Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 February 2007 to 01 July 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 21 September 1998
Deviations:
no
Remarks:
no hormonal measurements performed
GLP compliance:
yes
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid
EC Number:
617-769-9
Cas Number:
858956-08-8
Molecular formula:
C8H8ClN3O2
IUPAC Name:
6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 7 weeks
- Weight at study initiation: 253.0 - 256.4 g (males), 173.7 - 179.4 (females)
- Housing: individually in stainless steel, wire-mesh cages suspended above cage boards
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet 5002®, ad libitum
- Water: tap water, ad libitum
- Acclimation period: approximately 9 days

DETAILS OF FOOD AND WATER QUALITY: water samples were ananalyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 -26
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: test substance was added directly to the diet to achieve the target concentrations specified by the study design; the test diets were thoroughly mixed to ensure homogeneous distribution of the test substance throughout the diet.

DIET PREPARATION
- Rate of preparation of diet: weekly, except in two instances in which diets were prepared for 10 or 11 days, after stability was confirmed.
- Mixing appropriate amounts with: PMI® Nutrition International, LLC Certified Rodent LabDiet 5002®.
- Storage temperature of food: all diets were refrigerated until used.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate samples (concentration verification and homogeneity (from top, middle bottom of the sample) of all dietary concentrations were collected at the initial diet preparation and analyzed to verify the concentration (average of homogeneity samples) and homogeneity of test substance in the diets. Near the middle and end of the study, samples were taken to verify concentration. Duplicate samples were taken from the lowest (300 ppm) and the highest (18,000 ppm) dietary concentration of the initial diet preparation and were analyzed to verify the stability of the test substance in the diets. The 300 ppm concentration was included in the initial diet preparation to establish the stability to cover the dietary concentrations of the present study (600 to 18,000 ppm) as well as the concurrent mouse study (300 to 7000 ppm).

The certificate of analysis (COA) included in the original finalized report listed a purity of 92.2%, which did not fully account for all the inorganic impurities associated with this sample. Due to follow-up analysis of the test substance the COA was revised for the purity 90.5% that accounts for the full impurity profile. This reduction in determined purity of 1.7% had minimal impact on the reported doses determined by chemical analyses and on the values utilized for risk assessments. Therefore, the reported dose values were not adjusted.
Duration of treatment / exposure:
approximately 90 days (94 or 95 days in males and 96 or 97 days in females)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
600 ppm
Remarks:
Males: corresponding to 35 mg/kg bw/day
Females: corresponding to 45 mg/kg bw/day
Dose / conc.:
2 000 ppm
Remarks:
Males: corresponding to 114 mg/kg bw/day
Females: corresponding to 146 mg/kg bw/day
Dose / conc.:
6 000 ppm
Remarks:
Males: corresponding to 349 mg/kg bw/day
Females: corresponding to 448 mg/kg bw/day
Dose / conc.:
18 000 ppm
Remarks:
Males:corresponding to 1045 mg/kg bw/day
Females:corresponding to 1425 mg/kg bw/day
No. of animals per sex per dose:
15
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The test substance is a metabolite of methyl 6-amino-5-chloro-2-(cyclopropyl)-4-pyrimidinecarboxylate. A previous 28-day study in rats was conducted with methyl 6-amino-5-chloro-2-(cyclopropyl)-4-pyrimidinecarboxylate at dietary concentrations of 600, 2000, 6000, and 20,000 ppm. No test substance-related effects occurred on ophthalmogical observations, clinical observations, clinical pathology parameters, food consumption, organ weights, gross morphological observations, or survival. A test substance-related decrease in body weight, weight gain, and food efficiency occurred in 20,000 ppm males and females. Males in the 20,000 ppm group had thyroid follicular cell hypertrophy and pancreatic acinar cell apoptosis. Females in the 20,000 ppm group had induction of hepatic ß-oxidation activity. Based on these findings, the dietary concentrations selected for the current 90-day study were 600, 2000, 6000, and 18,000 ppm.

- Rationale for selecting satellite groups: on test day 56, blood was collected from satellite animals for evaluation of test substance and/or metabolite concentration in plasma (separate report was not available).


Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked: moribund/dead rats, abnormal behavior/appearance and acute clinical signs of systemic toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Day 0 and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- 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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretest and week 13
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 14
- Anaesthetic used for blood collection: not specified
- Animals fasted: not specified
- How many animals: all dose groups
- Parameters checked in table No. 1 in the "Any other information and methods incl. tables" section were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 14
- Animals fasted: not specified
- How many animals: all dose groups
- Parameters checked in table No.2 in the "Any other information and methods incl. tables" section were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: week 14
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.3 in the "Any other information and methods incl. tables" section were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pretest and weeks 4, 8, and 13
- Dose groups that were examined: all dose groups
- Battery of functions tested: motor activity / grip strength, rectal body temperature other: please refer to table No 4 in the "Any other information and methods incl. tables" section.

IMMUNOLOGY: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (Please refer to table 5 in the "Any other information and methods incl. tables" section.)

HISTOPATHOLOGY: Yes (Please refer to table 6 in the "Any other information and methods incl. tables" section.)
Organ weights determined for organs/tissues listed in table 7 in the "Any other information and methods incl. tables" section.)
Other examinations:
Subchronic Neurotoxicity:
On test days 94 and 95, rats were designated for neuropathology (5 rats/sex/group) analysis. Animals were anesthetized and underwent whole-body in situ perfusion.
(For tissues analysed and further details, please refer to table 8 in the "Any other information and methods incl. tables" section.)

Evaluation of test substance and/or metabolite concentration in plasma (satellite animals)
Approximately 0.5 mL of blood was collected from the satellite animals (5 rats/sex/concentration) on test day 56, for determination of plasma concentration of test substance and/or metabolites.
Statistics:
Significance was judged at p < 0.05. Separate analyses were performed on the data collected for each sex.
For details on statistics please refer to table 9 in the "Any other information and methods incl. tables" section.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
600 ppm and 2000 ppm: one female of each dose group had abnormal gait. However, abnormal gait was not observed in these animals during any of the neurobehavioral evaluations. Since no dose response was evident and occurrence was limited to 1 animal/group, these observations were considered to be spurious and not treatment-related.

Other clinical signs observed throughout the study were typical for rats of this age and strain, and included hair loss, eye, ear, or nose discharge hyperactivity, high posture, protrusion in abdomen, absent teeth, clipped teeth, maloccluded teeth, ear twitch, corneal opacity and wound. These clinical signs were observed at low incidence and with no dose response.

(Please refer to table 10 in the "Any other information on results incl. tables" section.)
Mortality:
mortality observed, non-treatment-related
Description (incidence):
control and 6000 ppm: one control male rat and one female rat in the 6000 ppm group were accidentally killed during blood collection just prior to necropsy (non-treatment-related).

(Please refer to table 10 in the "Any other information on results incl. tables" section.)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
18,000 ppm: significant decrease in mean body weights and body weight gains were observed in males and females. In males, mean body weight was 9% lower than that of controls on the final test day 91. Overall (test days 0-91) body weight gain in males was 15% lower (statistically significant) than that of controls. In females, mean body weight was 8% lower than that of controls on the final test day 91. Overall (test days 0-91) body weight gain in males was 18% lower (statistically significant) than that of controls.The effects were considered treatment-related and adverse.

6000 ppm: statistically significantly lower body weights and body weight gains compared to controls were observed in males and females over test days 21-28 and 14-21, respectively. (non-treatment-related and non-adverse)

600 ppm: statistically significantly lower body weights and body weight gains compared to controls were observed in females over test days 84-91 (non-treatment-related and non-adverse)..

Changes observed at 6000 and 600 ppm were considered spurious because they occurred only in one time interval and there was no dose response.

(Please refer to table 11 and 12 in the "Any other information on results incl. tables" section.)
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
18,000 ppm: mean daily food consumption was statistically significantly lower in males over test day intervals 0-7, 21-28, and 42-49 and in females over test day interval 70-77. These sporadic changes were considered incidental and not treatment-related, because overall mean food consumption was not significantly different from control.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
18,000 ppm: significant decrease in food efficiency was observed in males and females. The overall food efficiency (test days 0-91) in males and females of this dose group was 89% and 83% of control, respectively. In addition, statistically significantly lower mean food efficiency was noted in males over test day intervals 21-28, 70-77, and 77-84 and in females over test day interval 62-70. Since the changes in mean food efficiency were consistent with decreased body weight parameters, they were considered treatment-related and adverse.

6000 ppm: statistically significant reductions in food efficiency were noted in males over test days 21-28 (non-treatment-related and non-adverse).

600 ppm: statistically significant reductions in food efficiency were noted in females over test days 84-91 (non-treatment-related and non-adverse).

Since the findings at 6000 and 600 ppm occurred only over one weekly interval, they were considered spurious, non-treatment-related and non-adverse.

(Please refer to table 13 in the "Any other information on results incl. tables" section.)
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
600 ppm: one male had a posterior lens opacity and one female had a lens opacity and dislocation, retinal degeneration and iritis. Since effects were observed only in single animals and no dose-response relationship was visible, the observations were not considered treatment-related and non-adverse.
Haematological findings:
no effects observed
Description (incidence and severity):
(Please refer to table 14 in the "Any other information on results incl. tables" section.)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
6000 ppm: triglyceride (69% of control), total protein and albumin (94% and 93% of control, respectively) and calcium (95% of control) were decreased in female rats. The observed statistically significant changes in group mean clinical chemistry parameters at test days 96-97 in females were considered to be non-treatment-related and non-adverse, because the change did not occur in a dose-related manner.

(Please refer to table 15 in the "Any other information on results incl. tables" section.)
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
18,000 ppm: urine volume was decreased (52% of control) in male rats. However, urine volumes ranging from 2.0 to 14.6 mL in male rats were within the urine volume range of the male control group, 0.7 to 21.0 mL. In addition, there were no correlative clinical pathology or histopathology changes and a similar decrease in urine volume was not observed in females. Therefore, this change was considered non-treatment-related and non-adverse. Furthermore, pH was minimally decreased (91% of control) in female rats dosed. This change was considered non-adverse due to the minimal nature of the change and the lack of associated changes in other urinalysis or clinical chemistry parameters.

(Please refer to table 16 in the "Any other information on results incl. tables" section.)
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
18,000 ppm: in males, a small (9%) decrease in the mean final body weight, as compared to the control value, was associated with a similar decrease in the mean absolute liver, heart, spleen, and thymus weights. Some of these decreases were statistically significant. Since, there were no gross or microscopic pathology correlates observed and most mean relative (% body weight) organ weights were comparable to control values, the small decreases in mean absolute organ weights were interpreted to be a result of the decreased mean final body weight and not an indication of an organ-specific effect. There was a statistically significant decrease (12%) in the mean relative (% brain weight) liver weight in males, as compared to controls. This difference was also attributed to the decrease in final body weight and secondary to decrease in mean absolute liver weights.

