

1.94

6.55

0.31

0.12

0.06

16.82

25.28

±SD

0.68

1.48

0.09

0.07

0.04

1.78

1.76

n

5

5

5

5

5

5

5

G2, F & 0.001

Mean

2.51

6.70

0.25

0.13

0.07

17.50

20.36*

±SD

0.74

3.44

0.09

0.06

0.03

3.15

1.60

n

5

5

5

5

5

5

5

G3, F & 0.003

Mean

2.24

6.66

0.25

0.09

0.08

16.44

19.46*

±SD

0.82

2.75

0.21

0.01

0.06

2.49

2.35

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

2.66

6.69

0.28

0.15

0.04

17.50

17.86*

±SD

0.80

1.70

0.13

0.06

0.01

4.63

2.02

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

1.89

5.97

0.24

0.18

0.03

16.12

22.70

±SD

0.54

1.25

0.08

0.12

0.01

0.82

4.20

n

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

2.10

6.39

0.21

0.15

0.03

16.94

24.66

±SD

0.81

1.07

0.05

0.05

0.01

1.48

5.92

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

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

0.36

17.16

73.14

2.00

0.24

1.60

0.06

±SD

0.16

5.63

7.02

1.27

0.29

1.21

0.03

n

5

5

5

5

5

5

5

G2, M & 0.001

Mean

0.54

22.56

67.02

2.66

0.70

3.46

0.14

±SD

0.42

4.76

7.90

0.82

0.93

2.57

0.12

n

5

5

5

5

5

5

5

G3, M & 0.003

Mean

0.15

23.48

65.82

3.22

4.22*

8.06*

0.02

±SD

0.13

20.92

26.49

4.01

3.80

3.11

0.02

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

0.15

25.36

66.18

2.70

1.96

2.16

0.04

±SD

0.06

9.32

12.11

2.65

2.09

1.27

0.02

n

5

5

5

5

5

5

5

G1R, M & 0

Mean

0.34

17.08

57.66

1.10

0.90

5.56

0.06

±SD

0.12

3.07

20.72

0.49

1.33

3.29

0.03

n

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

0.40

17.76

56.22

1.78

0.88

3.66

0.07

±SD

0.22

9.61

12.23

1.80

0.75

2.14

0.06

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

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

0.26

0.01

0.00

0.01

63.76

322.20

±SD

0.11

0.00

0.00

0.01

27.81

108.59

n

5

5

5

5

5

5

G2, M & 0.001

Mean

0.34

0.01

0.00

0.02

83.96

291.48

±SD

0.24

0.01

0.00

0.01

53.17

142.06

n

5

5

5

5

5

5

G3, M & 0.003

Mean

0.12

0.00

0.00

0.01

52.34

306.36

±SD

0.10

0.00

0.00

0.01

45.59

105.45

n

5

5

5

5

5

5

G4, M & 0.03

Mean

0.10

0.00

0.00

0.00

34.26

281.34

±SD

0.05

0.01

0.00

0.00

9.63

105.25

n

5

5

5

5

5

5

G1R, M & 0

Mean

0.21

0.00

0.00

0.02

72.74

253.26

±SD

0.12

0.00

0.00

0.02

29.08

178.66

n

5

5

5

5

5

5

G4R, M & 0.03

Mean

0.22

0.01

0.00

0.01

149.78

200.61

±SD

0.11

0.01

0.00

0.01

122.02

89.26

n

5

5

5

5

5

5

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

TABLE 11 (Contd…). SUMMARY OF BALF RECORD

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

0.25

22.28

64.02

1.18

3.18

2.42

0.05

±SD

0.13

1.84

8.09

0.62

2.18

1.52

0.03

n

5

5

5

5

5

5

5

G2, F & 0.001

Mean

0.21

24.72

58.52

1.88

3.64

7.48

0.05

±SD

0.09

11.62

21.00

0.67

4.05

6.51

0.03

n

5

5

5

5

5

5

5

G3, F & 0.003

Mean

0.32

23.14

65.12

2.02

1.72

1.94

0.08

±SD

0.17

6.81

9.88

0.69

1.53

0.98

0.06

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

0.23

16.04

71.72

3.52*

3.48

4.42

0.04

±SD

0.17

8.52

11.69

1.93

2.34

4.54

0.03

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

0.22

21.06

53.16

0.98

1.72

6.22

0.04

±SD

0.09

6.39

7.11

1.31

