Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
28 days
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 29, 2016 - Feb 21, 2017 (28 days exposure)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Performed to GLP using OECD guidelines. However, some observations (not listed as a minimum gudeline requirement) were not recorded. This includes information relting to the colour of faeces and onlt limited details on the colour changes in urine. These effects are well recorded in the reproduction toxicity screening test 7.8.1

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
OECD 407:2008
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium [4-[[dihydroxy[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]benzenesulphonato(3-)]cuprate(1-)
EC Number:
305-729-0
EC Name:
Sodium [4-[[dihydroxy[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]benzenesulphonato(3-)]cuprate(1-)
Cas Number:
95009-01-1
Molecular formula:
C18H9CuN6O10S.Na
IUPAC Name:
sodium [4-[[dihydroxy[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]benzenesulphonato(3-)]cuprate(1-)
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Everlan SL65
- Substance type: Powder
- Composition of test material, percentage of components: 71.19%
- Lot/batch No.: 3010
- Storage condition of test material: Ambient

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: BioLASCO Taiwan Co., Ltd.
- Age at study initiation: about 4-week old
- Housing: Male and female rats were fed, respectively. Two rats per cage in an autoclaved polyethylene cage.
- Acclimation period: 1 week
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 15%
- Photoperiod: 12-hrs dark / 12-hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
- Rate of preparation of diet (frequency): everyday fresh preparation
- Concentration in vehicle: 6.25 mg/ml, 25.0 mg/ml and 100 mg/ml with water
Duration of treatment / exposure:
28 days
Frequency of treatment:
everyday
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Medium dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
For control group: 6 males and 6 females
For low dose group: 6 males and 6 females
For medium dose group: 6 males and 6 females
For high dose group: 6 males and 6 females
Control animals:
yes, concurrent vehicle

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
For male rats:
The body weight of medium dose (250 mg/kg bw) and high dose (1000 mg/kg bw) groups was significantly higher than control group at week 1 (p<0.05).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
For male rats:
The feed efficiency of high dose group was significantly higher than control group at week 3 and 4 (p < 0.05).
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
For male rats:
For medium dose group, the platelet count and monocyte were significantly higher than control group and lymphocyte was significantly lower than control group (p < 0.05). For high dose group, the WBC and neutrophil of were significantly higher than control group and lymphocyte and basophil were significantly lower than control group (p < 0.05).
For female rats:
For low dose group, the mean corpuscular volume (MCV) was significantly higher than control group and RBC, hemoglobin and mean corpuscular hemoglobin concentration (MCHC) were significantly lower than control group (p < 0.05). For medium dose group, the mean corpuscular hemoglobin (MCH) was significantly higher than control group and RBC, hemoglobin and hematocrit were significantly lower than control group (p < 0.05). For high dose group, the WBC, platelet count and neutrophil was significantly higher than control group and RBC, hemoglobin, hematocrit, lymphocyte and basophil were significantly lower than control group (p < 0.05).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
For male rats:
The Ca of medium dose was significantly higher than control group (p <0.05). The albumin and alkaline phosphatase (ALP) of high dose group were significantly lower than control group (p < 0.05).
For female rats:
The globulin of medium dose group was significantly lower than control group (p < 0.05). For high dose group, the P and Cl were significantly higher than control group (p < 0.05) and total protein, albumin and Na were significantly lower than control group (p < 0.05).
Urinalysis findings:
no effects observed
Description (incidence and severity):
No discolouration noted with same colour for controls and treated groups. Some high dose group fem
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
For male rats:
The brain absolute weight of low dose group was significantly lower than control group (p < 0.05). The spleen absolute weight of medium dose group, adrenal gland and spleen absolute weight of high dose group were significantly heavier than control group (p < 0.05). The adrenal gland, spleen and kidney relative weight of high dose group were significantly higher than control group (p < 0.05).
For female rats:
The spleen absolute weight of medium and high dose groups were significantly heavier than control group (p < 0.05). The heart relative weight of medium dose group and the spleen relative weight of medium and high dose group were significantly heavier than control group (p < 0.05).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Damage seen to cecum and colon in high dose group animals, typical of copper toxicity.
This local damage would impact on spleen activity and blood parameter changes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Specific histopathological findings:
Male and female rats in the high dose groups presented multifocal, minimal to moderate, ulcerative necrosis and inflammation in the cecum. Male rats in the high dose groups presented multifocal, moderate, ulcerative necrosis and inflammation in the colon. Male and female rats in the middle and high dose groups presented multifocal, minimal to moderate, inflammation in the terminal bronchial and alveoli of the lungs. Female rats in the high dose groups presented multifocal, minimal to moderate, acinar hyperplasia in the mammary gland.