(Please refer to table 17 in the "Any other information on results incl. tables" section.)
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All gross observations were consistent with normal background lesions in rats of this age and strain and thus non-treatment-related and non-adverse.

(Please refer to table 18 in the "Any other information on results incl. tables" section.)
Neuropathological findings:
no effects observed
Description (incidence and severity):
(Please refer to table 23 and 24 in the "Any other information on results incl. tables" section.)
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All microscopic findings were consistent with normal background lesions in rats of this age and strain and thus non-treatment-related and non-adverse.


(Please refer to table 19 in the "Any other information on results incl. tables" section.)
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Neurobehavioral evaluation

Functional Observational Battery: There were no treatment-related or statistically significant effects/differences on forelimb or hindlimb grip strength, in footsplay and in the number of rearing movements in the open field arena in males and females of any dose group. (Please refer to table 20, 21 and 22 in the "Any other information on results incl. tables" section.)

600 and 6000 ppm: occasional occurrence of abnormal gait in either the home cage or open field were noted in one 600 ppm male and one 6000 ppm female. Since a dose-response relationship was not evident for either males or females, these incidents were considered to be spurious, non-treatment-related and non-adverse.

Motor acitivity: There were no treatment-related or statistically significant effects on duration of movement or number of movements for males or females of any dose group.

Effect levels

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
18 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food efficiency
Remarks on result:
other: corresponding to 1045 and 1425 mg/kg bw/day in males and females, respectively.
Key result
Dose descriptor:
NOAEL
Effect level:
6 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect observed
Remarks on result:
other: corresponding to 349 and 448 mg/kg bw/day in males and females, respectively.

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 10: Summary of detailed clinical observations and mortality data

Males

Group

Concentration (ppm)

Control

600

2000

6000

18000

Number

15

15

15

15

15

Discharge - black – eye(s)/ear(s)

 

 

 

 

 

Number of Observations

2

 

 

7

4

Number of Animals

1

 

 

2

1

Days from - to

49 - 56

 

 

7 - 62

28 – 49

Discharge - brown - nose

 

 

 

 

 

Number of Observations

 

 

 

3

 

Number of Animals

 

 

 

1

 

Days from - to

 

 

 

7 – 21

 

Hair loss

 

 

 

 

 

Number of Observations

13

 

40

 

11

Number of Animals

1

 

4

 

2

Days from - to

7 - 91

 

7 - 91

 

21 - 91

Hyperreactive

 

 

 

 

 

Number of Observations

 

 

7

 

 

Number of Animals

 

 

1

 

 

Days from - to

 

 

49 - 91

 

 

Posture - high

 

 

 

 

 

Number of Observations

 

 

7

 

 

Number of Animals

 

 

1

 

 

Days from - to

 

 

49 - 91

 

 

Protrusion - abdomen

 

 

 

13

 

Number of Observations

 

 

 

1

 

Number of Animals

 

 

 

7 - 91

 

Days from - to

 

 

 

 

 

Teeth absent

 

 

 

 

 

Number of Observations

7

 

 

12

 

Number of Animals

1

 

 

1

 

Days from - to

49 - 91

 

 

14 – 91

 

Teeth clipped

 

 

 

 

 

Number of Observations

 

 

 

12

8

Number of Animals

 

 

 

1

1

Days from - to

 

 

 

14 – 91

42 – 91

Teeth maloccluded

 

 

 

 

 

Number of Observations

 

 

 

23

10

Number of Animals

 

 

 

2

1

Days from - to

 

 

 

7 - 91

28 - 91

Accidentally Killed   

 

 

 

 

 

Number of Observations

1

 

 

 

 

Number of Animals

1

 

 

 

 

Days from - to

95 - 95

 

 

 

 

Neuropathology sacrifice

 

 

 

 

 

Number of Observations

5

5

5

5

5

Number of Animals

5

5

5

5

5

Days from - to

94 - 95

94 - 95

94 - 95

94 - 95

94 - 95

Scheduled sacrifice

 

 

 

 

 

Number of Observations

9

10

10

10

10

Number of Animals

9

10

10

10

10

Days from - to

94 - 95

94 - 95

94 - 95

94 - 95

94 - 95

Females

Number

15

15

15

15

15

Abnormal gait

 

 

 

 

 

Number of Observations

 

1

4

 

 

Number of Animals

 

1

1

 

 

Days from - to

 

91 - 91

70 – 91

 

 

Ear twitch

 

 

 

 

 

Number of Observations

1

 

 

1

 

Number of Animals

1

 

 

1

 

Days from - to

7 -7

 

 

70 -70

 

Eye corneal opacity

 

 

 

 

 

Number of Observations

 

9

 

 

 

Number of Animals

 

1

 

 

 

Days from - to

 

35 - 91

 

 

 

Hair loss

 

 

 

 

 

Number of Observations

 

18

7

6

16

Number of Animals

 

2

1

1

2

Days from - to

 

7 - 91

49 - 91

56 - 91

14 – 91

Wound - superficial - neck

 

 

 

 

 

Number of Observations

 

 

 

 

3

Number of Animals

 

 

 

 

1

Days from - to

 

 

 

 

14 -28

Accidentally Killed

 

 

 

1

 

Number of Observations

 

 

 

1

 

Number of Animals

 

 

 

96 – 96

 

Days from - to

 

 

 

 

 

Neuropathology sacrifice

 

 

 

 

 

Number of Observations

5

5

5

5

5

Number of Animals

5

5

5

5

5

Days from - to

94 - 95

94 - 95

94 - 95

94 - 95

94 - 95

Scheduled sacrifice

 

 

 

 

 

Number of Observations

10

10

10

10

10

Number of Animals

10

10

10

10

10

Days from - to

96 - 97

96 - 97

96 - 97

96 - 97

96 - 97

 

Table 11: Mean body weights (g)

Males

Group

Concentration (ppm)

Control

600

2000

6000

18000

BW:DAY0

256.4

254.7

253.7

255.3

253.0

 

11.3(15)

13.3(15)

13.7(15)

13.0(15)

10.7(15)

BW:DAY7

311.7

310.4

308.9

309.4

300.6

 

15.8(15)

15.3(15)

17.6(15)

19.8(15)

11.8(15)

BW:DAY14

358.2

357.1

356.9

354.6

345.7

 

21.1(15)

17.6(15)

22.9(15)

36.2(15)

15.3(15)

BW:DAY21

390.4

390.3

390.2

384.3

371.9

 

27.3(15)

20.4(15)

27.8(15)

35.5(15)

17.7(15)

BW:DAY28

429.4

424.7

424.0

416.1

401.7*

 

31.9(15)

24.9(15)

30.6(15)

36.2(15)

17.2(15)

BW:DAY35

459.7

451.9

455.3

450.3

427.2*

 

36.0(15)

26.2(15)

35.6(15)

39.6(15)

19.7(15)

BW:DAY42

486.3

480.3

482.3

479.9

452.1*

 

38.7(15)

29.6(15)

39.0(15)

41.6(15)

23.8(15)

BW:DAY49

506.3

497.7

504.1

498.9

470.1*

 

41.9(15)

31.8(15)

42.8(15)

46.1(15)

25.3(15)

BW:DAY56

520.8

512.8

517.5

513.1

483.3

 

47.5(15)

36.5(15)

41.9(15)

49.7(15)

26.0(15)

BW:DAY62

538.7

531.2

534.6

533.7

500.2*

 

46.0(15)

36.2(15)

44.0(15)

50.2(15)

27.1(15)

BW:DAY70

551.6

543.6

551.7

549.5

514.2

 

46.4(15)

38.9(15)

46.7(15)

45.1(15)

26.4(15)

BW:DAY77

571.0

560.6

566.7

566.4

524.5*

 

48.1(15)

39.7(15)

50.2(15)

47.9(15)

30.4(15)

BW:DAY84

578.7

568.5

575.1

575.3

526.5*

 

48.1(15)

42.6(15)

51.2(15)

50.9(15)

31.9(15)

BW:DAY91

580.7

572.9

580.3

578.9

529.4*

 

50.4(15)

43.6(15)

51.4(15)

53.0(15)

34.9(15)

Females

Group

Concentration (ppm)

Control

600

2000

6000

18000

BW:DAY0

179.4

176.7

180.2

173.7

174.6

 

10.2(15)

8.8(15)

9.8(15)

10.1(14)

9.8(15)

BW:DAY7

196.5

193.9

200.3

189.1

188.7

 

13.0(15)

9.6(15)

16.3(15)

11.1(15)

11.7(15)

BW:DAY14

214.9

216.5

221.7

209.9

206.2

 

15.5(15)

14.8(15)

20.2(15)

12.6(15)

16.5(15)

BW:DAY21

226.9

228.0

236.3

214.5

213.9

 

17.2(15)

14.9(15)

21.8(15)

11.6(15)

16.5(15)

BW:DAY28

244.6

242.9

247.5

234.3

229.7

 

22.1(15)

18.2(15)

23.8(15)

14.6(15)

18.2(15)

BW:DAY35

254.0

251.7

261.1

243.5

239.3

 

22.9(15)

18.8(15)

28.5(15)

13.8(15)

22.4(14)

BW:DAY42

261.5

262.6

268.9

251.5

248.3

 

22.6(15)

20.2(15)

31.3(15)

13.7(15)

23.1(15)

BW:DAY49

267.1

268.3

273.5

254.6

250.0

 

22.5(15)

25.0(15)

29.5(15)

16.2(15)

19.3(15)

BW:DAY56

276.8

273.8

279.4

262.7

254.0*

 

24.1(15)

22.4(15)

27.9(15)

18.3(15)

22.4(15)

BW:DAY62

280.9

280.1

288.2

269.3

262.0

 

23.3(15)

20.0(15)

29.7(15)

20.9(15)

21.5(15)

BW:DAY70

287.4

289.6

293.6

276.1

261.7*

 

24.9(15)

24.1(15)

30.3(15)

23.2(15)

23.1(15)

BW:DAY77

293.7

292.2

302.9

279.9

264.7*

 

24.3(15)

26.1(15)

31.7(15)

25.9(15)

24.1(15)

BW:DAY84

293.4

294.9

301.9

282.9

269.6

 

29.8(15)

24.2(15)

32.9(15)

23.0(15)

21.2(15)

BW:DAY91

294.6

288.6

304.9

284.1

270.6

 

30.5(15)

24.6(15)