1.64

2.01

0.02

n

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

0.21

17.64

61.56

1.86

0.90

4.18

0.03

±SD

0.07

6.61

16.43

1.10

0.53

1.54

0.02

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

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

0.16

0.00

0.01

0.00

40.70

272.13

±SD

0.10

0.00

0.00

0.01

23.61

89.97

n

5

5

5

5

5

5

G2, F & 0.001

Mean

0.13

0.00

0.01

0.02

63.46

302.96

±SD

0.08

0.00

0.01

0.02

32.58

89.90

n

5

5

5

5

5

5

G3, F & 0.003

Mean

0.21

0.01

0.00

0.00

51.52

355.10

±SD

0.10

0.01

0.01

0.01

21.60

108.66

n

5

5

5

5

5

5

G4, F & 0.03

Mean

0.16

0.01

0.00

0.01

46.32

346.50

±SD

0.11

0.01

0.01

0.01

23.88

251.82

n

5

5

5

5

5

5

G1R, F & 0

Mean

0.12

0.00

0.00

0.01

46.52

235.94

±SD

0.06

0.00

0.00

0.01

12.39

96.20

n

5

5

5

5

5

5

G4R, F & 0.03

Mean

0.13

0.00

0.00

0.01

91.78*

151.51

±SD

0.05

0.00

0.00

0.00

26.14

37.86

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 CHEMISTRY RECORD

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

91.60

31.90

0.41

42.00

44.60

6.82

3.09

53.20

94.20

±SD

8.88

2.88

0.03

8.40

8.85

0.48

0.07

13.94

10.66

n

5

5

5

5

5

5

5

5

5

G2, M & 0.001

Mean

85.00

31.50

0.40

49.60

41.20

7.00

3.15

57.40

103.20

±SD

8.51

1.64

0.01

6.43

12.76

0.20

0.15

7.57

12.77

n

5

5

5

5

5

5

5

5

5

G3, M & 0.003

Mean

97.00

32.28

0.39

47.60

49.20

6.90

3.10

53.40

108.20

±SD

15.70

4.40

0.02

11.01

8.23

0.30

0.11

6.19

7.76

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

86.60

35.32

0.38

50.40

48.00

6.64

2.99

71.00

109.00

±SD

9.71

11.73

0.03

12.30

13.67

0.43

0.17

15.89

21.51

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

77.20

34.62

0.52

53.60

29.80

7.52

3.37

62.20

106.60

±SD

10.08

6.83

0.05

7.47

4.71

0.29

0.18

7.26

17.56

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

89.00

36.06

0.53

52.60

33.60

7.66

3.50

62.00

104.40

±SD

6.71

1.99

0.05

8.68

9.02

0.15

0.33

13.62

14.06

n

5

5

5

5

5

5

5

5

5

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

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

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

202.00

0.01

9.78

7.30

3.73

14.89

142.10

3.87

109.74

±SD

79.50

0.02

0.33

0.63

0.45

1.34

0.83

0.18

0.91

n

5

5

5

5

5

5

5

5

5

G2, M & 0.001

Mean

178.80

0.04

9.92

7.24

3.85

14.70

142.12

3.97

110.44

±SD

21.75

0.02

0.29

0.43

0.15

0.76

0.89

0.16

0.73

n

5

5

5

5

5

5

5

5

5

G3, M & 0.003

Mean

184.40

0.04

9.94

7.14

3.80

15.06

141.14

3.96

109.42

±SD

47.26

0.05

0.21

0.55

0.33

2.05

0.91

0.29

1.27

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

188.80

0.03

9.80

7.44

3.65

16.48

142.92

3.94

111.54*

±SD

57.73

0.02

0.12

0.36

0.27

5.47

0.91

0.09

0.52

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

142.60

0.05

9.60

6.08

4.15

16.16

144.46

3.54

105.24

±SD

31.91

0.01

0.28

0.47

0.26

3.19

0.27

0.34

1.42

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

163.80

0.05

9.52

5.36

4.16

16.83

144.52

3.55

106.68

±SD

44.64

0.01

0.36

0.92

0.30

0.93

0.64

0.12

0.63

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

