Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 Apr 2018 to 6 Jul 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Adopted 21st September 1998.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
21.8.2001
GLP compliance:
yes (incl. certificate)
Remarks:
Triskelion B.V., Utrechtseweg 48, 3704 HE, Zeist, The Netherlands
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar outbred rats (Crl:WI(Han)), (SPF).
Details on species / strain selection:
The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies.
The Han rat strain was used because it is routinely used at the test facility for this type of studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 to 7 weeks.
- Weight at study initiation: For males: 146 to 173g (mean 160 g); for females: 114 to 154 g (mean 133 g).
- Housing: The animals were housed under conventional conditions in one room. No other animals were housed in the same room during the study. The animals were kept in macrolon cages with wood shavings (Lignocel) as bedding material, and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. They were housed in groups of five, separated by sex.
- Diet: Cereal-based (closed formula) rodent diet (VRF1(FG)), ad libitum unless precluded by the performance of certain laboratory investigations
- Water: Tap water, ad libitum unless precluded by the performance of certain laboratory investigations
- Acclimation period: Quarantine period: 21 March to 23 March. Further acclimation until initiation of treatment on 5th of April.

DETAILS OF FOOD AND WATER QUALITY:
Each batch of diet was analysed by the supplier for nutrients and contaminants. Routine physical, chemical and microbiological examination of drinking water was conducted.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 45 to 65 except during brief periods associated with room cleaning
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
5 Apr 2018 to 6 Jul 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was weighed into a glass bottle for each day of the study and for each dosing group. The vials were stored at ambient temperature. Each dosing day, the corresponding amount of corn oil was added to obtain the final concentration of the test substance in corn oil. Before dosing, the suspension was stirred on a magnetic stirrer for at least 30 minutes, until visual homogeneity was obtained. All suspensions were continuously stirred throughout the dosing procedure, in order to maintain the homogeneity of the test substance in the vehicle. The concentration of the test substance were 8, 40 and 200 mg/mL for the low-dose, mid-dose and high-dose, respectively.

- VEHICLE
- Amount of vehicle: 5 mL/kg bw/day
- Batch no.: A1701528
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the homogeneity and content of the test substance in the gavage suspension was conducted using ICP-MS. The presence of the test substance in the test dilutions was determined by analysis of the Zinc content. The Zinc content was assumed to be proportional to the concentration of the test substance. The analyses were performed by inductively coupled plasma mass spectrometry (ICP-MS) after digestion with sulphuric acid and hydrogen peroxide.
Because the method involves the determination of the Zinc concentration only, the stability of the test substance could not be established. Therefore, fresh test dilutions were prepared daily.
The homogeneity (and content) of the test substance in the test dilutions were demonstrated in one batch prepared on 5 April 2018, by analysing three samples (taken at top-, mid- and bottom- of the vial) of each test dilution. The content of the test substance in each test dilution was determined in the batches prepared on 5 April, 30 April, 28 May and 27 June 2018.
Duration of treatment / exposure:
13 consecutive weeks.
Frequency of treatment:
Daily, seven days a week.
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Remarks:
Group 2, low-dose.
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Group 3, mid-dose.
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4, high-dose.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily in the morning and additionally in the afternoon for dead or moribund animals to minimize loss of animals from the study.
- All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: If necessary, rats were handled to detect signs of toxicity daily in the morning. In addition all rats were subjected to detailed clinical observations prior to the first exposure and then once weekly throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Day -1, 0 and subsequently once weekly. The animals were weighed on their scheduled necropsy date.

FOOD CONSUMPTION: Yes
- Food consumption was measured per cage by weighing the feeders. The consumption was measured over one-week periods throughout the treatment period for all animals in the cage. The results were expressed in g per animal per day.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Water consumption was measured per cage, by weighing the drinking bottles daily, during 5-day periods in or about weeks 1, 6 and 11. The results were expressed in g per animal per day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Day -8 (all males), day -7 (all females), and in all rats of the control group and the high-dose group in week 13 (day 88).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight (water was freely available).
- How many animals: All surviving animals.
- Parameters examined: haemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell count (lymphocytes, neutrophils, eosinophils, basophils and monocytes), prothrombin time, thrombocyte count (platelet count).
- The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight (water was freely available).
- How many animals: All surviving animals.
- Parameters examined: alkaline phosphatase activity (ALP), fasting glucose, aspartate aminotransferase activity (ASAT), bilirubin total, alanine aminotransferase activity (ALAT), cholesterol, gamma glutamyl transferase activity (GGT), triglycerides, total protein, calcium (Ca), albumin, sodium (Na), ratio albumin to globulin, potassium (K), urea, chloride (Cl), creatinine, inorganic phosphate, thyroxin (T4).

URINALYSIS: Yes
- Time schedule for collection of urine: In or around week 13.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for 16 hours.
- How many animals: All surviving animals.
- Parameters examined: volume, occult blood, density (specific gravity), ketones, appearance (colour and clarity), protein, pH, bilirubin, glucose, urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At the end of the study.
- Dose groups that were examined: All rats.
- Battery of functions tested: Functional Observation Battery and motor activity assessment (week 12 for males and week 13 for females).

IMMUNOLOGY: No

OTHER: ANALYSIS OF THYROID HORMONES (T4 AND TSH)
- Time schedule for collection of blood: At necropsy.
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia.
- Animals fasted: Yes, overnight (water was freely available).
- How many animals: All surviving animals.
Thyroid Stimulating Hormone (TSH) levels were assessed at necropsy in serum using commercially available enzyme-linked immunosorbent assays (ELISAs). Thyroxine (T4) was analysed in plasma collected at necropsy using the Siemens Dimension ExL
Sacrifice and pathology:
SACRIFICE:
Early in week 14, after overnight fasting (water was freely available), the surviving animals were killed on two successive working days (day 91 and 92 for males and females, respectively), in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under CO2/O2 anaesthesia and then examined grossly for pathological changes.