34.5(15)

23.4(15)

22.6(15)

Data arranged as: Mean Standard deviation (Number of values included in calculation)

* Parametric comparison to control (Dunnett/Tamhane-Dunnett) significant

@ Nonparametric comparison to control (Dunn’s) significant

 

Table 12: Mean body weight gains (g)

Males

Group

Concentration (ppm)

Control

600

2000

6000

18000

BWG:DAY0-DAY7

55.3

55.7

55.2

54.1

47.6*

 

8.1(15)

4.6(15)

6.0(15)

10.3(15)

5.4(15)

BWG:DAY7-DAY14

46.5

46.7

48.1

45.2

45.1

 

6.6(15)

5.9(15)

7.3(15)

21.0(15)

7.2(15)

BWG:DAY14-DAY21

32.2

33.2

33.3

29.7

26.2

 

9.1(15)

6.5(15)

9.1(15)

8.7(15)

6.0(15)

BWG:DAY21-DAY28

39.0

34.4

33.8

31.7*

29.7*

 

7.1(15)

6.6(15)

8.5(15)

5.5(15)

6.3(15)

BWG:DAY28-DAY35

30.3

27.3

31.3

34.2

25.5

 

6.0(15)

4.4(15)

6.4(15)

7.2(15)

6.5(15)

BWG:DAY35-DAY42

26.6

28.3

27.0

29.7

24.9

 

5.6(15)

5.8(15)

6.8(15)

4.9(15)

7.3(15)

BWG:DAY42-DAY49

19.9

17.5

21.8

18.9

17.9

 

6.8(15)

5.7(15)

6.5(15)

6.7(15)

8.2(15)

BWG:DAY49-DAY56

14.6

15.0

13.4

14.2

13.2

 

8.2(15)

8.9(15)

7.3(15)

7.9(15)

7.1(15)

BWG:DAY56-DAY62

17.9

18.5

17.0

20.6

16.9

 

4.2(15)

7.0(15)

4.5(15)

5.6(15)

6.8(15)

BWG:DAY62-DAY70

12.9

12.4

17.1

15.9

14.0

 

5.4(15)

6.8(15)

6.0(15)

9.0(15)

3.4(15)

BWG:DAY70-DAY77

19.4

17.0

15.1

16.9

10.3*

 

6.2(15)

5.8(15)

6.5(15)

4.5(15)

8.0(15)

BWG:DAY77-DAY84

7.6

7.9

8.4

8.9

1.9*

 

4.6(15)

5.5(15)

5.2(15)

7.6(15)

4.8(15)

BWG:DAY84-DAY91

2.0

4.3

5.2

3.7

2.9

 

6.4(15)

6.5(15)

6.3(15)

6.8(15)

7.2(15)

BWG:DAY0-DAY91

324.3

318.1

326.7

323.6

276.4*

 

45.0(15)

36.5(15)

41.5(15)

42.7(15)

30.0(15)

Females

Goup

Concentration (ppm)

Control

600

2000

6000

18000

BWG:DAY0-DAY7

17.1

17.3

20.1

16.0

14.1

 

8.6(15)

6.7(15)

7.6(15)

4.4(14)

7.0(15)

BWG:DAY7-DAY14

18.3

22.6

21.4

20.9

17.5

 

9.1(15)

9.2(15)

9.4(15)

5.2(15)

7.3(15)

BWG:DAY14-DAY21

12.0

11.5

14.6

4.6*

7.7

 

10.8(15)

6.4(15)

6.5(15)

5.8(15)

8.0(15)

BWG:DAY21-DAY28

17.7

14.9

11.2

19.8

15.7

 

9.6(15)

9.9(15)

8.1(15)

8.8(15)

6.5(15)

BWG:DAY28-DAY35

9.4

8.8

13.5

9.2

10.0

 

7.6(15)

7.2(15)

6.9(15)

5.9(15)

7.3(14)

BWG:DAY35-DAY42

7.5

10.9

7.9

8.0

8.9

 

9.6(15)

9.5(15)

6.1(15)

4.9(15)

4.8(14)

BWG:DAY42-DAY49

5.6

5.7

4.5

3.1

1.7

 

5.2(15)

7.9(15)

5.5(15)

6.5(15)

6.6(15)

BWG:DAY49-DAY56

9.7

5.5

6.0

8.1

4.0

 

8.7(15)

7.5(15)

8.7(15)

6.6(15)

9.8(15)

BWG:DAY56-DAY62

4.1

6.3

8.8

6.6

8.0

 

9.6(15)

6.5(15)

9.0(15)

10.1(15)

8.8(15)

BWG:DAY62-DAY70

6.5

9.5

5.4

6.8

-0.2@

 

4.5(15)

8.5(15)

5.4(15)

4.0(15)

5.6(15)

BWG:DAY70-DAY77

6.3

2.6

9.2

3.8

2.9

 

5.6(15)

8.3(15)

5.2(15)

9.0(15)

6.9(15)

BWG:DAY77-DAY84

-0.3

2.7

-1.0

3.0

4.9

 

8.0(15)

9.1(15)

6.4(15)

6.7(15)

7.9(15)

BWG:DAY84-DAY91

1.2

-6.3*

3.1

1.3

1.0

 

8.8(15)

7.1(15)

9.4(15)

6.5(15)

6.3(15)

BWG:DAY0-DAY91

115.2

111.9

124.7

112.5

94.9*

 

26.9(15)

19.3(15)

26.1(15)

17.5(14)

16.9(14)

Data arranged as: Mean Standard deviation (Number of values included in calculation)

* Parametric comparison to control (Dunnett/Tamhane-Dunnett) significant

@ Nonparametric comparison to control (Dunn’s) significant

 

Table 13: Food efficiency (body weight (g)/ food consumed (g))

Males

Group

Concentration (ppm)

Control

600

2000

6000

18000

FE:DAY0-DAY7

0.305

0.310

0.307

0.295

0.286

 

0.037(15)

0.021(15)

0.016(15)

0.045(15)

0.032(15)

FE:DAY7-DAY14

0.256

0.252

0.262

0.228

0.247

 

0.030(15)

0.029(15)

0.025(15)

0.151(15)

0.030(15)

FE:DAY14-DAY21

0.179

0.181

0.186

0.170

0.151

 

0.046(15)

0.032(15)

0.039(15)

0.052(15)

0.031(15)

FE:DAY21-DAY28

0.204

0.180

0.182

0.173*

0.168*

 

0.032(15)

0.026(15)

0.039(15)

0.029(15)

0.037(15)

FE:DAY28-DAY35

0.158

0.143

0.165

0.175

0.142

 

0.026(15)

0.023(15)

0.022(15)

0.028(15)

0.031(15)

FE:DAY35-DAY42

0.134

0.147

0.140

0.152

0.135

 

0.026(15)

0.026(15)

0.029(15)

0.020(15)

0.037(15)

FE:DAY42-DAY49

0.102

0.091

0.114

0.096

0.097

 

0.033(15)

0.028(15)

0.029(15)

0.028(15)

0.040(15)

FE:DAY49-DAY56

0.077

0.081

0.074

0.076

0.073

 

0.040(15)

0.043(15)

0.039(15)

0.036(15)

0.039(15)

FE:DAY56-DAY62

0.109

0.111

0.103

0.124

0.106

 

0.027(15)

0.041(15)

0.022(15)

0.033(15)

0.041(15)

FE:DAY62-DAY70

0.060

0.056

0.078

0.072

0.068

 

0.024(15)

0.030(15)

0.025(15)

0.039(15)

0.018(15)

FE:DAY70-DAY77

0.101

0.090

0.076

0.086

0.056*

 

0.027(15)

0.031(15)

0.028(15)

0.018(15)

0.042(15)

FE:DAY77-DAY84

0.041

0.042

0.046

0.045

0.011*

 

0.024(15)

0.026(15)

0.027(15)

0.035(15)

0.027(15)

FE:DAY84-DAY91

0.010

0.023

0.027

0.018

0.014

 

0.036(15)

0.036(15)

0.034(15)

0.033(15)

0.043(15)

FE:DAY0-DAY91

0.133

0.131

0.135

0.132

0.119*

 

0.015(15)

0.012(15)

0.007(15)

0.008(15)

0.009(15)

Females

Goup

Concentration (ppm)

Control

600

2000

6000

18000

FE:DAY0-DAY7

0.131

0.142

0.150

0.138

0.107

 

0.063(15)

0.052(15)

0.049(15)

0.040(14)

0.048(15)

FE:DAY7-DAY14

0.139

0.164

0.160

0.161

0.127

 

0.073(15)

0.059(15)

0.066(15)

0.043(15)

0.051(15)

FE:DAY14-DAY21

0.088

0.087

0.109

0.038

0.055

 

0.073(15)

0.052(15)

0.044(15)

0.048(15)

0.057(15)

FE:DAY21-DAY28

0.128

0.105

0.079

0.147

0.118

 

0.068(15)

0.068(15)

0.053(15)

0.065(15)

0.054(15)

FE:DAY28-DAY35

0.070

0.065

0.095

0.068

0.070

 

0.055(15)

0.058(15)

0.046(15)

0.041(15)

0.055(14)

FE:DAY35-DAY42

0.052

0.080

0.055

0.062

0.068

 

0.068(15)

0.069(15)

0.043(15)

0.038(15)

0.035(14)

FE:DAY42-DAY49

0.040

0.034

0.034

0.024

0.014

 

0.039(15)

0.049(15)

0.040(15)

0.049(15)

0.054(15)

FE:DAY49-DAY56

0.077

0.046

0.045

0.064

0.027

 

0.066(15)

0.063(15)

0.068(15)

0.052(15)

0.075(15)

FE:DAY56-DAY62

0.030

0.057

0.072

0.055

0.073

 

0.079(15)

0.060(15)

0.069(15)

0.084(15)

0.080(15)

FE:DAY62-DAY70

0.042

0.058

0.034

0.042

-0.005*

 

0.029(15)

0.047(15)

0.036(15)

0.021(15)

0.040(15)

FE:DAY70-DAY77

0.047

0.016

0.066

0.028

0.023

 

0.040(15)

0.059(15)

0.037(15)

0.070(15)

0.057(15)

FE:DAY77-DAY84

-0.008

0.020

-0.009

0.023

0.038

 

0.063(15)

0.067(15)

0.052(15)

0.054(15)

0.062(15)

FE:DAY84-DAY91

0.000

-0.050@

0.020

0.009

0.007

 

0.080(15)

0.065(15)

0.068(15)

0.050(15)

0.050(15)

FE:DAY0-DAY91

0.066

0.064

0.071

0.067

0.055*

 

0.010(15)