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

95.80

29.54

0.39

53.60

48.60

7.08

3.35

42.60

91.20

±SD

9.86

4.95

0.04

11.74

5.50

0.36

0.25

7.44

8.76

n

5

5

5

5

5

5

5

5

5

G2, F & 0.001

Mean

100.20

28.98

0.37

54.40

50.80

7.32

3.33

44.20

96.00

±SD

2.68

2.77

0.01

6.62

14.79

0.22

0.13

6.61

6.63

n

5

5

5

5

5

5

5

5

5

G3, F & 0.003

Mean

102.20

30.38

0.35

59.60

37.40

7.28

3.34

46.80

105.40

±SD

5.45

5.89

0.03

14.59

5.94

0.28

0.11

12.19

10.31

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

108.00

32.98

0.37

58.20

45.20

7.20

3.16

38.20

92.00

±SD

11.98

7.09

0.07

14.75

14.13

0.61

0.18

9.98

27.06

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

90.80

40.48

0.59

73.60

37.40

8.14

3.92

41.60

88.60

±SD

8.64

2.53

0.04

27.56

10.43

0.58

0.32

5.03

14.03

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

98.00

36.66

0.57

72.40

38.20

8.14

3.95

40.80

85.60

±SD

13.69

4.61

0.06

19.49

5.36

0.42

0.26

5.89

15.04

n

5

5

5

5

5

5

5

5

5

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

TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY

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

105.20

0.02

10.40

7.02

3.73

13.79

140.90

4.12

111.32

±SD

23.99

0.02

0.31

0.54

0.24

2.31

0.99

0.29

2.77

n

5

5

5

5

5

5

5

5

5

G2, F & 0.001

Mean

96.00

0.03

10.14

6.98

3.99

13.53

141.66

4.05

111.20

±SD

12.41

0.01

0.32

0.87

0.34

1.29

1.60

0.70

1.14

n

5

5

5

5

5

5

5

5

5

G3, F & 0.003

Mean

140.60

0.04

10.28

6.80

3.94

14.18

141.68

3.82

111.46

±SD

71.25

0.01

0.36

0.94

0.33

2.75

1.44

0.23

1.94

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

129.80

0.03

10.34

7.20

4.04

15.39

142.58

4.07

112.06

±SD

26.81

0.02

0.30

0.35

0.46

3.31

1.00

0.20

0.86

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

80.00

0.06

10.02

5.12

4.22

18.89

143.94

3.56

106.40

±SD

19.27

0.01

0.49

0.79

0.32

1.18

0.88

0.41

1.02

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

62.20

0.07

10.06

4.72

4.19

17.11

143.88

3.17

106.60

±SD

10.69

0.02

0.26

0.85

0.47

2.15

1.39

0.29

1.04

n

5

5

5

5

5

5

5

5

5

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

TABLE 13.  SUMMARY OF URINALYSIS

Examination

Group, Sex & Dose (mg/L)

G1, M & 0

G2, M & 0.001

G3, M & 0.003

G4, M & 0.03

G1R, M & 0

G4R, M & 0.03

Number of Animals

5

5

5

5

5

5

Physical

Colour

Pale Yellow

4

3

3

4

5

5

Yellow

1

2

2

1

-

-

Appearance

Clear

4

3

2

3

5

5

Turbid

1

2

3

2

-

-

Volume (mL)

Mean

5.5

5.9

5.7

5.4

6.5

6.5

±SD

0.9

0.7

1.3

0.4

0.6

0.8

Chemical

pH

Mean

6.4

6.0

6.8

6.1

7.5

7.4

±SD

1.6

0.9

1.3

1.0

0.6

0.5

Specific Gravity

Mean

1.009

1.007

1.007

1.006

1.018

1.016

±SD

0.002

0.004

0.003

0.002

0.007

0.007

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.2

0.2

0.2

0.4

±SD

0.0

0.0

0.0

0.0

0.0

0.4

Bilirubin (mg/dL)

Neg

5

5

5

5

5

5

Ketones (mg/dL)

Neg

2

4

2

2

-

2

5

2

1

3

3

5

2

15

1

-

-

-

-

1

Blood (Ery/µL)

Neg

-

-

-

-

1

1

Ca10

1

-

-

-

-

1

Ca25

3

2

1

-

2

2

Ca80

1

3

4

5

1

1

>=Ca200

-

-

-

-

1

-

Proteins (mg/dL)