Non-specific histopathological findings:
Only one male rat in the control group showed a focal, slight tubular cyst in the kidney. Only one male rat in high dose group presented focal, slight mononuclear cell infiltration in the prostate gland.
Histopathological findings: neoplastic:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: neoplastic

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
colon
other: cecum
Treatment related:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
respiratory system: lower respiratory tract
Organ:
alveoli
bronchi
Treatment related:
yes

Any other information on results incl. tables

Table 1: Mortality of the rats

 

 

Sex

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Male

Female

Male

Female

Male

Female

Male

Female

No of rate in each group

6

6

6

6

6

6

6

6

No of rat died during the study

0

0

0

0

0

0

0

0

Table 2: Incidence of abnormal clinical sign

 

 

Sex

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Male

Female

Male

Female

Male

Female

Male

Female

No of rate in each group

6

6

6

6

6

6

6

6

No of rat exhibited abnormal clinical sign

0

0

0

0

0

0

0

0

Table 3: Average body weight of male rats

Recording time

Average body weight (g)

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Before study

136.5±8.6

136.4±9.3

136.7±9.2

136.0±12.8

Week 1

168.8±16.4

183.6±12.9

194.3±6.5*

186.3±11.4*

Week 2

253.8±14.4

251.5±16.0

153.9±10.6

246.0±19.6

Week 3

311.9±15.2

301.8±16.5

305.0±17.2

298.2±19.9

Week 4

342.7±24.2

338.3±22.1

346.5±24.1

316.5±31.2

After fasting

312.9±25.3

310.5±20.2

320.5±23.0

296.9±27.8

*Significant different from control group (p < 0.05)

Table 4: Average body weight of female rats

Recording time

Average body weight (g)

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Before study

148.0±6.6

147.7±9.3

148.1±5.8

148.1±5.4

Week 1

172.0±12.6

172.2±13.9

164.1±8.8

162.0±5.8

Week 2

198.9±14.0

202.8±16.3

188.5±13.2

192.6±7.6

Week 3

220.9±15.7

225.6±21.5

211.1±14.2

215.0±6.2

Week 4

239.4±18.0

243.3±23.1

227.3±17.0

222.3±10.6

After fasting

212.6±16.0

217.3±20.7

208.9±15.7

205.1±8.8

Table 5: Average feed intake of male rats

Recording time

Average feed intake (g/rat/day)

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Week 1

16.2±1.5

17.8±0.5*

18.4±0.9*

18.0±0.9*

Week 2

25.7±0.6

24.9±1.6

24.7±0.8

25.6±1.9

Week 3

26.6±1.4

25.7±1.4

26.3±1.9

27.6±1.2

Week 4

24.4±3.2

24.7±1.4

27.0±1.8

25.6±2.8

*Significant different from control group (p < 0.05)

Table 6: Average feed intake of female rats

Recording time

Average feed intake (g/rat/day)

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Week 1

15.3±0.1

15.3±0.5

13.7±0.9*

14.7±0.9

Week 2

18.2±1.6

18.5±1.5

18.5±0.5

19.2±1.5

Week 3

19.5±1.1

18.6±1.8

17.8±0.7*

19.1±0.6

Week 4

18.8±1.2

18.9±0.3

18.1±0.3

18.5±2.0

*Significant different from control group (p < 0.05)

Table 7: Average daily Feed efficiency of male rats

Recording time

Average daily Feed efficiency (g/100 g body weight of rat/day)

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Week 1

9.6±0.6

9.8±0.7

9.5±0.3

9.6±0.2

Week 2

10.1±0.7

9.9±0.7

9.8±0.2

10.4±0.4

Week 3

8.6±0.5

8.6±0.4

8.6±0.3

9.3±0.5*

Week 4

7.1±0.7

7.3±0.3

7.8±0.4

8.1±0.8*

*Significant different from control group (p < 0.05)

 

Table 8: Average daily Feed efficiency of female rats

Recording time

Average daily Feed efficiency (g/100 g body weight of rat/day)