GROSS PATHOLOGY: Yes
- The following organs were weighed: adrenals, prostate, brain, seminal vesicles (with coagulating glands), epididymides, spleen, heart, testes, kidneys, thymus, liver, thyroids, ovaries, uterus.
- The following tissues were preserved: adrenals, oviducts (=fallopian tubes), aorta, pancreas, axillary lymph nodes, parathyroid, brain (brain stem, cerebrum, cerebellum), parotid salivary glands, cecum, pituitary, colon, prostate, duodenum, rectum, epididymides, seminal vesicles and coagulating glands, oesophagus, skeletal muscle (thigh), exorbital lachrymal glands*, skin (flank), eyes, spinal cord (cervical, mid-thoracic, lumbar), femur with joint*, spleen, GALT (gut associated lymphoid tissue, including Peyer's patches), sternum with bone marrow, stomach ('forestomach', fundus, pylorus), heart, sublingual salivary glands, ileum, submaxillary salivary glands, jejunum, testes, kidneys, thymus, liver, thyroid, lungs, trachea/bronchi, mammary gland, (females), urinary bladder, mandibular (cervical) lymph nodes*, uterus (with cervix), mesenteric lymph nodes, vagina, nerve-peripheral (sciatic), all gross lesions, ovaries.
* The tissues marked with * were preserved but not processed for histopathological examination, unless histopathological examination was considered necessary on the basis of gross observations.

HISTOPATHOLOGY: Yes
The tissues to be examined microscopically were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin. Histopathological examination (by light microscopy) were performed on all tissues and organs listed above - except those marked with an asterisk - of all animals of the control group and the high-dose group. Gross lesions were examined in rats of all dose groups.
Statistics:
A list of the statistical test performed can be found in Table 1 and 2 in 'Any other information on materials and methods incl. tables'.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical signs
Mortality:
no mortality observed
Description (incidence):
None of the rats died during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In the final stage of the study, mean body weights tended to be slightly (≤8%) higher in males of the mid- and high-dose groups, but there were no statistically significant differences between the treatment groups and the controls (Table 1 and 2 in 'Any other information on results incl. tables').
Incidental changes in body weight gain attained statistical significance in females of the low and mid-dose group in the pre-dose phase, and in males of the mid-dose group in week 5 (Table 3 and 4 in 'Any other information on results incl. tables'). The overall growth figures were, however, not adversely affected.
In conclusion; body weights and body weight gain were not adversely affected by the test substance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food intake was not affected by the treatment. Food intake was slightly though statistically significantly decreased in males in the low-dose groups in week 4. There were, however, no noticeable differences in overall food intake among the groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water intake was not affected by the treatment. On day 70-71, water intake was statistically significantly lower in female rats in the high dose groups than in controls, but there were no noticeable differences in overall weekly water consumption among the groups in the various weeks.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination of all animals in the control and high-dose group in week 13 did not reveal any changes.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
An overview of the results can be found in Table 5 to 8 in 'Any other information on results incl. tables'.
Red blood cell count, haemoglobin concentration and packed cell volume were statistically significantly decreased in females of the high dose group. Although the changes in this group were outside the range of historical control data, they were only slight (about -6%).
A slight, though statistically significant increase in the percentage of reticulocytes in low-dose males was not confirmed at the higher dose levels and is therefore considered an incidental finding.
The percentage of lymphocytes was statistically significantly increased and the percentage of neutrophils was decreased in males of the high-dose group. These findings were within the range of historical control data, and not reflected in significant changes in the absolute lymphocyte and neutrophil counts. Therefore these findings are of doubtful relevance and are not considered to be adverse.
An increase in the absolute number of neutrophils in mid-dose females was not confirmed at the high-dose level and therefore considered an incidental finding.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
An overview of the results can be found in Table 9 and 10 in 'Any other information on results incl. tables'.
Albumin and the albumin/globulin ratio were statistically significantly decreased in males of the mid- and high-dose group. The changes were within the range of historical control data.
Chloride concentration was statistically significantly decreased in high-dose females. The changes were within the range of historical control data.
A number of changes attained statistical significance but were not confirmed at higher dose level(s), and are therefore considered chance findings:
- Decrease in ALP activity in mid-dose males.
- Decreases in potassium concentration in low- and mid-dose males.
- Decrease in total protein in low-dose females.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No statistically significant differences were observed in urinary volume or density, or in semi-quantitative (dipstick) urinary measurements.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute weight of the liver was statistically significantly increased in male animals of the mid- and high-dose group (Table 11 and 12 in 'Any other information on results incl. tables'). The relative weight of this organ was not affected (Table 13 and 14 in 'Any other information on results incl. tables'). Therefore this finding is ascribed to the somewhat higher terminal body weights in these groups and not considered to be of toxicological significance.
A statistically significant decrease in the relative weight of the heart in mid-dose females was considered a chance finding.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy revealed no treatment-related abnormalities. The findings were considered unremarkable and part of the background pathology of rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination revealed no treatment-related abnormalities. The histopathological findings were considered unremarkable and part of the background pathology of rats of this strain and age.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
THYROID HORMONE LEVELS
No statistically significant effects in plasma T4 levels were noted between treatment groups and controls (Table 15 and 16 in 'Any other information on results incl. tables').
Thyroid stimulating hormone levels were slightly decreased in females of the high-dose group. This decrease in TSH levels was not corroborated by noticeable changes in T4 levels, thyroid weight or pathology, or by effects on plasma total cholesterol. Since, moreover the values were well within the range of historical control data and this variable is known to show considerable variation, the decrease in TSH levels in high-dose females was considered a chance finding (Table 15 and 16 in 'Any other information on results incl. tables').