0.009(15)

0.012(15)

0.012(14)

0.010(14)

Data arranged as: Mean Standard deviation (Number of values included in calculation)

* Parametric comparison to control (Dunnett/Tamhane-Dunnett) significant

@ Nonparametric comparison to control (Dunn’s) significant

 

 

Table 14 : Summary of haematology and coagulation values

 

Male

 

 

 

 

 

Groups

Concentration (ppm)

Control

600

2000

6000

18000

RBC (x106/µL)

 

 

 

 

 

DAY 94-95

8.80

8.82

8.91

8.60

8.88

 

0.34(10)

0.25(10)

0.23(10)

0.29(9)

0.51(10)

HGB (g/dL)

 

 

 

 

 

DAY 94-95

15.7

15.4

15.6

15.5

15.6

 

0.6(10)

0.7(10)

0.4(10)

0.5(9)

0.7(10)

HCT (%)

 

 

 

 

 

DAY 94-95

47.8

47.0

47.8

46.8

47.6

 

2.0(10)

2.0(10)

1.7(10)

1.6(9)

2.2(10)

MCV (fL)

 

 

 

 

 

DAY 94-95

54.3

53.4

53.7

54.5

53.7

 

1.7(10)

1.7(10)

1.6(10)

2.5(9)

1.2(10)

MCH (pg)

 

 

 

 

 

DAY 94-95

17.8

17.5

17.6

18.0

17.5

 

0.6(10)

0.5(10)

0.4(10)

0.6(9)

0.5(10)

MCHC (g/dL)

 

 

 

 

 

DAY 94-95

32.7

32.7

32.7

33.0

32.7

 

0.6(10)

0.5(10)

0.6(10)

0.5(9)

0.7(10)

RDW (%)

 

 

 

 

 

DAY 94-95

12.6

12.6

12.6

12.8

12.7

 

0.6(10)

0.6(10)

0.5(10)

0.5(9)

0.6(10)

ARET (x10³/µL)

 

 

 

 

 

DAY 94-95

153.0

142.8

159.8

169.2

148.4

 

32.0(10)

30.4(10)

23.6(10)

16.8(9)

36.0(10)

PLT (x10³/µL)

 

 

 

 

 

DAY 94-95

965

1030

1012

1074

1030

 

147(7)

149(10)

131(5)

96(6)

93(8)

WBC (x10³/µL)

 

 

 

 

 

DAY 94-95

10.18

10.55

11.66

12.05

9.11

 

2.00(10)

3.02(10)

3.70(10)

3.96(9)

2.78(10)

ANEU (x10³/µL)

 

 

 

 

 

DAY 94-95

1.71

1.50

1.80

1.64

1.64

 

0.39(10)

0.50(10)

0.76(10)

0.49(9)

0.87(10)

ALYM (x10³/µL)

 

 

 

 

 

DAY 94-95

7.96

8.54

9.26

9.81

7.03

 

1.91(10)

2.78(10)

3.36(10)

3.45(9)

2.47(10)

AMON (x10³/µL)

 

 

 

 

 

DAY 94-95

0.23

0.24

0.27

0.24

0.20

 

0.07(10)

0.10(10)

0.11(10)

0.10(9)

0.07(10)

AEOS (x10³/µL)

 

 

 

 

 

DAY 94-95

0.11

0.12

0.15

0.16

0.09

 

0.03(10)

0.06(10)

0.05(10)

0.08(9)

0.03(10)

ABAS (x10³/µL)

 

 

 

 

 

DAY 94-95

0.03

0.04

0.05

0.04

0.03

 

0.02(10)

0.01(10)

0.02(10)

0.02(9)

0.02(10)

ALUC (x10³/µL)

 

 

 

 

 

 

0.14

0.12

0.14

0.16

0.12

 

0.02(10)

0.06(10)

0.06(10)

0.07(9)

0.07(10)

Summary of coagulation values

DAY 94-95 PT (sec)

14.9

14.7

14.7

14.5

15.4

0.4(9)

0.8(10)

0.9(10)

0.6(10)

0.9(10)

 

APTT (sec)

 

 

 

 

 

DAY 94-95

16.3

15.6

15.5

15.0

16.0

1.8(9)

1.4(10)

1.4(10)

1.0(10)

1.8(10)

 

Female

 

 

 

 

 

Groups

Concentration (ppm)

Control

600

2000

6000

18000

RBC (x106/µL)

 

 

 

 

 

DAY 96-97

8.42

8.37

8.43

8.53

8.34

 

0.30(10)

0.25(10)

0.41(10)

0.29(10)

0.23(10)

HGB (g/dL)

 

 

 

 

 

DAY 96-97

15.6

15.4

15.5

15.7

15.4

 

0.6(10)

0.6(10)

0.7(10)

0.5(10)

0.5(10)

HCT (%)

 

 

 

 

 

DAY 96-97

47.9

46.8

47.0

47.6

46.6

 

1.8(10)

1.6(10)

2.2(10)

1.9(10)

1.5(10)

MCV (fL)

 

 

 

 

 

DAY 96-97

56.9

55.8

55.8

55.8

55.9

 

1.6(10)

1.5(10)

1.0(10)

1.9(10)

1.2(10)

MCH (pg)

 

 

 

 

 

DAY 96-97

18.6

18.3

18.4

18.5

18.5

 

0.6(10)

0.5(10)

0.4(10)

0.6(10)

0.5(10)

MCHC (g/dL)

 

 

 

 

 

DAY 96-97

32.7

32.8

33.0

33.1

33.1

 

0.3(10)

0.4(10)

0.5(10)

0.4(10)

0.3(10)

RDW (%)

 

 

 

 

 

DAY 96-97

11.9

11.8

11.8

11.4

11.8

 

0.5(10)

0.5(10)

0.4(10)

0.5(10)

0.4(10)

ARET (x10³/µL)

 

 

 

 

 

DAY 96-97

162.5

133.9

142.6

136.7

145.3

 

34.1(10)

37.1(10)

22.3(10)

20.1(10)

31.0(10)

PLT (x10³/µL)

 

 

 

 

 

DAY 96-97

1012

1081

940

1032

1012

 

63(9)

113(9)

92(9)

125(10)

62(8)

WBC (x10³/µL)

 

 

 

 

 

DAY 96-97

9.09

10.03

8.63

9.86

9.30

 

2.13(10)

2.39(10)

2.22(10)

1.74(10)

2.80(10)

ANEU (x10³/µL)

 

 

 

 

 

DAY 96-97

0.92

1.10

1.10

1.25

1.04

 

0.56(10)

0.90(10)

0.50(10)

0.78(10)

0.35(10)

ALYM (x10³/µL)

 

 

 

 

 

DAY 96-97

7.76

8.43

7.11

8.11

7.83

 

1.88(10)

2.33(10)

2.23(10)

1.19(10)

2.48(10)

AMON (x10³/µL)

 

 

 

 

 

DAY 96-97

0.17

0.20

0.18

0.19

0.19

 

0.05(10)

0.12(10)

0.10(10)

0.06(10)

0.07(10)

AEOS (x10³/µL)

 

 

 

 

 

DAY 96-97

0.10

0.10

0.10

0.12

0.09

 

0.05(10)

0.03(10)

0.03(10)

0.06(10)

0.03(10)

ABAS (x10³/µL)

 

 

 

 

 

DAY 96-97

0.04

0.06

0.05

0.04

0.05

 

0.03(10)

0.04(10)

0.03(10)

0.02(10)

0.03(10)

ALUC (x10³/µL)

 

 

 

 

 

DAY 96-97

0.10

0.13

0.09

0.15

0.11

 

0.05(10)

0.08(10)

0.03(10)

0.09(10)

0.05(10)

Summary of coagulation values

PT (sec)

 

 

 

 

 

DAY 96-97

13.5

13.6

13.3

13.8

13.3

0.5(9)

0.5(10)

1.0(10)

0.5(9)

0.7(10)

 

APTT (sec)

 

 

 

 

 

DAY 96-97

13.8

14.2

14.0

13.4

13.3

0.7(9)

2.1(10)

1.5(10)

1.4(9)

1.6(10)

 

Data summarized as: Mean Standard Deviation (n)

Statistical Analysis: Statistical significance is indicated by the following (p < 0.05):

* Parametric comparison to control (Dunnett/Tamhane-Dunnett) significant

@ Nonparametric comparison to control (Dunn's) significant

 

Table 15: Summary of clinical chemistry values

Male

 

 

 

 

 

Groups

Concentration (ppm)

Control

600

2000

6000

18000

AST (U/L)

 

 

 

 

 

DAY 94-95

72

70

84

78

70

 

10(10)

15(10)

21(10)

11(10)

16(10)

ALT (U/L)

 

 

 

 

 

DAY 94-95

26

26

27

30

24

 

3(10)

3(10)

6(10)

5(10)

2(10)

SDH (U/L)

 

 

 

 

 

DAY 94-95

14.8

12.9

12.5

13.2

12.0

 

4.2(10)

3.4(10)

8.2(10)

4.1(10)

3.6(9)

ALKP (U/L)

 

 

 

 

 

DAY 94-95

84

88

76

82

74

 

23(10)

16(10)

9(10)

20(10)

23(10)

BILI (mg/dL)

 

 

 

 

 

DAY 94-95

0.14

0.13

0.13

0.14

0.13

 

0.03(10)

0.02(10)

0.01(10)

0.02(10)

0.01(10)

BUN (mg/dL)

 

 

 

 

 

DAY 94-95

13

14

13

13

13

 

2(10)

2(10)

2(10)

2(10)

2(10)

CREA (mg/dL)

 

 

 

 

 

DAY 94-95

0.41

0.40

0.41

0.41

0.40

 

0.05(10)

0.05(10)

0.04(10)

0.06(10)

0.05(10)

CHOL (mg/dL)

 

 

 

 

 

DAY 94-95

54

51

49

55

60

 

9(10)

6(10)

10(10)

13(10)

17(10)

TRIG (mg/dL)

 

 

 

 

 

DAY 94-95

63

53

50

53

47

 

20(10)

29(10)

11(10)

14(10)

14(10)

GLUC (mg/dL)

 

 

 

 

 

DAY 94-95

132

136

145

142

131

 

23(10)

34(10)

35(10)

26(10)

27(10)

TP (g/dL)

 

 

 

 

 

DAY 94-95

6.7

6.6

6.7

6.6

6.5

 

0.3(10)

0.3(10)

0.2(10)

0.2(10)

0.2(10)

ALB (g/dL)

 

 

 

 

 

DAY 94-95

3.4

3.3

3.4

3.3

3.3

 

0.2(10)

0.1(10)

0.1(10)