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Week 1

8.9±0.7

8.9±0.7

8.4±0.7

9.1±0.4

Week 2

9.2±1.0

9.1±0.5

9.9±0.7

10.0±0.5

Week 3

8.9±0.8

8.3±0.6

8.5±0.7

8.9±0.2

Week 4

7.9±0.8

7.8±0.8

8.0±0.6

8.3±0.6

*Significant different from control group (p < 0.05)

 

Table 9: Ophthalmological examination before the study

 

 

Sex

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Male

Female

Male

Female

Male

Female

Male

Female

No of rate in each group

6

6

6

6

6

6

6

6

No of rat exhibited ophthalmic abnormality

0

0

0

0

0

0

0

0

Table 10: Ophthalmological examination at the end of the study

 

 

Sex

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Male

Female

Male

Female

Male

Female

Male

Female

No of rate in each group

6

6

6

6

6

6

6

6

No of rat exhibited ophthalmic abnormality

0

0

0

0

0

0

0

0

Table 11: Incidence of gross lesion

 

 

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Male

No of rate in each group

6

6

6

6

No of rat exhibited gross lesion

0

0

0

0

Female

No of rate in each group

6

6

6

6

No of rat exhibited gross lesion

0

0

0

0

Table 12: Absolute organ weight of male rats\

Item

Absolute organ weight (g)

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Testis

3.060±0.236

2.927±0.233

3.044±0.123

3.198±0.114

Adrenal gland

0.054±0.006

0.056±0.004

0.058±0.007

0.064±0.007*

Spleen

0.613±0.130

0.662±0.134

0.784±0.103*

0.848±0.066*

Kidney

3.028±0.193

2.978±0.302

3.131±0.278

3.238±0.515

Heart

1.302±0.063

1.283±0.043

1.302±0.106

1.344±0.154

Brain

2.047±0.027

1.952±0.071*

1.997±0.091

2.007±0.057

Liver

11.685±1.211

10.974±1.410

11.945±0.931

12.189±1.902

Thymus

0.552±0.106

0.518±0.086

0.523±0.082

0.553±0.158

Epididymis

0.750±0.073

0.723±0.060

0.766±0.049

0.827±0.089

Coagulating glands

2.053±0.311

1.999±0.169

2.240±0.216

1.930±0.490

*Significant different from control group (p < 0.05)

 

Table 13: Absolute organ weight of female rats

Item

Absolute organ weight (g)

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Ovary

0.095±0.019

0.093±0.020

0.080±0.018

0.085±0.013

Adrenal gland

0.073±0.013

0.076±0.017

0.069±0.009

0.074±0.012

Spleen

0.507±0.075

0.548±0.067

0.691±0.099*

0.720±0.150*

Kidney

2.216±0.270

2.278±0.274

2.157±0.161

1.994±0.168

Heart

0.877±0.080

0.942±0.105

0.939±0.055

0.881±0.065

Brain

1.881±0.104

1.906±0.073

1.892±0.046

1.916±0.024

Liver

8.500±1.391

9.060±1.102

8.757±1.364

8.716±0.970

Thymus

0.433±0.049

0.472±0.074

0.454±0.085

0.424±0.109

Uterus

0.793±0.214

0.792±0.131

0.847±0.258

0.672±0.052

*Significant different from control group (p < 0.05)

 

Table 14:Relative organ weight of male rats

Item

Absolute organ weight (g)

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Testis

0.985±0.116

0.944±0.067

0.954±0.073

1.083±0.090

Adrenal gland

0.017±0.002

0.018±0.002

0.019±0.002

0.022±0.003*

Spleen

0.197±0.042

0.214±0.043

0.247±0.045

0.287±0.029*

Kidney

0.972±0.080

0.959±0.070

0.977±0.058

1.091±0.143*

Heart

0.419±0.042

0.415±0.028

0.407±0.026

0.458±0.081

Brain

0.658±0.051

0.630±0.030

0.627±0.067

0.681±0.062

Liver

3.735±0.271

3.530±0.336

3.730±0.186

4.131±0.772

Thymus

0.176±0.030

0.167±0.029

0.163±0.019

0.189±0.065

Epididymis

0.241±0.030

0.233±0.024

0.241±0.033

0.280±0.040

Coagulating glands

0.662±0.124

0.647±0.077

0.704±0.105

0.659±0.210

*Significant different from control group (p < 0.05)