Neg

-

-

-

2

-

1

Trace

2

4

3

1

-

1

30

3

1

1

2

2

3

100

-

-

1

-

2

-

>=300

-

-

-

-

1

-

Nitrite

Neg

3

5

4

5

4

5

Pos

2

-

1

-

1

-

Leucocytes (Leu/µL)

Neg

2

3

-

1

-

1

Ca15

3

2

5

4

2

1

Ca70

-

-

-

-

3

2

Ca125

-

-

-

-

-

1

Glucose (mg/dL)

Neg

5

5

5

5

5

5

Micro albumin (MALB) (mg/dL)

Neg

-

-

-

2

-

-

>15

5

5

5

3

5

4

15

-

-

-

-

-

1

Microscopic

Epithelial cells

0

1

-

1

1

4

3

0-1

2

4

3

3

1

2

0-2

1

-

-

-

-

-

1-2

1

1

1

1

-

-

Casts

Absent

5

5

5

5

5

5

Crystals

Absent

2

1

1

2

-

-

Present

3

4

4

3

5

5

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

TABLE 13 (Contd…). SUMMARY OF URINALYSIS

Examination

Group, Sex & Dose (mg/L)

G1, F & 0

G2, F & 0.001

G3, F & 0.003

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

4

4

4

5

5

Yellow

-

1

1

1

-

-

Appearance

Clear

5

3

4

4

5

5

Turbid

-

2

1

1

-

-

Volume (mL)

Mean

6.1

6.6

7.6

6.6

6.6

6.9

±SD

2.3

0.8

2.3

2.9

1.0

1.3

Chemical

pH

Mean

6.6

7.1

6.8

6.4

7.0

7.6

±SD

1.7

1.3

1.2

1.0

0.4

0.7

Specific Gravity

Mean

1.006

1.005

1.005

1.005

1.013

1.009*

±SD

0.002

0.000

0.000

0.000

0.003

0.002

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

5

5

5

5

5

5

Ketones (mg/dL)

Neg

5

5

5

5

5

5

Blood (Ery/µL)

Neg

4

1

1

-

4

4

Ca10

-

-

-

3

1

-

Ca25

-

3

4

2

-

1

Ca80

1

1

-

-

-

-

Proteins (mg/dL)

Neg

1

3

2

4

5

3

Trace

3

2

3

1

-

1

30

1

-

-

-

-

-

100

-

-

-

-

-

1

Nitrite

Neg

4

4

4

5

3

5

Pos

1

1

1

-

2

-

Leucocytes (Leu/µL)

Neg

1

-

-

-

4

4

Ca15

4

5

5

5

1

-

Ca70

-

-

-

-

-

1

Glucose (mg/dL)

Neg

5

5

5

5

5

5

Micro albumin (MALB) (mg/dL)

Neg

1

3

2

4

3

3

>15

4

2

3

1

-

2

15

-

-

-

 

2

-

Microscopic

Epithelial cells

0

1

1

2

2

5

4

0-1

3

1

1

3

-

1

1-2

1

3

2

-

-

-

Casts

Absent

4

5

4

5

5

5

Present

1

-

1

-

-

-

Crystals

Absent

2

1

-

3

-

-

Present

3

4

5

2

5

5

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

*: Statistically significant (p<0.05).

TABLE 14.      SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g)