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1. Body weights males – days relative to start date

Sex: Male

 

Bodywt day -x

(g)

[G]

Bodywt (g)

 

[G]

Bodywt (g)

 

[G1]

Bodywt (g)

 

[G1]

Bodywt (g)

 

[G1]

Bodywt (g)

 

[G1]

Bodywt (g)

 

[G1]

Bodywt (g)

 

[G1]

Bodywt (g)

 

[G1]

Bodywt (g)

 

[G1]

Bodywt (g)

 

[G1]

Bodywt (g)

 

[G1]

Bodywt (g)

 

[G1]

Bodywt (g)

 

[G1]

Bodywt (g)

 

[G1]

 

 

-1

0

7

14

21

28

35

42

49

56

63

70

77

84

90

0 mg/kg

Mean

SD

N

156.01

8.84

10

159.82

8.60

10

197.22

7.39

10

231.42

7.03

10

256.04

9.75

10

275.02

15.25

10

288.00

17.15

10

298.59

17.36

10

308.30

18.90

10

315.75

19.25

10

323.28

21.05

10

331.75

21.69

10

338.57

21.93

10

334.59

21.47

10

349.08

21.20

10

40 mg/kg

Mean

SD

N

155.79

9.20

10

160.03

8.38

10

197.35

10.62

10

228.61

14.66

10

253.51

18.79

10

268.33

24.09

10

281.39

27.80

10

293.11

28.87

10

304.26

29.72

10

312.59

29.99

10

320.69

31.39

10

329.98

32.59

10

337.57

32.78

10

332.47

32.33

10

349.96

33.14

10

200 mg/kg

Mean

SD

N

155.70

9.43

10

160.13

9.00

10

200.23

9.11

10

235.66

9.37

10

264.63

12.54

10

285.48

16.67

10

305.17

20.48

10

319.88

23.04

10

330.93

23.79

10

340.05

26.39

10

346.83

29.44

10

357.75

31.01

10

363.36

31.53

10

357.73

30.07

10

373.58

33.31

10

1000 mg/kg

Mean

SD

N

155.94

9.36

10

160.21

9.47

10

199.91

11.62

10

233.85

13.11

10

263.57

14.20

10

283.98

17.55

10

301.84

17.61

10

315.39

18.95

10

328.75

19.36

10

337.88

19.34

10

348.12

20.65

10

358.94

20.28

10

365.88

19.45

10

361.50

21.33

10

376.86

22.16

10

[G] - Kruskal-Wallis & Dunnett on Ranks

[G1] - Ancova/Anova & Dunnett

 

Table 2. Body weights females – days relative to start date

Sex: Female

 

Bodywt day -x

(g)

 

[G]

Bodywt (g)

[G]

Bodywt (g)

[G]

Bodywt (g)

[G]

Bodywt (g)

[G]

Bodywt (g)

[G]

Bodywt (g)

[G]

Bodywt (g)

[G]

Bodywt (g)

[G]

Bodywt (g)

[G]

Bodywt (g)

[G]

Bodywt (g)

[G]

Bodywt (g)

[G1]

Bodywt (g)

[G]

Bodywt (g)

[G]

 

 

-1

0

7

14

21

28

35

42

49

56

63

70

77

84

90

0 mg/kg

Mean

SD

N

129.72

8.32

10

133.67

8.61

10

151.92

7.01

10

167.35

9.44

10

176.66

10.75

10

189.11

13.48

10

198.34

14.12

10

201.25

13.03

10

206.38

12.38

10

215.16

14.44

10

217.58

12.18

10

218.22

13.99

10

219.36

13.43

10

223.60

12.23

10

223.85

15.28

10

40 mg/kg

Mean

SD

N

130.73

9.57

10

132.71

9.21

10

151.78

10.39

10

168.01

11.74

10

180.11

13.69

10

190.67

15.94

10

198.38

14.80

10

202.20

15.71

10

205.08

15.01

10

211.81

15.11

10

214.01

16.10

10

216.43

17.00

10

216.44

15.43

10

217.46

15.76

10

222.42

17.07

10

200 mg/kg

Mean SD

N

131.44

10.64

10

132.47

12.57

10

152.11

11.62

10

168.43

12.61

10

181.47

11.41

10

192.00

16.47

10

203.13

16.08

10

208.81

15.77

10

213.06

14.26

10

218.77

16.92

10

221.63

15.02

10

223.10

14.32

10

224.65

13.10

10

227.78

18.00

10

230.86

14.93

10

1000 mg/kg

Mean SD

N

127.84

9.85

10

131.18

8.44

10

149.11

10.00

10

163.33

9.19

10

176.02

10.22

10

184.29

11.18

10

195.25

10.85

10

199.77

10.03

10

206.95

12.67

10

209.55

11.79

10

212.01

11.93

10

215.15

10.94

10

216.70

12.77

10

218.94

12.43

10

220.09

12.21

10

[G] - Ancova/Anova & Dunnett

[G1] - Kruskal-Wallis & Dunnett on Ranks

 

Table 3. Body weight change males – days relative to the start date.

Sex: Male

 

Wgt change last -x to 0

(g)

[G1]

Body wt change

(g)

 

 

[G1]

Body wt change

(g)

 

 

[G1]

Body wt change

(g)

 

 

[G1]

Body wt change

(g)

 

 

[G1]

Body wt change

(g)

 

 

[G1]

Body wt change

(g)

 

 

[G1]

Body wt change

(g)

 

 

[G1]

Body wt change

(g)

 

 

[G]

Body wt change

(g)

 

 

[G1]

Body wt change

(g)

 

 

[G1]

Body wt change

(g)

 

 

[G1]

Body wt change

(g)

 

 

[G1]

Body wt change

(g)

 

 

[G2]

Body wt change

(g)

 

 

[G1]

 

 

-x - 0

0 - 7

7 - 14

14 - 21

21 - 28

28 - 35

35 - 42

42 - 49

49 - 56

56 - 63

63 - 70

70 - 77

77 - 84

84 - 90

0 - 90

0 mg/kg

Mean

SD

N

3.81

1.60

10

37.40

3.75

10

34.20

4.45

10

24.62

5.24

10

18.98

6.83

10

12.98

6.42

10

10.59

3.78

10

9.71

2.72

10

7.45

2.81

10

7.53

2.87

10

8.47

3.43

10

6.82

1.77

10

-3.98

2.84

10

14.49

1.98

10

189.26

25.54

10

40 mg/kg

Mean

SD

N

4.24

2.84

10

37.32

3.59

10

31.26

6.79

10

24.90

5.85

10

14.82

6.69

10

13.06

5.92

10

11.72

3.51

10

11.15

3.90

10

8.33

2.28

10

8.10

2.66

10

9.29

2.02

10

7.59

2.08

10

-5.10

4.84

10

17.49

4.57

10

189.93

28.07

10

200 mg/kg

Mean

SD

N

4.43

1.52

10

40.10

3.11

10

35.43

3.75

10

28.97

5.09

10

20.85

5.86

10

19.69 * 4.64

10

14.71

4.58

10

11.05

2.73

10

9.12

3.42

10

6.78

3.47

10

10.92

3.57

10

5.61

1.63

10

-5.63

4.12

10

15.85

4.84

10

213.45

32.73

10

1000 mg/kg

Mean

SD

N

4.27

1.32

10

39.70

3.50

10

33.94

3.84

10

29.72

6.24

10

20.41

4.67

10

17.86

4.67

10

13.55

3.34

10

13.36

2.89

10

9.13

2.58

10

10.24

3.64

10

10.82

3.31

10

6.94

2.56

10

-4.38

3.28

10

15.36

3.18

10

216.65

21.43

10

[G] - Ancova/Anova & Dunnett(Log)

[G1] - Ancova/Anova & Dunnett: * = p < 0.05

[G2] - Kruskal-Wallis & Dunnett on Ranks

 

Table 4. Body weight change females – days relative to the start date.