 

Table 15: Relative organ weight of female rats

Item

 

Absolute organ weight (g)

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Ovary

0.044±0.007

0.042±0.006

0.038±0.006

0.042±0.007

Adrenal gland

0.034±0.005

0.035±0.008

0.033±0.003

0.036±0.006

Spleen

0.238±0.028

0.252±0.015

0.332±0.056*

0.352±0.077*

Kidney

1.041±0.079

1.047±0.065

1.034±0.068

0.972±0.080

Heart

0.412±0.016

0.434±0.022

0.450±0.013*

0.430±0.027

Brain

0.887±0.040

0.883±0.068

0.910±0.077

0.935±0.038

Liver

3.979±0.344

4.162±0.203

4.172±0.343

4.257±0.530

Thymus

0.203±0.013

0.218±0.038

0.218±0.042

0.206±0.046

Uterus

0.373±0.099

0.365±0.053

0.409±0.136

0.328±0.023

*Significant different from control group (p < 0.05)

 

Table 16: Hematology of male rats

Item

Hematology

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

WBC (10^3/ul)

11.59±1.90

12.46±2.39

13.56±1.91

18.58±7.34*

RBC (10^6/ul)

9.20±0.34

8.69±0.37

8.67±0.68

8.75±0.81

Hemoglobin (g/dl)

17.60±0.43

16.97±1.16

16.65±0.94

16.72±1.06

Hematocrit (%)

53.53±0.96

51.85±3.19

51.28±2.12

51.35±3.27

MCV (fL)

58.23±2.09

59.65±1.37

59.33±2.42

58.87±3.28

MCH (pg)

19.12±0.50

19.53±0.54

19.23±0.52

19.15±0.79

MCHC (g/dl)

32.87±0.40

32.73±0.33

32.43±0.59

32.55±0.71

Platelet (10^3/ul)

923.8±88.6

958.3±139.5

1136.8±83.4*

1061.2±154.7

Neutrophil (%)

11.92±3.97

10.78±2.77

15.27±2.33

17.40±4.73*

Lymphocyte (%)

84.22±4.01

84.68±3.67

79.07±1.83*

78.27±4.93*

Monocyte (%)

2.87±0.56

3.62±0.89

4.87±1.41*

3.58±0.98

Eosinophil (%)

0.75±0.43

0.70±0.19

0.62±0.17

0.63±0.34

Basophil (%)

0.25±0.05

0.22±0.10

0.18±0.08

0.12±0.04*

Reticulocyte (%)

2.00±0.81

1.72±0.44

1.42±0.39

1.33±0.53

PT (sec)

15.80±1.28

15.27±2.85

16.03±0.96

14.90±1.97

*Significant different from control group (p < 0.05)

 

Table 17: Hematology of female rats

Item

Hematology

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

WBC (10^3/ul)

12.67±2.21

12.93±3.89

12.23±2.34

18.15±5.41*

RBC (10^6/ul)

8.90±0.40

8.21±0.31*

7.89±0.23*

8.03±0.41*

Hemoglobin (g/dl)

17.35±0.82

16.18±0.64*

15.85±0.46*

15.30±0.48*

Hematocrit (%)

53.07±2.37

51.20±1.63

48.12±1.09*

46.58±1.55*

MCV (fL)

59.60±0.76

62.40±2.16*

61.02±2.37

58.07±1.47

MCH (pg)

19.50±0.19

19.72±0.62

20.10±0.44*

19.08±0.42

MCHC (g/dl)

32.70±0.23

31.62±0.40*

32.93±0.83

32.83±0.31

Platelet (10^3/ul)

976.8±119.0

908.2±63.0

1005.2±70.2

1111.8±102.3*

Neutrophil (%)

9.07±2.20

9.75±2.37

11.83±1.05

16.77±6.38*

Lymphocyte (%)

86.78±3.04

86.75±2.97

83.70±1.56

78.92±6.63*

Monocyte (%)

3.20±0.90

2.60±0.59

3.62±0.71

3.78±1.34

Eosinophil (%)

0.70±0.26

0.70±0.21

0.60±0.27

0.42±0.22

Basophil (%)