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Testes

Heart

Kidneys

Brain

Liver

Lungs

G1, M & 0

Mean

0.0546

0.3164

1.0157

3.2814

0.9708

1.9636

1.9756

8.1699

0.5472

±SD

0.0079

0.0341

0.0992

0.2444

0.0559

0.1238

0.1009

0.6837

0.0516

n

5

5

5

5

5

5

5

5

5

G2, M & 0.001

Mean

0.0507

0.2643

1.1574

2.8052*

1.0410

1.9459

1.9383

8.4021

0.7314*

±SD

0.0057

0.0492

0.2344

0.2205

0.0990

0.1978

0.1199

0.5872

0.0658

n

5

5

5

5

5

5

5

5

5

G3, M & 0.003

Mean

0.0525

0.2908

1.1117

2.9190

0.9237

1.7474

1.8780

7.0657

0.5565

±SD

0.0037

0.0359

0.1191

0.1732

0.0443

0.0918

0.0720

0.4982

0.0609

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

0.0542

0.2789

0.9743

3.4094

1.0249

1.9576

1.9070

7.4683

0.7014*

±SD

0.0049

0.0236

0.0965

0.4453

0.0507

0.1801

0.0340

0.9456

0.0711

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

0.0609

0.2117

1.3692

3.6598

1.4328

2.8445

2.0602

11.2475

0.7140

±SD

0.0042

0.0517

0.0633

0.3034

0.1040

0.5484

0.0512

1.7149

0.0767

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

0.0631

0.2829*

1.1172*

3.3671

1.3194

2.3350

2.0685

10.1517

0.6762

±SD

0.0048

0.0365

0.1003

0.2875

0.0756

0.1815

0.0608

0.0759

0.0505

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

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

0.0623

0.3702

0.6049

0.1205

0.4089

0.7567

1.4280

1.7656

6.2227

0.5146

±SD

0.0037

0.0701

0.1064

0.0129

0.0518

0.0741

0.1903

0.0929

0.4228

0.0453

n

5

5

5

5

5

5

5

5

5

5

G2, F & 0.001

Mean

0.0594

0.3682

0.4791

0.1181

0.5253

0.7450

1.3006

1.8402

5.5576

0.5163

±SD

0.0080

0.0627

0.1005

0.0094

0.1533

0.0599

0.1535

0.0842

0.3985

0.0639

n

5

5

5

5

5

5

5

5

5

5

G3, F & 0.003

Mean

0.0561

0.2862

0.3462*

0.0987

0.3390

0.7369

1.2861

1.7616

5.6863

0.5077

±SD

0.0070

0.0741

0.0372

0.0138

0.0602

0.1018

0.1160

0.0763

0.3377

0.0388

n

5

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

0.0560

0.2707

0.4679

0.1156

0.3450

0.7066

1.2147

1.7537

6.0287

0.6040

±SD

0.0056

0.0276

0.0857

0.0180

0.0603

0.0985

0.1357

0.0714

0.6349

0.0824

n

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

0.0809

0.2637

0.6039

0.1321

0.7511

0.9010

1.5550

1.8811

7.9632

0.4801

±SD

0.0111

0.0441

0.0799

0.0168

0.2097

0.0950

0.0643

0.0875

0.4811

0.0316

n

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

0.0778

0.2352

0.5206

0.1647*

0.7788

0.9655

1.5697

1.8020

7.2701

0.4812

±SD

0.0035

0.0293

0.0647

0.0077

0.2014

0.0968

0.1387

0.1353

0.9500

0.0610

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

Group, Sex & Dose (mg/L)

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Testes

Heart

Kidneys

Brain

Liver

Lungs

G1, M & 0

Mean

229.55

0.0237

0.1385

0.4480

1.4392

0.4259

0.8641

0.8683

3.5879

0.2396

±SD

20.95

0.0018

0.0167

0.0808

0.1679

0.0480

0.1288

0.1105

0.4917

0.0270

n

5

5

5

5

5

5

5

5

5

5

G2, M & 0.001

Mean

225.52

0.0225

0.1174

0.5200

1.2504

0.4629

0.8621

0.8604

3.7368

0.3261*

±SD

15.25

0.0022

0.0207

0.1333

0.1495

0.0523

0.0501

0.0388

0.3325

0.0437

n

5

5

5

5

5

5

5

5

5

5

G3, M & 0.003

Mean

228.80

0.0230

0.1278

0.4869

1.2795

0.4045

0.7658

0.8230

3.1008

0.2440

±SD

14.36

0.0012

0.0204

0.0550

0.1057

0.0231

0.0593

0.0548

0.3321

0.0305

n

5

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

229.21

0.0236

0.1220

0.4252

1.4888

0.4480

0.8539

0.8344

3.2547

0.3068*

±SD

15.64

0.0009

0.0117

0.0314

0.1825

0.0220

0.0491

0.0454

0.3036

0.0339

n

5

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

423.01

0.0144

0.0499

0.3236

0.8657

0.3385

0.6744

0.4873

2.6686

0.1692

±SD

11.01

0.0008

0.0116

0.0088

0.0748

0.0194

0.1410

0.0183

0.4805

0.0227

n

5

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

418.89

0.0151

0.0676*

0.2670*

0.8047

0.3150

0.5574

0.4939

2.4239

0.1613

±SD

6.76

0.0013

0.0089

0.0268

0.0797

0.0187

0.0407

0.0156

0.0366

0.0098

n

5

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

Group, Sex & Dose (mg/L)

