Sodium [4-[[dihydroxy[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]benzenesulphonato(3-)]cuprate(1-)

Repeated dose toxicity: oral

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

Test material information

Constituent 1

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:

Reaction products of diazotized 4-aminobenzenesulfonic acid coupled with resorcinol subsequently coupled with diazotised 2-amino-4,6-dinitrophenol, copper (2+) complexes, sodium salts

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 / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Medium dose

Doses / concentrations 3

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

1

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

2

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..