0.25±0.10

0.20±0.09

0.25±0.10

0.12±0.04*

Reticulocyte (%)

2.72±0.80

2.48±0.65

2.48±1.23

2.78±0.84

PT (sec)

9.75±0.30

9.68±0.26

10.00±0.68

9.48±0.26

*Significant different from control group (p < 0.05)

 

Table 18: Clinical chemistry of male rats

Item

Hematology

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Glucose (mg/dl)

165.17±39.36

144.83±39.30

181.50±53.44

145.67±64.08

BUN (mg/dl)

13.00±0.54

13.05±2.46

12.85±1.99

12.85±1.74

Creatinine (mg/dl)

0.65±0.05

0.63±0.08

0.68±0.04

0.62±0.04

AST (U/L)

90.17±14.51

105.83±11.37

120.00±51.56

114.67±40.13

ALT (U/L)

36.83±4.12

39.17±6.37

38.17±13.23

32.33±5.47

Total protein (g/dl)

6.43±0.41

6.32±0.29

6.77±0.30

6.03±0.28

Albumin (g/dl)

4.73±0.18

4.65±0.24

4.95±0.24

4.27±0.19*

ALP (U/L)

218.50±47.34

212.00±40.06

219.00±46.65

131.00±37.16*

r-GT (U/L)

< 2.0

< 2.0

< 2.0

< 2.0

Cholesterol (mg/dl)

60.17±16.92

57.67±10.78

62.83±8.52

68.33±11.17

Triglyceride (mg/dl)

54.83±29.37

63.83±20.06

50.33±9.33

51.83±14.58

Calcium (mg/dl)

11.42±0.49

11.62±0.44

12.30±0.66*

11.62±0.81

Phosphorus (mg/dl)

13.60±0.70

13.18±0.65

14.15±0.71

13.73±0.92

Sodium (meq/dl)

145.83±2.79

145.50±2.07

146.83±0.75

144.17±2.23

Potassium (meq/dl)

7.85±0.95

8.05±0.94

7.85±1.10

9.27±1.58

Chloride (meq/dl)

101.67±1.63

101.33±1.75

101.00±1.10

102.00±2.45

Globulin (g/dl)

1.70±0.30

1.67±0.10

1.82±0.18

1.77±0.18

Total bilirubin (ug/dl)

< 0.04

< 0.04

< 0.04

< 0.04

Total bile acid (umol/dl)

12.12±3.13

16.05±12.19

18.33±11.11

9.73±2.75

*Significant different from control group (p < 0.05)

 

Table 19: Clinical chemistry of female rats

Item

Hematology

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Glucose (mg/dl)

126.00±58.15

134.83±39.11

99.50±19.55

100.67±25.32

BUN (mg/dl)

14.23±1.65

14.60±1.80

12.53±1.51

20.70±9.91

Creatinine (mg/dl)

0.70±0.00

0.67±0.05

0.68±0.04

0.67±0.08

AST (U/L)

108.67±27.90

104.83±14.88

107.00±28.98

168.17±103.97

ALT (U/L)

30.00±3.90

30.67±6.80

27.17±6.01

37.83±15.54

Total protein (g/dl)

7.18±0.30

6.88±0.42

6.77±0.25

5.98±0.44*

Albumin (g/dl)

5.47±0.25

5.23±0.34

5.27±0.26

4.45±0.40*

ALP (U/L)

97.67±23.02

94.67±16.35

96.83±14.92

92.00±11.21

r-GT (U/L)

< 2.0

< 2.0

< 2.0

< 2.0

Cholesterol (mg/dl)

80.33±17.28

90.17±20.52

85.83±9.62

69.67±7.31

Triglyceride (mg/dl)

29.50±8.60

25.50±8.64

37.17±15.38

33.67±7.53

Calcium (mg/dl)

12.30±0.30

12.58±0.26

12.50±0.27

12.18±0.40

Phosphorus (mg/dl)

12.42±1.08

12.25±1.00

12.37±0.82

15.57±1.36*

Sodium (meq/dl)

143.50±1.38

142.00±1.67

142.67±1.75

140.83±1.94*

Potassium (meq/dl)

9.82±0.74

9.47±1.32

9.72±1.00

11.02±1.20

Chloride (meq/dl)

101.50±1.05

100.33±0.52

102.50±1.52

104.33±1.21*

Globulin (g/dl)

1.72±0.12

1.65±0.10

1.50±0.14*

1.53±0.08

Total bilirubin (ug/dl)

< 0.04

< 0.04

< 0.04

< 0.04

Total bile acid (umol/dl)

25.88±19.94

19.07±10.95

16.52±3.60

18.63±8.73

Table 20. Microscopic examination of urinary sediments of male rats.