Sex: Female

 

Wgt change last -x to 0

(g)

[G]

Body wt change

(g)

  

[G1]

Body wt change

(g)

 

 [G2]

Body wt change

(g)

  

[G2]

Body wt change

(g)

 

 [G2]

Body wt change

(g)

 

 [G2]

Body wt change

(g)

 

 [G2]

Body wt change

(g)

 

 [G2]

Body wt change

(g)

 

 [G2]

Body wt change

(g)

 

 [G2]

Body wt change

(g)

 

 [G2]

Body wt change

(g)

 

 [G2]

Body wt change

(g)

 

 [G2]

Body wt change

(g)

 

 [G]

Body wt change

(g)

  

[G2]

 

 

-x - 0

0 - 7

7 - 14

14 - 21

21 - 28

28 - 35

35 - 42

42 - 49

49 - 56

56 - 63

63 - 70

70 - 77

77 - 84

84 - 90

0 - 90

0 mg/kg

Mean

SD

N

3.95

2.79

10

18.25

3.52

10

15.43

4.94

10

9.31

9.35

10

12.45

4.77

10

9.23

3.43

10

2.91

3.91

10

5.13

7.26

10

8.78

6.57

10

2.42

3.72

10

0.64

3.62

10

1.14

6.99

10

4.24

4.46

10

0.25

5.97

10

90.18

14.98

10

40 mg/kg

Mean

SD

N

1.98 *

1.87

10

19.07

2.47

10

16.23

3.65

10

12.10

6.18

10

10.56

7.46

10

7.71

2.95

10

3.82

3.95

10

2.88

5.84

10

6.73

7.53

10

2.20

4.66

10

2.42

3.12

10

0.01

5.03

10

1.02

5.27

10

4.96

6.48

10

89.71

12.04

10

200 mg/kg

Mean SD

N

1.03 ** 2.94

10

19.64

3.16

10

16.32

2.50

10

13.04

6.60

10

10.53

6.16

10

11.13

4.79

10

5.68

5.37

10

4.25

7.37

10

5.71

6.59

10

2.86

3.87

10

1.47

3.97

10

1.55

8.17

10

3.13

5.99

10

3.08

7.24

10

98.39

8.53

10

1000 mg/kg

Mean SD

N

3.34

3.09

10

17.93

3.67

10

14.22

5.29

10

12.69

5.50

10

8.27

4.48

10

10.96

4.18

10

4.52

5.33

10

7.18

5.64

10

2.60

6.07

10

2.46

4.87

10

3.14

4.99

10

1.55

5.17

10

2.24

5.99

10

1.15

6.40

10

88.91

8.53

10

[G] - Kruskal-Wallis & Dunnett on Ranks: * = p < 0.05; ** = p < 0.01

[G1] - Ancova/Anova & Dunnett(Log)

[G2] - Ancova/Anova & Dunnett

 

Table 5. Red blood cell and coagulation parameters males – day 91 relative to start date.

Sex: Male

 

RBC (10E12

/L)

[G]

Hb (mmol

/L)

[G]

PCV (L/L)

 

[G]

MCV (fL)

 

 

[G]

MCH

(fmol)

 

[G]

MCHC

(mmol

/L)

[G]

Reticulo cytes

(%)

[G]

Thrombo cytes

(10E9/L)

[G]

Prothrom Time

(s)

[G]

0 mg/kg

Mean

SD

N

8.885

0.311

10

9.61

0.26

10

0.4748

0.0140

10

53.45

0.61

10

1.082

0.024

10

20.24

0.31

10

1.811

0.223

10

699.0

65.4

10

19.33

0.49

10

40 mg/kg

Mean SD

N

8.916

0.315

10

9.59

0.31

10

0.4793

0.0149

10

53.77

1.03

10

1.076

0.024

10

20.01

0.39

10

2.178 ** 0.178

10

767.2

112.5

10

19.71

0.80

10

200 mg/kg

Mean SD

N

8.879

0.324

10

9.52

0.26

10

0.4722

0.0162

10

53.20

1.17

10

1.073

0.018

10

20.17

0.31

10

1.989

0.233

10

748.4

88.2

10

19.57

0.65

10

1000 mg/kg

Mean SD

N

8.967

0.403

10

9.64

0.31

10

0.4800

0.0170

10

53.56

1.26

10

1.076

0.026

10

20.09

0.35

10

1.862

0.194

10

732.0

86.8

10

19.56

0.89

10

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

 

Table 6. Red blood cell and coagulation parameters females – day 92 relative to start date.

Sex: Female

 

RBC (10E12

/L)

[G]

Hb (mmol

/L)

[G]

PCV (L/L)

 

[G]

MCV (fL)

 

 

[G]

MCH

(fmol)

 

[G]

MCHC

(mmol

/L)

[G1]

Reticulo cytes

(%)

[G1]

Thrombo cytes

(10E9/L)

[G]

Prothrom Time

(s)

[G1]

0 mg/kg

Mean

SD

N

8.483

0.467

10

9.57

0.46

10

0.4691

0.0266

10

55.30

0.99

10

1.129

0.018

10

20.41

0.27

10

2.419

0.369

10

738.0

53.6

10

19.39

1.03

10

40 mg/kg

Mean SD

N

8.320

0.339

10

9.21

0.28

10

0.4505

0.0137

10

54.17

0.84

10

1.107

0.020

10

20.44

0.17

10

2.504

0.463

10

749.3

92.9

10

19.71

0.60

10

200 mg/kg

Mean SD

N

8.383

0.386

10

9.31

0.28

10

0.4556

0.0168

10

54.38

1.42

10

1.112

0.040

10

20.44

0.50

10

2.531

0.400

10

718.4

58.8

10

19.43

0.69

10

1000 mg/kg

Mean SD

N

7.940 **

0.341

10

8.98 ** 0.31

10

0.4391 **

0.0144

10

55.34

1.61

10

1.132

0.039

10

20.45

0.32

10

2.534

0.176

10

716.0

73.6

10

19.60

0.33

10

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

[G1] - Kruskal-Wallis & Dunnett on Ranks

 

Table 7. Total and differential white blood cell counts males – day 91 relative to start date.