Fasting Body

Weight (g)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

183.73

0.0340

0.2021

0.3302

0.0659

0.2228

0.4117

0.7780

0.9621

3.3918

0.2801

±SD

10.58

0.0031

0.0399

0.0616

0.0093

0.0267

0.0288

0.0967

0.0446

0.2396

0.0176

n

5

5

5

5

5

5

5

5

5

5

5

G2, F & 0.001

Mean

184.83

0.0323

0.1989

0.2584

0.0639

0.2833

0.4037

0.7078

0.9991

3.0179

0.2812

±SD

11.27

0.0057

0.0301

0.0468

0.0023

0.0773

0.0323

0.1163

0.0846

0.3169

0.0489

n

5

5

5

5

5

5

5

5

5

5

5

G3, F & 0.003

Mean

181.71

0.0308

0.1592

0.1918*

0.0546

0.1868

0.4045

0.7093

0.9707

3.1331

0.2800

±SD

11.37

0.0027

0.0487

0.0310

0.0094

0.0339

0.0379

0.0690

0.0360

0.1629

0.0240

n

5

5

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

180.35

0.0312

0.1505

0.2599

0.0643

0.1925

0.3923

0.6760

0.9740

3.3548

0.3360

±SD

8.36

0.0040

0.0179

0.0485

0.0111

0.0407

0.0568

0.0940

0.0590

0.4502

0.0522

n

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

313.71

0.0258

0.0840

0.1930

0.0423

0.2400

0.2871

0.4958

0.5996

2.5426

0.1531

±SD

9.95

0.0035

0.0141

0.0286

0.0065

0.0704

0.0272

0.0176

0.0176

0.2084

0.0095

n

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

315.75

0.0247

0.0746

0.1648

0.0522*

0.2452

0.3060

0.4971

0.5706

2.2994

0.1527

±SD

10.67

0.0011

0.0098

0.0187

0.0037

0.0568

0.0317

0.0403

0.0362

0.2605

0.0212

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

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Testes

Heart

Kidneys

Liver

Lungs

G1, M & 0

Mean

2.7788

16.0226

51.3579

166.1722

49.2449

99.5469

413.9069

27.8307

±SD

0.4854

1.5547

3.4144

10.1365

3.9319

7.2237

33.9594

3.9036

n

5

5

5

5

5

5

5

5

G2, M & 0.001

Mean

2.6222

13.6994

60.4701

145.6095

53.8465

100.3085

435.2792

37.9309*

±SD

0.3220

2.6851

15.6939

19.4023

5.9632

6.4199

47.2753

5.0597

n

5

5

5

5

5

5

5

5

G3, M & 0.003

Mean

2.7980

15.4923

59.2262

155.4821

49.2030

93.0155

376.2989

29.7121

±SD

0.2108

1.8488

6.3794

8.1774

2.0680

1.8959

23.2915

3.8733

n

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

2.8385

14.6201

51.0374

178.7947

53.7343

102.6157

391.1467

36.7839*

±SD

0.2285

1.1804

4.1897

23.3721

2.2678

8.8050

44.0085

3.7514

n

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

2.9629

10.3105

66.5051

177.9219

69.6012

137.7537

545.6673

34.6491

±SD

0.2716

2.6969

3.8161

17.8584

5.7242

24.4075

79.4291

3.5038

n

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

3.0525

13.6603*

53.9801*

162.9183

63.8029

112.8276

491.1223

32.6866

±SD

0.2426

1.5808

4.1095

15.1124

3.4811

7.0959

14.6572

2.1792

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

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Liver

Lungs

G1, F & 0

Mean

3.5292

20.9151

34.2411

6.8497

23.1571

42.7947

80.6817

352.3827

29.1361

±SD

0.1907

3.3835

5.4867

0.8875

2.5721

2.2916

7.4942

13.8956

1.8218

n

5

5

5

5

5

5

5

5

5

G2, F & 0.001

Mean

3.2258

20.0988

26.2348

6.4392

28.7329

40.5900

70.6182

302.3913*

28.0970

±SD

0.4068

3.9272

6.4878

0.7038

9.0904

4.1657

6.8405

23.1828

3.5362

n

5

5

5

5

5

5

5

5

5

G3, F & 0.003

Mean

3.1860

16.3556

19.7471*

5.6262

19.2871

41.7060

73.1094

323.1543

28.8421

±SD

0.3606

4.8290

2.9899

0.9687

3.6096

4.1403

7.2204

21.4424

2.1647

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

3.1976

15.4181

26.5831

6.6137

19.7461

40.2033

69.2865

343.4061

34.3678*

±SD

0.3393

1.1681

3.8553

1.1811

3.8810

4.5301

7.6436

28.5407

3.6477

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

4.2971

14.0269

32.2042

7.0595

40.0258

47.8431

82.7173

424.6109

25.5294

±SD

0.4989

2.4052

4.8172