Item

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Cell type

RBC

0 – 1 (hpf)

6/6

5/6

0/6

0/6

2 – 5 (hpf)

0/6

1/6

6/6

6/6

6 – 15 (hpf)

0/6

0/6

0/6

0/6

WBC

0 – 1 (hpf)

1/6

1/6

0/6

0/6

2 – 5 (hpf)

5/6

5/6

6/6

5/6

6 – 15 (hpf)

0/6

0/6

0/6

1/6

EP

0 – 1 (hpf)

4/6

4/6

3/6

4/6

2 – 5 (hpf)

2/6

2/6

3/6

2/6

6 – 15 (hpf)

0/6

0/6

0/6

0/6

Crystal

None found

1/6

3/6

1/6

5/6

Triple phosphates

5/6

2/6

5/6

1/6

Am.urate

0/6

1/6

0/6

0/6

Am.phos

0/6

0/6

0/6

0/6

Table 21. Microscopic examination of urinary sediments of female rats.

Item

Control

0 mg/kg

Low dose

62.5 mg/kg

Middle dose

250 mg/kg

High dose

1,000 mg/kg

Cell type

RBC

0 – 1 (hpf)

0/6

0/6

0/6

0/6

2 – 5 (hpf)

6/6

6/6

6/6

6/6

6 – 15 (hpf)

0/6

0/6

0/6

0/6

WBC

0 – 1 (hpf)

0/6

0/6

0/6

0/6

2 – 5 (hpf)

6/6

6/6

6/6

4/6

6 – 15 (hpf)

0/6

0/6

0/6

2/6

EP

0 – 1 (hpf)

3/6

3/6

2/6

2/6

2 – 5 (hpf)

3/6

3/6

4/6

4/6

6 – 15 (hpf)

0/6

0/6

0/6

0/6

Crystal

None found

2/6

2/6

3/6

4/6

Triple phosphates

4/6

4/6

3/6

2/6

Am.urate

0/6

0/6

0/6

0/6

Am.phos

0/6

0/6

0/6

0/6

Table 22. Urinalysis of male rats.

Item

Control    0 mg/kg

Low dose 62.5 mg/kg

Middle dose 250 mg/kg

High dose 1,000 mg/kg

Color

Yellow

6/6

5/6

6/6

6/6

Pale yellow

0/6

1/6

0/6

0/6

Clarity

Clear

0/6

0/6

0/6

0/6

Light turbid

6/6

6/6

6/6

5/6

Turbid

0/6

0/6

0/6

1/6

Glucose

Negative

6/6

6/6

6/6

6/6

Trace

0/6

0/6

0/6

0/6

Bilirubin

Negative

6/6

6/6

6/6

6/6

1+

0/6

0/6

0/6

0/6

Ketone

Negative

6/6

6/6

6/6

5/6

Trace

0/6

0/6

0/6

1/6

1+

0/6

0/6

0/6

0/6

Specific gravity

<= 1.015

6/6

6/6

6/6

4/6

1.016 – 1.020

0/6

0/6

0/6

2/6

1.021 – 1.025

0/6

0/6

0/6

0/6

1.026 – 1.030

0/6

0/6

0/6

0/6

1.031 – 1.035

0/6

0/6

0/6

0/6

>= 1.036

0/6

0/6

0/6

0/6

pH

<= 6.0

0/6

0/6

0/6

0/6

6.5

0/6

3/6

0/6

2/6

7.0

5/6

3/6

3/6

2/6

7.5

1/6

0/6

2/6

2/6

>= 8.0

0/6

0/6

1/6

0/6

Protein

Negative

5/6

5/6

2/6

4/6

Trace

1/6

1/6

4/6

1/6

1+

0/6

0/6

0/6

1/6

2+

0/6

0/6

0/6

0/6

Urobilinogen

<= 1.0

6/6

6/6

6/6

5/6

2

0/6

0/6

0/6

1/6

Nitrite

Negative

6/6

5/6

1/6

4/6

Positive

0/6

1/6

3/6

2/6

2+

0/6

0/6

2/6

0/6

Occult Blood

Negative

6/6

6/6

6/6

5/6

Trace

0/6

0/6

0/6

1/6

1+

0/6

0/6

0/6

0/6

Leukocyte esterase (Leu/uL)

Negative

0/6

1/6

0/6

4/6

Trace

5/6

5/6

5/6

1/6

1+

1/6

0/6

1/6

0/6

2+

0/6

0/6

0/6

1/6

3+

0/6

0/6

0/6

0/6

Table 23. Urinalysis of female rats.