0.1(10)

0.2(10)

GLOB (g/dL)

 

 

 

 

 

DAY 94-95

3.3

3.2

3.4

3.3

3.2

 

0.2(10)

0.2(10)

0.2(10)

0.2(10)

0.2(10)

CALC (mg/dL)

 

 

 

 

 

DAY 94-95

10.5

10.6

10.5

10.5

10.4

 

0.4(10)

0.4(10)

0.3(10)

0.3(10)

0.4(10)

IPHS (mg/dL)

 

 

 

 

 

DAY 94-95

7.8

7.8

7.8

8.3

8.3

 

1.3(10)

0.9(10)

1.3(10)

1.8(10)

1.5(10)

NA (mmol/L)

 

 

 

 

 

DAY 94-95

144.8

144.3

144.4

144.5

144.3

 

1.7(10)

1.4(10)

1.9(10)

1.6(10)

1.1(10)

K (mmol/L)

DAY 94-95

5.85

5.85

5.69

5.57

6.20

 

0.43(10)

0.60(10)

0.53(10)

0.55(10)

0.49(10)

CL (mmol/L)

 

 

 

 

 

DAY 94-95

101.3

101.4

100.9

101.8

101.0

 

1.3(10)

1.6(10)

1.6(10)

2.6(10)

1.4(10)

Female

 

 

 

 

 

Groups

Concentration (ppm)

Control

600

2000

6000

18000

AST (U/L)

 

 

 

 

 

DAY 96-97

91

88

96

94

102

 

51(10)

38(10)

55(10)

38(10)

51(10)

ALT (U/L)

 

 

 

 

 

DAY 96-97

46

40

47

43

39

 

40(10)

24(10)

32(10)

17(10)

15(10)

SDH (U/L)

 

 

 

 

 

DAY 96-97

19.8

17.2

18.7

16.9

19.8

 

7.6(10)

5.4(10)

6.5(10)

6.1(10)

7.0(10)

ALKP (U/L)

 

 

 

 

 

DAY 96-97

51

54

59

59

46

 

12(10)

13(10)

17(10)

24(10)

9(10)

BILI (mg/dL)

 

 

 

 

 

DAY 96-97

0.19

0.20

0.18

0.19

0.18

 

0.02(10)

0.03(10)

0.03(10)

0.04(10)

0.03(10)

BUN (mg/dL)

 

 

 

 

 

DAY 96-97

15

15

16

15

15

 

2(10)

2(10)

2(10)

2(10)

2(10)

CREA (mg/dL)

 

 

 

 

 

DAY 96-97

0.46

0.44

0.47

0.45

0.46

 

0.05(10)

0.04(10)

0.03(10)

0.05(10)

0.04(10)

CHOL (mg/dL)

 

 

 

 

 

DAY 96-97

98

92

79

76

91

 

25(10)

15(10)

16(10)

25(10)

25(10)

TRIG (mg/dL)

 

 

 

 

 

DAY 96-97

62

49

52

43*

57

 

16(10)

17(10)

13(10)

9(10)

22(10)

GLUC (mg/dL)

 

 

 

 

 

DAY 96-97

110

112

113

105

100

 

15(10)

23(10)

22(10)

9(10)

10(10)

TP (g/dL)

 

 

 

 

 

DAY 96-97

8.0

7.7

7.7

7.5@

7.8

 

0.4(10)

0.4(10)

0.4(10)

0.4(10)

0.6(10)

ALB (g/dL)

 

 

 

 

 

DAY 96-97

4.2

4.1

4.0

3.9@

4.1

 

0.3(10)

0.3(10)

0.3(10)

0.2(10)

0.4(10)

GLOB (g/dL)

 

 

 

 

 

DAY 96-97

3.7

3.6

3.7

3.6

3.7

 

0.2(10)

0.2(10)

0.2(10)

0.2(10)

0.2(10)

CALC (mg/dL)

 

 

 

 

 

DAY 96-97

11.2

11.0

10.9

10.6*

11.0

 

0.5(10)

0.4(10)

0.2(10)

0.3(10)

0.5(10)

IPHS (mg/dL)

 

 

 

 

 

DAY 96-97

5.9

5.5

5.2

5.2

5.0

 

1.7(10)

1.0(10)

0.9(10)

1.0(10)

0.6(10)

NA (mmol/L)

 

 

 

 

 

DAY 96-97

144.9

144.6

144.0

144.4

144.8

 

1.5(10)

2.1(10)

1.6(10)

1.3(10)

2.0(10)

K (mmol/L)

DAY 96-97

5.67

5.40

5.60

5.50

5.44

 

0.46(10)

0.29(10)

0.38(10)

0.44(10)

0.41(10)

CL (mmol/L)

 

 

 

 

 

DAY 96-97

99.8

99.4

99.2

99.4

99.5

 

2.1(10)

1.6(10)

1.6(10)

2.0(10)

2.9(10)

Data summarized as: Mean Standard Deviation (n)

Statistical Analysis: Statistical significance is indicated by the following (p < 0.05):

* Parametric comparison to control (Dunnett/Tamhane-Dunnett) significant

@ Nonparametric

 

Table 16 : Summary of urinalysis values

Male

 

 

 

 

 

Groups

Concentration (ppm)

Control

600

2000

6000

18000

UVOL (mL)

 

 

 

 

 

DAY 94-95

12.0

9.7

10.2

8.3

6.2@

6.1(10)

6.4(10)

4.9(10)

5.6(10)

3.7(10)

 

UOSM (mOsm/kg)

 

 

 

 

 

DAY 94-95

1276

1552

1168

1728

1693

1163(10)

1079(10)

479(10)

997(10)

791(10)

 

pH

 

 

 

 

 

DAY 94-95

6.7

6.6

7.0

6.5

6.5

0.3(10)

0.3(10)

0.3(10)

0.4(10)

0.3(10)

 

URO (EU/dL)

 

 

 

 

 

DAY 94-95

0.2

0.4

0.2

0.4

0.3

0.0(10)

0.3(10)

0.0(10)

0.4(10)

0.3(10)

 

UMTP (mg/dL)

 

 

 

 

 

DAY 94-95

107

81

67

89

85

121(10)

60(10)

29(10)

55(10)

31(10)

 

Female

 

 

 

 

 

Groups

Concentration (ppm)

Control

600

2000

6000

18000

UVOL (mL)

 

 

 

 

 

DAY 96-97

5.7

6.8

6.5

7.7

4.3

 

2.3(10)

4.2(10)

4.2(10)

3.9(10)

2.4(9)

UOSM (mOsm/kg)

 

 

 

 

 

DAY 96-97

1211

1375

1175

1140

1860

 

767(10)

974(10)

477(10)

1177(10)

1627(9)

pH

 

 

 

 

 

DAY 96-97

6.8

6.6

6.5

6.9

6.2@

 

0.4(10)

0.5(10)

0.4(10)

0.6(10)

0.3(9)

URO (EU/dL)

 

 

 

 

 

DAY 96-97

0.2

0.2

0.2

0.2

0.2

 

0.0(10)

0.0(10)

0.0(10)

0.0(10)

0.0(9)

UMTP (mg/dL)

 

 

 

 

 

DAY 96-97

19

19

17

47

27

 

15(10)

14(10)

11(10)

95(10)

25(9)

Data summarized as: Mean Standard Deviation (n)

Statistical Analysis: Statistical significance is indicated by the following (p < 0.05):

* Parametric comparison to control (Dunnett/Tamhane-Dunnett) significant

@ Nonparametric comparison to control (Dunn's) significant

 

Table 17: Summary of organ weight values

MALES

Groups Concentration (ppm)

 

 

 

 

 

0

600

2000

6000

18000

Mean final body weight and absolute organ weights (g)

Final body weight

 

565

 

556

 

567

 

568

 

509*

 

55(9)

42(10)

35(10)

62(10)

35(10)

Adrenal glands

0.060

0.060

0.063

0.064

0.060

 

0.009(9)

0.012(10)

0.013(10)

0.012(10)

0.010(10)

Brain

2.164

2.164

2.185

2.173

2.125

 

0.067(9)

0.121(10)

0.075(10)

0.085(10)

0.073(10)

Epididymides

1.633

1.569

1.654

1.600

1.623

 

0.116(9)

0.187(10)

0.215(10)

0.133(10)

0.150(10)

Heart

1.792

1.753

1.744

1.787

1.601*

 

0.148(9)

0.147(10)

0.178(10)

0.201(10)

0.113(10)

Kidneys

4.218

4.109

4.186

4.146

4.012

 

0.555(9)

0.415(10)

0.349(10)

0.405(10)

0.193(10)

Liver

14.992

14.991

15.100

15.226

12.865*

 

1.433(9)

1.839(10)

1.376(10)

1.802(10)

0.839(10)

Spleen

0.896

0.854

0.871

0.880

0.780

 

0.163(9)

0.149(10)

0.074(10)

0.099(10)

0.096(10)

Testes

3.735

3.484

3.552

3.553

3.512

 

0.330(9)

0.302(10)

0.240(10)

0.361(10)

0.243(10)

Thymus

0.299

0.359

0.346

0.370

0.270

 

0.058(9)

0.133(10)

0.081(10)

0.094(10)

0.072(10)

Mean relative organ weight (% of brain weight)

Adrenal glands/ brain * 100

 

2.780

 

2.765

 

2.891

 

2.950

 

2.802

 

0.414(9)

0.587(10)

0.623(10)

0.528(10)

0.447(10)

Epididymides/ brain * 100

 

75.601

 

72.377

 

75.794

 

73.708

 

76.498

 

6.756(9)

6.128(10)

10.315(10)

6.211(10)

7.671(10)

Heart/ brain * 100

 

82.835

 

81.166

 

79.826

 

82.347

 

75.480

 

6.639(9)

7.342(10)

8.029(10)

9.590(10)

6.747(10)

Kidneys/ brain * 100

 

194.760

 

189.998

 

191.839

 

190.738

 

188.992

 

23.783(9)

16.345(10)

18.312(10)

16.191(10)

10.279(10)

Liver/ brain * 100

 

691.668

 

693.084

 

691.514

 

701.519

 

606.506*

 

50.435(9)

77.623(10)

63.465(10)

85.394(10)

49.654(10)

Spleen/ brain * 100

 

41.318

 

39.697

 

39.874

 

40.504

 

36.810

 

6.825(9)

7.907(10)

3.292(10)

4.386(10)

5.222(10)

Testes/ brain * 100

 

172.749

 

161.169

 

162.633

 

163.641

 

165.368

 

16.499(9)

12.615(10)

11.079(10)

16.434(10)

10.550(10)

Thymus/ brain * 100

 