Sex: Male

 

WBC (10E9/L)

 

 

[G]

Lympho Absolute

(10E9/L)

[G]

Neutro Absolute

(10E9/L)

[G]

Eosino Absolute

(10E9/L)

[G]

Baso Absolute

(10E9/L)

[G]

Mono Absolute

(10E9/L)

[G1]

Lympho cytes

(%)

 

[G]

Neutro phils

(%)

 

[G]

Eosino phils

(%)

 

[G]

Baso phils

(%)

 

[G]

Mono cytes

(%)

 

[G1]

0 mg/kg

Mean

SD

N

6.16

1.00

10

4.76

0.87

10

1.14

0.20

10

0.125

0.046

10

0.015

0.007

10

0.095

0.028

10

77.00

2.89

10

18.74

3.46

10

2.02

0.63

10

0.24

0.12

10

1.53

0.32

10

40 mg/kg

Mean SD

N

5.72

1.76

10

4.25

1.37

10

1.22

0.38

10

0.105

0.045

10

0.011

0.007

10

0.115

0.075

10

73.87

4.78

10

21.77

4.42

10

1.90

0.71

10

0.18

0.08

10

1.88

0.74

10

200 mg/kg

Mean SD

N

6.32

1.74

10

5.01

1.46

10

1.06

0.30

10

0.105

0.038

10

0.014

0.007

10

0.100

0.038

10

79.02

3.59

10

16.97

2.93

10

1.73

0.83

10

0.21

0.07

10

1.56

0.39

10

1000 mg/kg

Mean SD

N

6.35

1.21

10

5.19

1.09

10

0.92

0.25

10

0.097

0.031

10

0.014

0.006

10

0.097

0.031

10

81.56 * 3.81

10

14.74 * 3.39

10

1.55

0.46

10

0.21

0.07

10

1.52

0.34

10

[G] - Ancova/Anova & Dunnett: * = p < 0.05

[G1] - Ancova/Anova & Dunnett(Log)

 

Table 8. Total and differential white blood cell counts females – day 92 relative to start date.

Sex: Female

 

WBC (10E9/L)

 

 

[G]

Lympho Absolute

(10E9/L)

[G]

Neutro Absolute

(10E9/L)

[G]

Eosino Absolute

(10E9/L)

[G1]

Baso Absolute

(10E9/L)

[G1]

Mono Absolute

(10E9/L)

[G2]

Lympho cytes

(%)

 

[G]

Neutro phils

(%)

 

[G]

Eosino phils

(%)

 

[G1]

Baso phils

(%)

 

[G2]

Mono cytes

(%)

 

[G]

0 mg/kg

Mean

SD

N

4.60

1.45

10

3.79

1.16

10

0.62

0.30

10

0.083

0.072

10

0.014

0.010

10

0.076

0.054

10

82.89

5.83

10

13.05

4.39

10

1.91

1.96

10

0.29

0.14

10

1.53

0.75

10

40 mg/kg

Mean SD

N

4.56

1.26

10

3.62

1.12

10

0.74

0.20

10

0.086

0.028

10

0.012

0.009

10

0.087

0.045

10

78.57

5.88

10

16.94

5.79

10

1.96

0.61

10

0.26

0.18

10

1.89

0.64

10

200 mg/kg

Mean SD

N

5.39

1.16

10

4.16

1.07

10

1.02 *

0.34

10

0.075

0.030

10

0.013

0.006

10

0.097

0.040

10

76.74

6.24

10

19.31

6.17

10

1.38

0.46

10

0.23

0.08

10

1.77

0.48

10

1000 mg/kg

Mean SD

N

4.91

1.03

10

3.85

0.85

10

0.86

0.31

10

0.086

0.041

10

0.012

0.006

10

0.083

0.027

10

78.53

5.62

10

17.48

5.14

10

1.74

0.67

10

0.25

0.12

10

1.69

0.38

10

[G] - Ancova/Anova & Dunnett: * = p < 0.05

[G1] - Kruskal-Wallis & Dunnett on Ranks

[G2] - Ancova/Anova & Dunnett(Log)

 

Table 9. Clinical chemistry males – day 91 relative to start date.

Sex: Male

 

ALP (U/L)

[G]

ASAT (U/L)

[G]

ALAT (U/L)

[G1]

GGT (U/L)

[G]

Bilirub Total

(umol/L)

[G]

Creatin ine

(umol/L)

[G1]

Total Protein

(g/L)

[G2]

Albumin (g/L)

[G1]

Albumin/ Globulin

[G2]

Glucose Plasma

(mmol/L)

[G]

Cholest erol

(mmol/L)

[G]

Triglyc erides

(mmol/L)

[G1]

Urea (mmol/L)

[G]

PO4

(mmol/L)

[G]

Ca (mmol/L)

[G]

Cl  (mmol/L)

[G]

K

(mmol/L)

[G]

Na (mmol/L)

[G]

T4

(ng/ml)