Item

Control    0 mg/kg

Low dose 62.5 mg/kg

Middle dose 250 mg/kg

High dose 1,000 mg/kg

Color

Yellow

5/6

6/6

5/6

2/6

Pale yellow

1/6

0/6

0/6

0/6

Orange

0/6

0/6

1/6

4/6

Clarity

Clear

0/6

0/6

0/6

0/6

Light turbid

6/6

6/6

4/6

3/6

Turbid

0/6

0/6

2/6

3/6

Glucose

Negative

6/6

6/6

6/6

6/6

Trace

0/6

0/6

0/6

0/6

Bilirubin

Negative

6/6

6/6

6/6

6/6

1+

0/6

0/6

0/6

0/6

Ketone

Negative

5/6

6/6

6/6

6/6

Positive

0/6

0/6

0/6

0/6

Trace

1/6

0/6

0/6

0/6

1+

0/6

0/6

0/6

0/6

2+

0/6

0/6

0/6

0/6

Specific gravity

<= 1.015

4/6

3/6

4/6

3/6

1.016 – 1.020

1/6

0/6

0/6

3/6

1.021 – 1.025

1/6

3/6

2/6

0/6

1.026 – 1.030

0/6

0/6

0/6

0/6

1.031 – 1.035

0/6

0/6

0/6

0/6

>= 1.036

0/6

0/6

0/6

0/6

pH

<= 6.0

0/6

0/6

0/6

0/6

6.5

1/6

2/6

0/6

2/6

7.0

3/6

3/6

3/6

2/6

7.5

1/6

0/6

2/6

1/6

>= 8.0

1/6

1/6

1/6

2/6

Protein

Negative

5/6

4/6

4/6

4/6

Trace

0/6

2/6

1/6

1/6

1+

1/6

0/6

1/6

1/6

2+

0/6

0/6

0/6

0/6

Urobilinogen

<= 1.0

6/6

6/6

6/6

5/6

2

0/6

0/6

0/6

1/6

Nitrite

Negative

2/6

0/6

6/6

3/6

Positive

3/6

4/6

0/6

1/6

2+

1/6

2/6

0/6

2/6

Occult Blood

Negative

6/6

6/6

6/6

6/6

Trace

0/6

0/6

0/6

0/6

1+

0/6

0/6

0/6

0/6

Leukocyte esterase (Leu/uL)

Negative

3/6

3/6

3/6

3/6

Trace

1/6

1/6

2/6

3/6

1+

2/6

2/6

1/6

0/6

2+

0/6

0/6

0/6

0/6

3+

0/6

0/6

0/6

0/6

Applicant's summary and conclusion

Conclusions:
According to OECD 407 test method, the NOAEL of Everlan SL65 for the rats was 62.5 mg/kg B.W..
Executive summary:

This test used the procedures and OECD 407 (2008). The test article was administered to the three treatment groups by oral gavage in a dose of 62.5, 250 and 1000 mg/kg B.W. for 28 consecutive days. There were six male and six female Sprague-Dawley rat in each group. Clinical observation of the rats was carried out daily and the body weight and feed intake of the rats were recorded once a week. Results of the study indicated that rats in all group gained weight normally and did not show any abnormal clinical signs and ophthalmological examination during the study period. At end of the study, there were no significant abnormalities of the urinalysis parameters between the treatment and control groups. In haematological analysis, clinical biochemistry analysis and organ weight, results indicated that there was enlargement of the spleen with corresponding changes in the hematological profile of blood, probably as a response to the damage to the cecum and colon .

Necropsy and histopathological examination indicated that treatment-related change was found in high or middle dose group. On the basis of the test results given above, the NOAEL of Everlan SL65 for the rats was 62.5 mg/kg B.W..