13.784

 

16.519

 

15.862

 

17.036

 

12.727

 

2.566(9)

5.800(10)

3.787(10)

4.467(10)

3.572(10)

Mean relative organ weight (% of body weight)

Adrenal glands/ final body * 100

 

0.011

 

0.011

 

0.011

 

0.011

 

0.012

 

0.002(9)

0.002(10)

0.002(10)

0.002(10)

0.002(10)

Brain/

Final body * 100

 

0.386

 

0.391

 

0.387

 

0.386

 

0.419

 

0.032(9)

0.033(10)

0.024(10)

0.037(10)

0.035(10)

Epididymides/ final body * 100

 

0.292

 

0.283

 

0.292

 

0.284

 

0.321

 

0.038(9)

0.034(10)

0.037(10)

0.038(10)

0.042(10)

Heart/ final body * 100

Final body * 100

 

0.318

 

0.316

 

0.307

 

0.316

 

0.315

 

0.020(9)

0.020(10)

0.021(10)

0.035(10)

0.018(10)

Kidneys/ final body * 100

Final body * 100

 

0.746

 

0.741

 

0.739

 

0.732

 

0.791

 

0.056(9)

0.065(10)

0.059(10)

0.051(10)

0.054(10)

Liver/ final body * 100

Final body * 100

 

2.657

 

2.694

 

2.664

 

2.682

 

2.529

 

0.152(9)

0.195(10)

0.175(10)

0.177(10)

0.084(10)

Spleen/ final body * 100

Final body * 100

 

0.159

 

0.153

 

0.154

 

0.156

 

0.153

 

0.024(9)

0.021(10)

0.009(10)

0.019(10)

0.017(10)

Testes/ final body * 100

Final body * 100

 

0.664

 

0.629

 

0.628

 

0.631

 

0.693

 

0.065(9)

0.061(10)

0.045(10)

0.081(10)

0.065(10)

Thymus/ final body * 100

Final body * 100

 

0.053

 

0.065

 

0.061

 

0.066

 

0.053

 

0.007(9)

0.024(10)

0.013(10)

0.018(10)

0.012(10)

Feales

 

 

 

 

 

Groups Concentration (ppm)

 

 

 

 

 

0

600

2000

6000

18000

Mean final body weight and absolute organ weights (g)

 

 

 

 

 

Final body weight

 

285

 

283

 

291

 

269

 

262

 

30(10)

22(10)

38(10)

22(9)

19(10)

Adrenal glands

0.066

0.069

0.071

0.062

0.068

 

0.010(10)

0.010(10)

0.014(10)

0.015(9)

0.013(10)

Brain

1.971

1.956

1.961

1.957

1.907

 

0.086(10)

0.052(10)

0.099(10)

0.087(9)

0.075(10)

Heart

1.097

1.118

1.101

0.994

1.026

 

0.167(10)

0.154(10)

0.129(10)

0.068(9)

0.080(10)

Kidneys

2.104

2.053

2.053

1.980

2.056

 

0.279(10)

0.157(10)

0.232(10)

0.237(9)

0.186(10)

Liver

8.006

7.784

7.857

7.306

7.459

 

0.837(10)

0.672(10)

0.899(10)

0.590(9)

0.779(10)

Ovaries

0.124

0.127

0.137

0.118

0.142

 

0.028(10)

0.029(10)

0.022(10)

0.022(9)

0.056(10)

Spleen

0.520

0.571

0.566

0.548

0.531

 

0.042(10)

0.093(10)

0.081(10)

0.039(9)

0.078(10)

Thymus

0.268

0.298

0.257

0.271

0.261

 

0.059(10)

0.068(10)

0.067(10)

0.066(9)

0.057(10)

Uterus

0.720

0.697

0.666

0.709

0.698

 

0.186(10)

0.233(10)

0.219(10)

0.196(9)

0.182(10)

Mean relative organ weight (% of brain weight)

Adrenal glands/ brain * 100

 

3.366

 

3.542

 

3.633

 

3.197

 

3.551

 

0.514(10)

0.533(10)

0.746(10)

0.825(9)

0.658(10)

Heart/ brain * 100

 

55.717

 

57.198

 

56.201

 

50.900

 

53.898

 

8.439(10)

8.008(10)

6.274(10)

4.226(9)

4.775(10)

Kidneys/ brain * 100

 

106.548

 

105.078

 

104.856

 

101.386

 

107.740

 

11.471(10)

9.205(10)

11.605(10)

12.737(9)

7.858(10)

Liver/ brain * 100

 

405.992

 

398.564

 

402.451

 

373.319

 

391.640

 

36.623(10)

39.299(10)

55.286(10)

23.693(9)

43.691(10)

Ovaries/ brain * 100

 

6.272

 

6.476

 

6.982

 

6.008

 

7.453

 

1.377(10)

1.520(10)

1.145(10)

0.963(9)

3.074(10)

Spleen/ brain * 100

 

26.404

 

29.161

 

28.966

 

28.071

 

27.851

 

2.479(10)

4.486(10)

4.650(10)

2.381(9)

4.180(10)

Thymus/ brain * 100

 

13.618

 

15.240

 

13.138

 

13.822

 

13.673

 

2.948(10)

3.387(10)

3.327(10)

3.091(9)

2.684(10)

Uterus/ brain * 100

 

36.604

 

35.559

 

34.009

 

36.328

 

36.585

 

9.742(10)

11.690(10)

11.128(10)

10.018(9)

9.381(10)

Mean relative organ weight (% of body weight)

Adrenal glands/ final body * 100

 

0.024

 

0.025

 

0.024

 

0.024

 

0.026

 

0.005(10)

0.004(10)

0.004(10)

0.007(9)

0.005(10)

Brain/ final body * 100

Final body * 100

 

0.698

 

0.694

 

0.686

 

0.732

 

0.731

 

0.063(10)

0.055(10)

0.120(10)

0.062(9)

0.054(10)

Heart/ final body * 100

Final body * 100

 

0.386

 

0.395

 

0.380

 

0.371

 

0.392

 

0.043(10)

0.045(10)

0.036(10)

0.023(9)

0.020(10)

Kidneys/ final body * 100

Final body * 100

 

0.741

 

0.726

 

0.709

 

0.740

 

0.786

 

0.088(10)

0.044(10)

0.062(10)

0.092(9)

0.050(10)

Liver/ final body * 100

Final body * 100

 

2.815

 

2.755

 

2.705

 

2.738

 

2.852

 

0.182(10)

0.234(10)

0.125(10)

0.343(9)

0.261(10)

Ovaries/ final body * 100

Final body * 100

 

0.043

 

0.045

 

0.048

 

0.044

 

0.054

 

0.009(10)

0.011(10)

0.010(10)

0.008(9)

0.020(10)

Spleen/ final body * 100

Final body * 100

 

0.184

 

0.202

 

0.196

 

0.205

 

0.202

 

0.020(10)

0.028(10)

0.025(10)

0.022(9)

0.025(10)

Thymus/ final body * 100

Final body * 100

 

0.094

 

0.105

 

0.088

 

0.102

 

0.099

 

0.016(10)

0.022(10)

0.016(10)

0.029(9)

0.016(10)

Uterus/ final body * 100

Final body * 100

 

0.255

 

0.245

 

0.232

 

0.266

 

0.270

 

0.076(10)

0.075(10)

0.083(10)

0.080(9)

0.086(10)

Data summarized as: Mean Standard Deviation (n)

Statistical Analysis: Statistical significance is indicated by the following (p < 0.05):

* Parametric comparison to control (Dunnett/Tamhane-Dunnett) significant

@ Nonparametric comparison to control (Dunn's) significant

 

Table 18: Selected incidences of gross observations in males and females

Groups Concentration (ppm)

Sex

0

600

2000

6000

18000

Pancreas

m

 

 

 

 

 

no visible lesion

 

10

10

10

10

9

discoloration

 

0

0

0

0

1

Eye(s)

f

 

 

 

 

 

no visible lesion

 

9

9

10

10

10

discoloration

 

0

1

0

0

0

rupture

 

1

0

0

0

0

Kidneys

f

 

 

 

 

 

no visible lesion

 

10

10

10

9

10

dilatation

 

0

0

0

1

0

No further visible lesions observed in males and females at any concentrations

 

Table 19: Selected incidences of microscopic findings (neoplastic and non-neoplastic lesions)

Groups Concentration (ppm)

Sex

0

600

2000

6000

18000

Bone marrow

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

9

0

0

0

10

necrosis, focal

 

1

0

0

0

0

Cecum

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

6

0

0

0

6

inflammation, mucosal

 

4

0

0

0

4

Colon

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

9

0

0

0

10

inflammation, mucosal

 

1

0

0

0

0

Eye(s)

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

9

0

0

0

8

fold/rosette, retinal

 

1

0

0

0

2

Heart

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

5

0

0

0

5

cardiomyopathy

 

4

0

0

0

5

fibrosis, endocardial, focal/multifocal

 

1

0

0

0

0

Kidneys

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

6

0

0

0

2

chronic progressive nephropathy

 

4

0

0

0

7

aggregates, lymphoid

 

0

0

0

0

1

hydronephrosis, unilateral

 

1

0

0

0

0

hyaline droplets

 

1

0

0

0

0

Spleen

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

2

0

0

0

2

inflammation, subacute to chronic

 

7

0

0

0

8

fatty change, median cleft

 

3

0

0

0

1

fatty change, periportal

 

1

0

0

0

0

Lungs

 

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

0

0

0

0

8

inflammation, alveolar

 

0

0

0

0

2

Mandibular lymph node

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

9

0

0

0

9

hyperplasia, lymphoid

 

0

0

0

0

1

hyperplasia, plasma cell

 

1

0

0

0

0

Mesenteric lymph node

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

0

0

0

0

9

erythrocytosis/hemosiderosis, sinus

 

0

0

0

0

1

Nose

 

 

 

 

 

 

examined

m

(10)

(0)

(0)

(0)

(10)

within normal limits

 

9

0

0

0

9

odontodysplasia; bilateral

 

1

0

0

0

1

inflammation, teeth

 

1

0

0

0

0

erosion/ulcer, oral mucosa

 

1

0

0

0

1

Optic nerve

m

 

 

 

 

 

examined

 

(9)

(0)

(0)

(0)

(10)

within normal limits

 

9

0

0

0

0

not examined, missing

 

1

0

0

0

0

Pancreas

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

9

0

0

0

9

atrophy, lobular

 

1

0

0

0

1

Parathyroid glands

m

 

 

 

 

 

examined

 

(9)

(0)

(0)

(0)

(10)

within normal limits

 

9

0

0

0

0

not examined: missing

 