0 mg/kg

Mean

SD

N

99.9

23.3

10

65.8

10.6

10

53.5

14.3

10

6.80

1.03

10

1.04

0.31

10

34.5

5.5

10

64.3

2.3

10

12.6

1.3

10

0.244

0.029

10

6.750

0.827

10

1.826

0.192

10

0.762

0.199

10

5.32

0.55

10

2.380

0.387

10

2.727

0.064

10

105.2

0.8

10

5.79

0.38

10

149.1

1.2

10

532.37

103.37

10

40 mg/kg

Mean SD

N

95.4

16.0

10

61.8

18.7

10

59.7

25.6

10

6.90

0.99

10

1.21

0.52

10

37.3

5.6

10

64.7

2.1

10

11.7

0.8

10

0.221

0.014

10

6.515

1.265

10

1.701

0.275

10

1.030

0.506

10

5.10

0.63

10

2.055

0.351

10

2.718

0.061

10

105.3

1.2

10

5.39 *

0.26

10

148.7

0.9

10

470.93

88.37

10

200 mg/kg

Mean SD

N

73.5 ** 16.6

10

57.0

14.4

10

50.2

11.6

10

7.30

1.25

10

1.23

0.38

10

35.6

5.5

10

65.6

2.8

10

11.4 *

0.7

10

0.210 ** 0.009

10

7.262

0.885

10

1.730

0.290

10

0.854

0.271

10

4.94

0.60

10

2.082

0.318

10

2.732

0.054

10

105.8

0.8

10

5.39 *

0.37

10

148.9

0.6

10

471.04

62.42

10

1000 mg/kg

Mean SD

N

86.4

17.8

10

61.0

10.0

10

40.3

6.4

10

6.70

1.16

10

0.98

0.25

10

38.3

4.4

10

65.3

3.7

10

11.4 *

0.8

10

0.212 ** 0.013

10

7.306

1.136

10

1.893

0.182

10

0.978

0.464

10

5.12

0.52

10

2.208

0.393

10

2.782

0.082

10

105.7

0.9

10

5.56

0.33

10

149.2

0.9

10

570.67

99.57

10

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

[G1] - Ancova/Anova & Dunnett(Log): * = p < 0.05

[G2] - Kruskal-Wallis & Dunnett on Ranks: ** = p < 0.01

 

Table 10. Clinical chemistry females – day 92 relative to start date.

Sex: Female

 

ALP (U/L)

[G]

ASAT (U/L)

[G1]

ALAT (U/L)

[G]

GGT (U/L)

[G1]

Bilirub Total

(umol/L)

[G2]

Creatin ine

(umol/L)

[G1]

Total Protein

(g/L)

[G1]

Albumin (g/L)

[G1]

Albumin/ Globulin

 

[G1]

Glucose Plasma

(mmol/L)

[G1]

Cholest erol

(mmol/L)

[G2]

Triglyc erides

(mmol/L)

[G1]

Urea (mmol/L)

[G2]

PO4

(mmol/L)

[G3]

Ca (mmol/L)

[G2]

Cl  (mmol/L)

[G2]

K

(mmol/L)

[G1]

Na (mmol/L)

[G3]

T4

(ng/ml)

0 mg/kg

Mean

SD

N

49.4

15.0

10

79.7

10.8

10

57.2

21.6

10

7.70

0.82

10

0.80

0.25

10

40.4

4.1

10

69.1

2.8

10

13.7

1.6

10

0.248

0.031

10

5.450

0.804

10

1.414

0.315

10

0.771

0.231

10

6.20

0.66

10

2.209

0.260

10

2.783

0.050

10

105.9

0.7

10

5.62

0.46

10

146.6

1.3

10

468.30

93.41

10

40 mg/kg

Mean SD

N

47.3

15.6

10

74.2

7.1

10

42.9

8.4

10

7.10

1.37

10

1.00

0.30

10

37.4

7.2

10

65.5 *

2.0

10

13.7

0.9

10

0.265

0.020

10

5.512

0.767

10

1.446

0.208

10

0.778

0.376

10

6.38

1.16

10

2.268

0.426

10

2.745

0.066

10

105.9

1.7

10

5.72

0.30

10

147.5

1.5

10

435.96

139.27

10

200 mg/kg

Mean SD

N

41.1

8.7

10

71.1

5.7

10

44.7

14.1

10

6.60

0.84

10

1.18

0.81

10

43.0

5.9

10

69.5

3.2

10

13.9

1.9

10

0.250

0.034

10

5.568

0.738

10

1.456

0.461

10

0.813

0.341

10

6.94

1.57

10

2.094

0.271

10

2.777

0.045

10

105.4

1.2

10

5.74

0.33

10

146.6

1.5

10

474.06

82.02

10

1000 mg/kg

Mean SD

N

38.0

5.3

10

77.9

7.7

10

41.7

10.7

10

7.00

1.25

10

1.07

0.59

10

42.8

6.0

10

69.7

3.5

10

14.5

1.7

10

0.262

0.025

10

5.110

0.562

10

1.496

0.290

10

0.869

0.283

10

7.28

0.67

10

2.141

0.315

10

2.793

0.087

10

104.4 *

1.1

10

5.75

0.28

10

145.4

0.7

10

388.54

110.98

10

[G] - Ancova/Anova & Dunnett(Log)

[G1] - Ancova/Anova & Dunnett: * = p < 0.05

[G2] - Kruskal-Wallis & Dunnett on Ranks

[G3] - Ancova/Anova & Dunnett

 

Table 11. Absolute organ weight males – day 92 relative to start date.

Sex: Male

 

Terminal

body wgt (g)

 

[G]

Brain

(g)

 

[G]

Heart

(g)

 

[G]

Adrenals

(g)

 

[G]

Kidneys

(g)

 

[G]

Liver

(g)

 

[G]

Spleen

(g)

 

[G]

Thymus

(g)

 

[G]

Thyroid

(g)

 

[G1]

Testes

(g)

 

[G]

Epididymides (g)

[G2]

Prostate

(g)

 

[G]

Seminal

vesicles (g)

 

[G]