1.1360

11.8751

3.5805

2.8224

39.7625

1.4101

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

4.3383

13.1595

29.1223

9.1710*

43.1295

53.6468

87.3998

403.2624

26.7929

±SD

0.3998

2.2296

5.0961

0.7043

10.4359

4.6557

8.9537

40.2997

3.4916

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


Group & Conc.

(mg/L)

 

Temp.

 (°C)

Rh

(%)

O2

(%)

CO2

 (mg/L)

Air inlet flow rate

(L/min)*

BZC

(mg/L)

G1/G1R & 0

Mean

22.55

55.52

20.39

617.48

20.00

-

±SD

0.31

1.90

0.19

5.43

0.00

-

G2 & 0.001

Mean

22.66

55.95

20.47

619.67

20.00

0.0011

±SD

0.31

1.35

0.21

4.84

0.00

0.0001

G3 & 0.003

Mean

22.64

55.88

20.50

619.73

20.00

0.0032

±SD

0.31

1.32

0.23

5.31

0.00

0.0001

G4/G4R & 0.03

Mean

22.59

55.10

20.38

617.74

20.00

0.032

±SD

0.31

1.59

0.20

5.12

0.00

0.001

    *: 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.

TABLE 18.    SUMMARY OF NOMINAL CONCENTRATION

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)

1150

20

360

0.16

4370

0.61

5230

0.73

2

G1/G1R

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1120

20

360

0.16

4440

0.62

5110

0.71

 3

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1200

20

360

0.17

4380

0.61

5140

0.71

4

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1160

20

360

0.16

4350

0.60

5160

0.72

5

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1180

20

360

0.16

4430

0.62

5200

0.72

6

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1090

20

360

0.15

4340

0.60

5180

0.72

7

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1170

20

360

0.16

4430

0.62

5190

0.72

TABLE 18 (Contd…). SUMMARY OF NOMINAL CONCENTRATION

Day

Group

Test item Used (mg) (a)

Air flow rate

(L/minute)(b)

Minute

(min)

(c)

Nominal Concentration

(mg/L)

(a)    / (b) × (c)

8

G1/G1R 

(Air Only)

-

20

    360

-

G2, G3 and G4/G4R 

(Test Item)

1140

20

360

0.16

4570

0.63

5200

0.72

9

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1150

20

360

0.16

4330

0.60

5280

0.73

 10

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1190

20

360

0.17

4410

0.61

5110

0.71

11

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1130

20

360

0.16

4440

0.62

5210

0.72

12

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1170

20

360

0.16

4580

0.64

5290

0.73

13

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1190

20

360

0.17

4320

0.60

5150

0.72

14

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1160

20

360

0.16

4440

0.62

5120

0.71

TABLE 18 (Contd…). SUMMARY OF NOMINAL CONCENTRATION

Day

Group

Test item Used (mg) (a)

Air flow rate

(L/minute)(b)

Minute

(min)

(c)

Nominal Concentration

(mg/L)

(a)    / (b) × (c)

15

G1/G1R 

(Air Only)

-

20

    360

-

G2, G3 and G4/G4R 

(Test Item)

1170

20

360

0.16

4400

0.61

5170

0.72

16

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1170

20

360

0.16

4460

0.62

5210

0.72

 17

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1190

20

360

0.17

4400

0.61

5270

0.73

18

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1140

20

360

0.16

4360

0.61

5200

0.72

19

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1190

20

360

0.17

4590

0.64

5230

0.73

20

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1160

20

360

0.16

4590

0.64

5270

0.73

TABLE 19.     SUMMARY OF GROSS PATHOLOGY FINDINGS

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 Concentration (mg/L)

0

0.001*

0.003*

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 Concentration (mg/L)

0

0.001*

0.003*

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

-

-

1

-

-

-

Uterus– small in size

-

-

1

-

-

-

R: Recovery; *: Above doses were provided by the sponsor; - : No incidence.