1

0

0

0

0

Pituitary gland

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

9

0

0

0

0

cyst

 

1

0

0

0

0

Prostate

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

3

0

0

0

3

aggregates, lymphoid

 

7

0

0

0

7

Skeletal muscle

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

0

0

0

0

9

Inflammation, subacute to chronic

 

0

0

0

0

1

Thymus

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

9

0

0

0

0

hyperplasia, epithelial

 

1

0

0

0

0

Thyroid gland

m

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(10)

within normal limits

 

9

0

0

0

0

hypertrophy, follicular cell

 

1

0

0

0

0

 

 

 

 

 

 

 

FEMALES

 

 

 

 

 

 

Cecum

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(10)

within normal limits

 

8

0

0

1

8

inflammation, mucosal

 

8

0

0

0

2

Eye(s)

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(10)

within normal limits

 

8

0

0

1

10

fold/rosette, retinal

 

1

0

0

0

0

Ruptured (iatrogenic), unilateral

 

1

 

 

 

0

Heart

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1

(10)

within normal limits

 

10

0

0

0

9

cardiomyopathy

 

0

0

0

1

1

Kidneys

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(10)

within normal limits

 

10

0

0

1

8

chronic progressive nephropathy

 

0

0

0

0

2

dilated tubules, medullary

 

0

0

0

0

1

Spleen

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(10)

within normal limits

 

5

0

0

1

4

inflammation, subacute to chronic

 

4

0

0

0

5

fatty change, median cleft

 

1

0

0

0

2

necrosis, focal

 

0

0

0

0

2

Mandibular lymph node

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(10)

within normal limits

 

5

0

0

1

6

hyperplasia, lymphoid

 

2

0

0

0

1

hyperplasia, plasma cell

 

1

0

0

0

0

erythrocytosis/hemosiderosis, sinus

 

3

0

0

0

3

Mesenteric lymph node

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(10)

within normal limits

 

8

0

0

1

9

erythrocytosis/hemosiderosis, sinus

 

1

0

0

0

0

hyperplasia, lymphoid

 

1

0

0

 

1

Nose

 

 

 

 

 

 

examined

f

(10)

(0)

(0)

(1)

(10)

within normal limits

 

9

0

0

1

10

erosion/ulcer, oral mucosa

 

1

0

0

0

0

Ovaries

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(10)

within normal limits

 

9

0

0

1

8

corpora lutea decreased

 

1

0

0

0

1

cyst, unilateral

 

0

0

0

0

0

Pancreas

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(10)

within normal limits

 

10

0

0

1

9

atrophy, lobular

 

0

0

0

0

1

Parathyroid glands

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(0)

(8)

within normal limits

 

10

0

0

0

8

not examined: missing

 

0

0

0

0

2

Peyers patch

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(9)

within normal limits

 

10

0

0

1

8

not examined: missing

 

0

0

0

0

1

mineralization

 

0

0

0

0

1

Sciatic nerve

f

 

 

 

 

 

examined

 

(9)

(0)

(0)

(1)

(10)

within normal limits

 

9

0

0

1

10

not examined: missing

 

1

0

0

0

0

Skeletal muscle

f

 

 

 

 

 

examined

 

(9)

(0)

(0)

(1)

(10)

within normal limits

 

9

0

0

1

10

not examined: missing

 

1

0

0

0

0

Spleen

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(10)

within normal limits

 

10

0

0

1

9

aggregates, macrophage

 

0

0

0

0

1

Thymus

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(10)

within normal limits

 

9

0

0

1

9

hyperplasia, epithelial

 

1

0

0

0

1

Thyroid gland

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(10)

within normal limits

 

9

0

0

1

10

cyst, ultimobranchial

 

1

0

0

0

0

Urinary bladder

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(10)

within normal limits

 

8

0

0

1

10

aggregates, lymphoid

 

2

0

0

0

0

Vagina

f

 

 

 

 

 

examined

 

(10)

(0)

(0)

(1)

(10)

within normal limits

 

0

0

0

0

0

Estrus stage: metestrus

 

3

0

0

0

4

Estrus stage: estrus

 

5

0

0

0

5

Estrus stage: proestrus

 

2

0

0

1

1

No further visible lesions observed in males and females at any concentrations

Table 20: Mean forelimb and hindlimb grip strength, footsplay, body temperature, and rearing

Males

 

 

Group

Concentration

(ppm)

Number

Forelimb grip strength

(kg)      

              

              

              

 

Hindlimb grip strength

(kg)

Footsplay

(cm)

Body temperature

(ºC)

Rearing

(number)

Baseline

 

 

 

 

 

 

 

1

0

10

0.81 (0.18)

0.34 (0.06)

6.0 (1.4)

36.4 (0.8)

5

2

600

10

0.81 (0.11)

0.33 (0.03)

6.9 (1.6)

36.2(0.6)

4

3

2000

10

0.86 (0.15)

0.38 (0.06)

6.9 (1.7)

36.2 (0.6)

5

4

6000

10

0.78 (0.13)

0.37 (0.07)

6.9 (18)

36.1(0.5)

5

5

18,000

10

0.85 (0.12)

0.34 (0.06)

6.4 (1.8)

35.9 (0.7)

3

Week 4

 

 

 

 

 

 

 

1

0

10

1.26 (0.27)

0.35 (0.05)

7.3 (2.6)

35.5 (1.0)

4

2

600

10

1.17 (0.15)

0.39(0.09)

8.4 (2.0)

35.6 (1.1)

4

3

2000

10

1.17(0.23)

0.38 (0.07)

7.1 (2.3)

35.8 (0.9)

3

4

6000

10

1.18 (0.12)

0.37 (0.05)

8.0 (1.8

36.0 (1.0)

3

5

18,000

10

1.18 (0.08)

0.40 (0.09)

6.9 (1.6)

35.4 (1.0)

4

Week 8

1

 

0

 

10

 

1.10 (0.21)

 

0.45 (0.06)

 

7.3 (2.8)

 

34.8 (0.6)

 

4

2

600

10

1.12 (0.14)

0.41 (0.10)

8.0 (1.3)

35.1 (0.8)

6

3

2000

10

1.19 (0.23)

0.40 (0.10)

7.1 (2.2)

35.0 (0.5)

4

4

6000

10

1.20 (0.13)

0.46 (0.14)

8.6 (1.5)

34.8 (0.4)

4

5

18,000

10

1.22 (0.16)

0.42 (0.07)

7.0 (2.1)

34.9 (0.5)

3

 

WEEK 13

 

 

 

 

 

 

 

1

0

10

0.93 (0.23)

0.40 (0.06)

7.4 (2.4)

35.9 (0.9)

4

2

600

10

1.06 (0.19)

0.45 (0.06)

7.9 (2.1)

35.5 (0.6)

4

3

2000

10

1.06 (0.24)

0.50 (0.07)

7.7 (1.9)

36.0 (0.9)

3

4

6000

10

1.15 (0.17)

0.45 (0.10)

8.3 (2.4)

35.7 (0.8)

3

5

18,000

10

1.05 (0.15)

0.41 (0.06)

7.9 (2.1)

35.8 (0.9)

3

Females

 

 

 

 

 

 

 

Baseline

 

 

 

 

 

 

 

1

0

10

0.74 (0.18)

0.36 (0.08)

5.4 (1.8)

36.6 (1.0)

6

2

600

10

0.74 (0.17)

0.34 (0.07)

5.2 (2.1)

36.2(1.0)

5

3

2000

10

0.77 (0.14)

0.34 (0.10)

6.4 (2.1)

36.7 (0.5)

6

4

6000

10

0.74 (0.21)

0.32 (0.06)

5.5 (1.5)

36.4(1.0)

6

5

18,000

10

0.72(0.11)

0.30 (0.04)

6.3 (1.4)

36.3 (0.4)

6

Week 4

 

 

 

 

 

 

 

1

0

10

1.02 (0.18)

0.36 (0.08)

5.6 (1.7)

36.2 (0.4)

8

2

600

10

0.93 (0.20)

0.34(0.04)

6.1 (2.1)

35.7 (0.5)

7

3

2000

10

0.94 (0.16)

0.35 (0.08)

6.4 (2.3)

35.8 (1.1)

6

4

6000

10

0.99 (0.16)

0.39 (0.09)

5.6 (1.4)

36.2 (0.8)

7

5

18,000

10

0.93 (0.18)

0.37 (0.08)

5.8 (2.2)

36.1(1.0)

7

 

WEEK 8

 

 

 

 

 

 

 

1

0

10

0.96 (0.26)

0.34 (0.08)

5.3 (1.8)

35.7 (0.4)

10

2

600

10

0.92 (0.20)

0.39 (0.08)

5.2 (1.5)

35.8 (0.4)

8

3

2000

10

0.93 (0.18)

0.38 (0.08)

6.5 (2.7)

36.0 (0.6)

8

4

6000

10

0.94 (0.11)

0.40 (0.07)

5.6 (1.3)

35.8 (0.4)

9

5

18,000

10

0.96 (0.19)

0.37 (0.10)

6.0 (2.3)

35.7 (0.4)

8

Week 13

 

 

 

 

 

 

 

 

1

0

10

0.72 (0.21)

0.37 (0.11)

5.8 (1.7)

37.3 (1.0)

10

2

600

10

0.70 (0.16)

0.36 (0.07)

6.0 (1.8)

37.3 (1.0)

8

3

2000

10

0.84 (0.26)

0.39 (0.08)

6.4 (2.4)

36.9 (1.1)

7

4

6000

10

0.80 (0.22)

0.37 (0.09)

5.8 (1.2)

36.5 (1.4)

9

5

18,000

10

0.82 (0.25)

0.39 (0.08)

6.9 (1.8)

36.5 (0.9)#

8

Data arranged as: Mean (Standard Deviation).