0 mg/kg

Mean

SD

N

337.41

21.86

10

2.053

0.081

10

0.911

0.078

10

0.0441

0.0069

10

1.891

0.149

10

7.473

0.633

10

0.5049

0.0710

10

0.3266

0.0421

10

0.0099

0.0026

10

3.442

0.294

10

1.118

0.072

10

0.843

0.188

10

1.019

0.155

10

40 mg/kg

Mean

SD

N

339.70

32.37

10

1.991

0.044

10

0.936

0.076

10

0.0463

0.0073

10

1.832

0.194

10

7.792

0.731

10

0.4905

0.0585

10

0.2989

0.0611

10

0.0108

0.0020

10

3.278

0.261

10

1.103

0.172

10

0.883

0.118

10

1.185

0.163

10

200 mg/kg

Mean

SD

N

362.47

31.69

10

2.043

0.095

10

0.994

0.084

10

0.0487

0.0064

10

1.986

0.168

10

8.565 **

0.948

10

0.5348

0.0539

10

0.3595

0.0886

10

0.0122

0.0031

10

3.520

0.370

10

1.189

0.141

10

0.860

0.108

10

1.038

0.203

10

1000 mg/kg

Mean

SD

N

365.57

22.67

10

2.045

0.073

10

0.953

0.080

10

0.0508

0.0068

10

1.904

0.146

10

8.335 *

0.754

10

0.5232

0.0633

10

0.3489

0.0639

10

0.0130

0.0050

10

3.389

0.348

10

1.215

0.091

10

0.929

0.122

10

1.100

0.162

10

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

[G1] - Ancova/Anova & Dunnett(Log)

[G2] - Kruskal-Wallis & Dunnett on Ranks

 

Table 12. Absolute organ weight females – day 92 relative to start date.

Sex: Female

 

Terminal body wgt

(g)

[G]

Brain (g)

[G]

Heart (g)

[G]

Adrenals (g)

[G]

Kidneys (g)

[G]

Liver (g)

[G1]

Spleen (g)

[G]

Thymus (g)

[G]

Thyroid (g)

[G]

Ovaries (g)

[G2]

Uterus (g)

[G1]

0 mg/kg

Mean

SD

N

216.93

12.73

10

1.920

0.071

10

0.739

0.077

10

0.0588

0.0107

10

1.373

0.092

10

5.429

0.514

10

0.4268

0.0470

10

0.2654

0.0496

10

0.0098

0.0021

10

0.0889

0.0136

10

0.9341

0.5332

10

40 mg/kg

Mean SD

N

213.64

15.96

10

1.908

0.067

10

0.706

0.068

10

0.0642

0.0113

10

1.399

0.080

10

5.575

0.448

10

0.4479

0.0507

10

0.3210

0.0489

10

0.0115

0.0023

10

0.0931

0.0158

10

0.8480

0.2649

10

200 mg/kg

Mean SD

N

222.49

15.51

10

1.904

0.081

10

0.693

0.062

10

0.0594

0.0076

10

1.428

0.135

10

5.713

0.658

10

0.4399

0.0575

10

0.2973

0.0623

10

0.0105

0.0022

10

0.0915

0.0163

10

0.8288

0.2644

10

1000 mg/kg

Mean SD

N

214.08

10.86

10

1.874

0.037

10

0.673

0.065

10

0.0591

0.0073

10

1.316

0.099

10

5.464

0.279

10

0.4530

0.0568

10

0.3135

0.0556

10

0.0093

0.0018

10

0.0961

0.0262

10

0.6687

0.2828

10

[G] - Ancova/Anova & Dunnett

[G1] - Kruskal-Wallis & Dunnett on Ranks

[G2] - Ancova/Anova & Dunnett(Log)

 

Table 13. Relative organ weight males – day 92 relative to start date.

 

 

Terminal

body wgt (g)

[G]

Brain

rel.wgt (g/kg body wgt) [G]

Heart

rel.wgt (g/kg body wgt) [G]

Adrenals

rel.wgt (g/kg body wgt) [G]

Kidneys

rel.wgt (g/kg body wgt) [G]

Liver

rel.wgt (g/kg body wgt) [G]

Spleen

rel.wgt (g/kg body wgt) [G1]

Thymus

rel.wgt (g/kg body wgt) [G]

Thyroid

rel.wgt (g/kg body wgt) [G1]

Testes

rel.wgt (g/kg body wgt) [G1]

Epididy

rel.wgt (g/kg body wgt) [G1]

Prostate

rel.wgt (g/kg body wgt) [G2]

Sem ves

rel.wgt (g/kg body wgt) [G]

0 mg/kg

Mean

SD

N

337.41

21.86

10

6.108

0.476

10

2.700

0.155

10

0.1314

0.0235

10

5.619

0.504

10

22.15

1.16

10

1.502

0.228

10

0.969

0.119

10

0.0297

0.0092

10

10.197

0.457

10

3.321

0.238

10

2.503

0.554

10

3.027

0.481

10

40 mg/kg

Mean

SD

N

339.70

32.37

10

5.908

0.564

10

2.772

0.279

10

0.1363

0.0163

10

5.411

0.504

10

22.97

1.09

10

1.445

0.110

10

0.878

0.148

10

0.0322

0.0077

10

9.745

1.325

10

3.290

0.680

10

2.637

0.569

10

3.528

0.646

10

200 mg/kg

Mean

SD

 N

362.47

31.69

10

5.670

0.505

10

2.749

0.189

10

0.1347

0.0157

10

5.485

0.227

10

23.67

2.10

10

1.478

0.112

10

0.984

0.181

10

0.0336

0.0079

10

9.749

1.064

10

3.291

0.387

10

2.372

0.204

10

2.875

0.591

10

1000 mg/kg

Mean

SD

N

365.57

22.67

10

5.606

0.274

10

2.613

0.238

10

0.1399

0.0232

10

5.224

0.484

10

22.78

1.26

10

1.435

0.184

10

0.957

0.187

10

0.0360

0.0150

10

9.262

0.639

10

3.327

0.212

10

2.549

0.364

10

3.020

0.497

10

[G] - Ancova/Anova & Dunnett

[G1] - Kruskal-Wallis & Dunnett on Ranks

[G2] - Ancova/Anova & Dunnett(Log)

 

Table 14. Relative organ weight females – day 92 relative to start date.