TABLE 20.      SUMMARY OF HISTOPATHOLOGY FINDINGS – MAIN GROUP

Route of administration

Inhalation

Treatment

Air only

High Dose

Nominal Target Concentration (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

4

3

4

Cyst

Present

1

1

2

-

Cast

Minimal

1

-

-

-

Basophilia, tubules

Minimal

-

-

1

-

Dilatation, 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(left lobe)

Number examined

5

5

5

5

Within normal limits

5

5

5

5

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

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

TABLE 20 (Contd..). SUMMARY OF HISTOPATHOLOGY FINDINGS – MAIN GROUP

Route of administration

Inhalation

Treatment

Air only

High Dose

Nominal Target Concentration (mg/L)

0

0.03

Group

G1

G4

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

4

X

Inflammation

Mild

-

X

1

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

4

X

5

X

Atrophy, tubules

Minimal

1

X

-

X

Thymus

Number examined

5

5

5

5

Within normal limits

5

3

5

4

Cyst, epithelial

Present

-

2

-

1

Thyroid

Number examined

5

5

5

5

Within normal limits

5

3

3

5

Cyst, ultimobranchial

Present

-

1

2

-

Ectopic tissue, thymus

Present

-

1

-

-

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

5

X

4

Dilatation, lumen

Minimal

X

-

X

1

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

TABLE 21. SUMMARY OF HISTOPATHOLOGY FINDINGS – RECOVERY GROUP

Route of administration

Inhalation

Treatment

Air only

High Dose Recovery

Nominal Target Concentration (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

4

5

4

5

Ectopic tissue, adrenocortical

Present

1

-

1

-

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

4

5

5

5

Cellularity, increased, lymphocytes

Mild

1

-

-

-

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

2

5

5

Infiltrate, mononuclear cells

Minimal

1

1

-

-

Cyst

Present

-

1

-

-

Dilatation, 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(left lobe)

Number examined

5

5

5

5

Within normal limits

3

3

3

3

Pigment (background finding-likely hemosiderin)

Minimal

2

2

2

1

Mild

-

-

-

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

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

TABLE 21 (Contd…). SUMMARY OF HISTOPATHOLOGY FINDINGS – RECOVERY GROUP

Route of administration

Inhalation

Treatment

Air only

High Dose Recovery

Nominal Target Concentration (mg/L)

0

0.03

Group

G1R

G4R

Sex

M

F

M

F

Number of Animals

5

5

5

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

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

3

4

3

Cyst, epithelial

Present

-

2

1

2

Cellularity, increased, epithelial cells

Minimal

-

-

1

-

Thyroid

Number examined

5

5

5

5

Within normal limits

4

4

3

4

Cyst, ultimobranchial

Present

1

1

1

1

Ectopic tissue, thymus

Present

-

-

1

-

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

3

Dilatation, lumen

Minimal

X

1

X

-

Mild

X

-

X

2

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

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 was found to be 0.03 mg/L when exposed for 6 hours/day, 5 days/week, for 04 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 04 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 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 Hostaperm Yellow H6G for 6 hours per day, 5 days per week, for 04 consecutive weeks, at a nominal target concentration of 0.001, 0.003 and 0.03 mg/L. Animals of the control group (G1/G1R) received air only inhalation for 6 hours/day for 04 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 observed once daily for clinical signs and twice daily for mortality. Body weight was recorded before exposure (day 1) and weekly twice first two weeks during exposure period and weekly once for remaining two weeks during exposure period and recovery period. Feed consumption was recorded weekly. Ophthalmoscopic examinations were performed during the acclimatization period for all groups, and during Week4 for main groups (G1and G4), and duringWeek12 for recovery groups (G1Rand G4R). Neurological/Functional tests were performed during Week4 for G1 and G4 groups and during Week12 for recovery group animals.

At the end of treatment and recovery periods, all animals were fasted overnight (water was available ad 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, absolute organ weight, or relative organ weight.

There were no test item-related, microscopic findings in animals evaluated after terminal and recovery period in the study.

Few microscopic findings observed in this study such as ultimobranchial cyst in thyroid gland, epithelial cysts in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected in laboratory rats.

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