# Statistically significant trend compared to the control at p < 0.05 by Jonckheere-Terpstra trend test.

 

Table 21: Summary of selected functional observational battery findings

Males

Baseline

Week 4

Week 8

Week 13

Concentration

control

600

2000

6000

18000

control

600

2000

6000

18000

control

600

2000

6000

18000

control

600

2000

6000

18000

Number examined

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Home cage - Posture

Limbs spread out or lying on one side

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Curled up

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Sitting, standing or rearing normally, alert

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Jumping

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Removal from cage:

Ease of removal:

Too easy (rat sits quietly, no resistance)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Some resistance (rears, follows observer's hand)

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Difficult (runs around cage, may attack)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Ease of handling:

Too easy

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Easy (alert, limbs pulled up against body)

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Difficult

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Arousal:

Very low (stupor, little or no responsiveness)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Low

0

1

2

1

2

1

2

3

2

2

0

0

0

1

2

1

3

1

2

0

Normal (alert, exploratory movements)

10

9

8

9

8

9

8

7

7

8

10

10

10

9

8

9

7

9

8

10

High (slight excitement, tense, sudden movements)

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

0

Manipulations:

Approach & touch:

No reaction

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

0

Normal

10

10

8

10

8

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Increased reaction (jumps away or attacks)

0

0

2

0

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Tail pinch:

no response

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

normal

10

10

10

10

9

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

exaggerate response

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Auditory stimulus:

 

No reaction

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

0

Normal reaction(rat flinches or flicks ear)

10

10

10

10

8

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Exaggerated reaction (rat jumps, flips)

0

0

0

0

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Posture:

normal

0

0

0

0

0

0

0

0

0

9

0

0

0

0

0

0

0

0

0

0

abnormal

10

10

10

10

10

10

10

10

10

1

10

10

10

10

10

10

10

10

10

10

Gait/coordinations

normal

10

10

10

10

10

10

9

10

10

10

10

10

10

10

10

10

9

10

10

10

unbalance, swaying, uncoordinated

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

1

0

0

0

ataxic

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

unable to move

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Additional observations:

Waddling

absent

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

9

10

10

10

present

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

High posture

absent

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

9

present

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Females

Baseline

Week 4

Week 8

Week 13

Concentration

control

600

2000

6000

18000

control

600

2000

6000

18000

control

600

2000

6000

18000

control

600

2000

6000

18000

Number examined

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Home Cage - Posture

Limbs spread out or lying on one side

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Curled up

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Sitting, standing or rearing normally, alert

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Jumping

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Removal from cage:

Ease of removal:

Too easy

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Some resistance

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Difficult

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Ease of handling:

Too easy

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Easy (alert, limbs pulled up against body)

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Difficult

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Posture:

normal

0

0

0

0

0

9

0

0

8

8

0

0

0

0

0

9

0

8

9

0

abnormal

10

10

10

10

10

1

10

10

2

2

10

10

10

10

10

1

10

2

1

10

Arousal:

Very low (stupor, little or no responsiveness)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Low

0

0

1

0

0

1

0

1

0

0

1

0

1

0

0

0

0

1

0

0

Normal

10

10

9

9

10

6

10

8

8

9

8

10

8

8

9

8

9

8

9

10

High (slight excitement, tense, sudden movements)

0

0

0

1

0

3

0

1

2

1

1

0

1

2

1

2

1

1

1

0

Manipulations:

Approach & touch:

No reaction

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

0

Normal

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Increased reaction (jumps away or attacks)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Auditory stimulus:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

No reaction

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

0

Normal reaction(rat flinches or flicks ear)

10

10

10

10

10

10

10

9

10

10

10

10

10

10

10

10

10

10

10

10

Exaggerated reaction (rat jumps, flips)

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

Tail pinch:

no response

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

normal

10

9

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

exaggerate response

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Additional observations:

Ear twitch

absent

10

10

10

10

10

10

10

10

8

10

10

10

10

10

10

10

10

10

10

10

present

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

0

0

0

Extra digit left hindlimb:

absent

0

0

0

0

0

10

9

0

0

0

0

9

0

0

0

0

9

0

0

0

present

0

0

0

0

0

0

1

0

0

0

0

1

0

0

0

0

1

0

0

0

High posture:

absent

0

0

0

0

0

9

10

10

8

8

0

0

10

10

10

10

0

8

9

10

present

0

0

0

0

0

1

0

0

2

2

0

0

0

0

0

0

0

2

1

0

Corneal opacity – left eye:

absent

0

0

0

0

0

0

0

0

0

0

0

9

0

0

0

0

9

0

0

0

present

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

1

0

0

0

Bent tail

absent

0

0

0

0

0

10

10

10

10

0

0

0

0

0

0

0

0

0

9

0

present

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

 

Table 22: Motor activity assessment: mean number of movements

Male

Group

Concentration

Successive 10 minute intervals

 

ppm

 N

 1

 

 2

 

 3

 

 4

 

 5

 

 6

 

 Total

Baseline

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

0

10

136

(18)

137

(17)

110

(28)

72

(44)

45

(64)

29

(40)

529

(150)

2

600

10

122

(18)

117

(43)

8 8

(43)

40

(51)

13

(17)

17

(33)

396

(108)

3

2000

10

123

(20)

126

(25)

120

(23)

88

(51)

45

(54)

23

(47)

525

(148

4

6000

10

131

(19)

133

(25)

104

(42)

55

(37)

8

(6)

10

(8)

441

(86)

5

18,000

10

143

(11)

142

(17)

107

(49)

71

(50)

41

(60)

22

(45)

526

(175)

Week 4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

0

10

130

(12)

133

(17)

117

(23)

112

(28)

102

(41)

96

(47)

689

(93)

2

600

10

118

(17)

127

(8)

119

(28)

113

(31)

114

(42)

86

(50)

676

(106

3

2000

10

124

(15)

128

(14)

121

(19)

126

(25)

121

(46)

107

(48)

725

(134)

4

6000

10

43

(15)

137

(17)

137

(21)

130

(8)

107

(45)

86

(48)

740

(110)

5

18,000

10

146

(18)

133

(25)

119

(34)

98

(61)

63

(65)

68

63)

627

(224)

Week 8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

0

10

132

(14)

131

(25)

121

35)

114

(25)

106

(20)

99

(36)

702

(87)

2

600

10

131

(19)

138

(18)

132

27)

126

(31)

110

(41)

105

(37)

742

(142)

3

2000

10

135

(20)

139

(22)

133

13)

128

(15)

119

(37)

118

(43)

771

(117)

4

6000

0

3

(19)

115

(28)

115

32)

101

(45)

86

(51)

71

(51)

622

(158)

5

18,000

10

149

(15)

146

(17)

117

37)

118

(33)

87

(58)

94

(55)

711

(161)

Week 13

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

0

10

133

(18)

127

(22)

110

(32)

99

(26)

91

(43)

79

(44)

638

(75)

2

600

10

132

(21)

134

(18)

113

(27)

107

(30)

96(

(42)

95

(40)

676

(132)

3

2000

10

131

(12)

129

(16)

116

(29)

96

(36)

93

(34)

97

(46)

662

(126)

4

6000

10

137

(14)

129

(20)

102

(37)

102

(29)

94

(38)

79

(37)

642

(113)

5

18,000

10

149

(14)

138

(21)

93

(37)

116

(23)

79

(51)

75

(42)

649

(104)

Female

Group

Concentration

Successive 10 minute intervals

 

ppm

 N

 1

 

 2

 

 3

 

 4

 

 5

 

 6

 

Total

Baseline

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

0

10

138

(15)

117

(43)

108

(42)

80

(34)

50

(51)

52

(49)

545

(138)

2

600

10

134

(12)

109

(42)

72

(41)

74

(64)

36

(46)

26

(49)

452

(162)

3

2000

10

134

(13)

119

(29)

82

(53)

80

(55)

65

(52)

65

(32)

505

(150)

4

6000

10

132

(11)

125

(25)

99

(42)

89

(44)

49

(47)

49

(36)

528

(111)

5

18,000

10

130

(19)

134

(21)

103

(38)

87

(45)

47

(44)

32

(36)

533

(112)

Week 4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

0

10

137

(22)

124

(38)

124

(23)

101

(39)

90

(56)

89

(53)

664

(133)

2

600

10

132

(19)

120

(28)

105

(44)

106

(54)

92

(51)

95

(60)

650

(154)

3

2000

10

134

(10)

133

(21)

118

(41)

95

(49)

82

(48)

84

(62)

626

(168)

4

6000

10

127

(16)

122

(46)

108

(34)

90

(58)

95

(64)

126

(35)

679

(190)

5

18,000

10

129

(20)

133

(16)

105

(33)

90

(47)

96

(43)

76

(42)

630

(107)

Week 8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

0

10

137

(16)

133

(18)

110

(28)

124

(22)

105

(45)

89

(41)

698

(120)

2

600

10

142

(20)

137

(22)

118

(43)

119

(35)

120

(46)

101

(62)

736

(168)

3

2000

10

135

(14)

148

(17)

96

(49)

113

(33)

103

(45)

83

(55)

679

(142)

4

6000

0

137

(18)

123

(41)

112

(50)

118

(47)

86

(45)

81

(63)

657

(144)

5

18,000

10

127

(24)

141

(17)

106

(39)

109

(29)

101

(45)

107

(47)

691

(92)

Week 13

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

0

10

142

(15)

129

(14)

93

(38)

118

(23)

104

(42)

97

(50)

682

(117)

2

600

10

143

(23)

124

(11)

97

(32)

98

(26)

109

(47)

103

(52)

674

(119)

3

2000

10

131

(12)

119

(27)

108

(48)

95

(45)

93

(45)

101

(45)

647

(183)

4

6000

10

131

(18)

127

(12)

113

(44)

110

(23)

106

(35)

83

(53)

670

(96)

5

18,000

10

140

(15)

132

(28)

107

(29)

78

(51)

101

(46)

63

(43)

620

(128)

Data arranged as: Mean (Standard Deviation).

N = Number examined.

There were no statistically significantdifferencescompared to the control group at p < 0.05.

 

 

Table 23: Incidences of gross observations in neuropathology

Males

 

 

 

 

 

Concentration

Control

600

2000

6000

18000

Number of animals of study

5

5

5

5

5

Kidneys

 

 

 

 

 

no visble lesions

5

5

5

4

5

dilatation

0

0

0

1

0

Males

 

 

 

 

 

Concentration

Control

600

2000

6000

18000

Number of animals of study

5

5

5

5

5

No gross lesions were observed in any of the female neuropathology rats.

 

Table 24: Selected incidences and lesion grades of microscopic findings (neoplastic and non-neoplastic lesions) in neuropathology analysis

Males

 

 

 

 

 

 

Concentration

Control

600

2000

6000

18000

 

Number of animals of study

5

5

5

5

5

 

Gastrocnemius muscle

 

 

 

 

 

 

examined

5

0

0

0

5

 

within normal limits

5

0

0

0

4

 

aggregates, macrophage

0

0

0

0

1

 

minimal

0

0

0

0

1

 

Eye

 

 

 

 

 

 

examined

5

0

0

0

5

 

within normal limits

5

0

0

0

4

 

fold/rosette, retinal

0

0

0

0

1

 

minimal

0

0

0

0

1

 

Females

 

 

 

 

 

 

Concentration

Control

600

2000

6000

18000

 

Number of animals of study

5

5

5

5

5

 

All observations within normal limits

 

 

Applicant's summary and conclusion