Sex: Female

 

Terminal body wgt

(g)

 

[G]

Brain rel.wgt

(g/kg body wgt)

[G]

Heart rel.wgt

(g/kg body wgt)

[G]

Adrenals rel.wgt

(g/kg body wgt)

[G]

Kidneys rel.wgt

(g/kg body wgt)

[G]

Liver rel.wgt

(g/kg body wgt)

[G]

Spleen rel.wgt

(g/kg body wgt)

[G]

Thymus rel.wgt

(g/kg body wgt)

[G]

Thyroid rel.wgt

(g/kg body wgt)

[G]

Ovaries rel.wgt

(g/kg body wgt)

[G1]

Uterus rel.wgt

(g/kg body wgt)

[G2]

0 mg/kg

Mean

SD

N

216.93

12.73

10

8.865

0.357

10

3.402

0.230

10

0.2710

0.0463

10

6.330

0.237

10

25.05

2.15

10

1.967

0.192

10

1.233

0.268

10

0.0451

0.0090

10

0.4098

0.0582

10

4.267

2.413

10

40 mg/kg

Mean SD

N

213.64

15.96

10

8.970

0.659

10

3.315

0.349

10

0.3017

0.0565

10

6.571

0.480

10

26.15

1.92

10

2.101

0.225

10

1.515

0.290

10

0.0539

0.0104

10

0.4367

0.0725

10

3.976

1.241

10

200 mg/kg

Mean SD

N

222.49

15.51

10

8.581

0.452

10

3.116 * 0.188

10

0.2675

0.0325

10

6.415

0.373

10

25.76

3.06

10

1.980

0.241

10

1.344

0.304

10

0.0470

0.0088

10

0.4108

0.0679

10

3.775

1.379

10

1000 mg/kg

Mean SD

N

214.08

10.86

10

8.770

0.384

10

3.140

0.210

10

0.2758

0.0283

10

6.150

0.390

10

25.57

1.68

10

2.114

0.207

10

1.469

0.276

10

0.0436

0.0090

10

0.4461

0.1018

10

3.137

1.366

10

[G] - Ancova/Anova & Dunnett: * = p < 0.05

[G1] - Ancova/Anova & Dunnett(Log)

[G2] - Kruskal-Wallis & Dunnett on Ranks

Table 15. Thyroid hormone levels in males and females – Day 91 and 92 relative to start date, respectively.

 

Male

Male

Female

Female

TSH

(pg/ml)

[G]

T4

(ng/mL)

TSH

(pg/ml)

[G1]

T4

(ng/mL)

0 mg/kg

Mean SD

N

2419.25

728.82

10

532.37

103.37

10

2977.77

551.55

10

468.30

93.41

10

40 mg/kg

Mean

2406.56

470.93

3604.39

435.96

 

SD

736.17

88.37

1405.95

139.27

 

N

10

10

10

10

200 mg/kg

Mean

1947.21

471.04

2716.86

474.06

 

SD

505.32

62.42

709.22

82.02

 

N

10

10

10

10

1000 mg/kg

Mean

2498.93

570.67

2216.54 *

388.54

 

SD

994.38

99.57

296.92

110.98

 

N

10

10

10

10

[G] - Ancova/Anova & Dunnett(Log)

[G1] - Kruskal-Wallis & Dunnett on Ranks: * = p < 0.05

Table 16. Historical control data for TSH in serum samples.

 

Males

Females

Study type

OECD 443

OECD 443

OECD 408

OECD 443

OECD 443

OECD 408

Approx. age of rats

22 weeks

13 weeks

19 weeks

19 weeks

13 weeks

19 weeks

Mean

2661

3872

2969

1923

983

3683

SD

1174.6

1047.5

798.6

692.0

567.6

1134.2

CV

44.1%

27.1%

26.9 %

36.0%

57.7%

30.8 %

Count

10

8

10

10

9

10

Applicant's summary and conclusion

Conclusions:
In this GLP complaint oral repeated dose toxicity study, performed according to OECD 408, the no-observed-adverse effect level (NOAEL) was placed at the highest level tested, namely 1000 mg/kg bw/day because the test substance did not induce any toxicologically relevant changes in any test group.
Based on this OECD 408 study and preliminary data from an OECD 414 study, a need for changes to the study design of the OECD 443 study is identified. Considerations for this change can be found in section 7.8.1 of this dossier under ‘Toxicity to reproduction – waiver/change in study design’ in the 'Justification for type of information' field.
Executive summary:

In this GLP compliant 90 day gavage study performed according to OECD 408, the safety of ZBEC was examined in Wistar rats of both sexes. ZBEC was administered to the rats by daily oral gavage as a suspension in corn oil during 13 weeks at levels of 0, 40, 200 and 1000 mg/kg body weight/day. Clinical signs, body weight, food and water consumption were monitored throughout the study. Ophthalmoscopic examinations were performed before dosing commenced for all animals and towards the end of treatment in the control and high-dose group. Behavioural endpoints (Functional Observation Battery and motor activity assessment) were investigated in all rats at the end of the study. Haematology, clinical chemistry (including thyroid hormones TSH and T4) and urinalysis were performed at the end of the study. All animals were killed, subjected to necropsy and post mortem examination, major organs were weighed and a full range of tissues were examined microscopically.

The administration of ZBEC was well tolerated at all dose levels, and did not induce any relevant changes in general condition, growth, feed or water intake, neurobehavioral observations, ophthalmoscopy, urinalysis, organ weights or in macroscopy and microscopy of organs and tissues. A few differences with the control reached statistical significance in the high-dose group, but were not considered to be adverse as discussed below: In females of the high dose group, red blood cell count, haemoglobin concentration and packed cell volume were somewhat lower than in controls. Because these changes were only slight (about -6%) they were not considered to be adverse. Furthermore, the percentage of lymphocytes was increased and the percentage of neutrophils was decreased in males of the high-dose group. Because these findings were within the range of historical control data and not reflected by significant changes in the absolute lymphocyte and neutrophil counts, they were not considered to be adverse. Albumin and the albumin/globulin ratio were decreased in males of the mid- and high-dose groups and chloride concentration was decreased in high-dose females. Because these changes were within the range of historical control data and not corroborated by any effects on relative organ weight or pathology, they were not considered to be adverse. TSH levels were slightly decreased in females of the high-dose group. This slight decrease in TSH levels was not corroborated by noticeable changes in T4 levels, growth, thyroid weight or pathology. Moreover the values were well within the range of historical control data and this variable is known to show considerable variation. Therefore the decrease in TSH levels in high-dose females was considered to be a chance finding.

Because ZBEC did not induce any relevant changes in any test group, the no-observed-adverse effect level (NOAEL) was placed at the highest level tested, namely 1000 mg/kg bw/day.