Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: ‘‘Technical Guideline for Long Term Toxicity Test of chemical drugs’’ (SFDA, 2005 - PR China)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Animals dosed 6 days/week. Doses tested 50, 250, 1250 mg/kg.

GLP compliance:

yes

Remarks:

Good Laboratory Practice (GLP) Regulations of the State Food and Drug Administration of China.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

7-(2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyloxy)-2,3-dihydro-4',5,7-trihydroxyflavone

EC Number:

233-566-4

EC Name:

7-(2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyloxy)-2,3-dihydro-4',5,7-trihydroxyflavone

Cas Number:

10236-47-2

Molecular formula:

C27H32O14

IUPAC Name:

5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranoside

Test material form:

solid

Specific details on test material used for the study:

Naringin (batch No. 20080203) was extracted and purified in the laboratory. Prepared from pulverized Citrus grandis 'Tormentosa' by the following procedures: extracted with water, precipitated by ethanol, and filtered; and then collected and further concentrated the filtrate; the filtrate, on standing, deposited crystals; the precipitate was separated and recrystallized from mixtures of ethanol and water at different ratios; the recrystallized precipitate was dried at 110 C.
Naringin was obtained, and identification was performed by ultraviolet–visible spectroscopy (UV/Vis), electron spray ionization–mass spectrometry, proton nuclear magnetic resonance (1H NMR) and carbon-13 nuclear magnetic resonance (13C NMR) spectroscopy. The purity analysis was performed on a Shimadzu (HPLC) LC-6A instrument (Shimadzu Corp., Kyoto, Japan) with a Dionex C18 column (5 µm, 4.6 mm 250 mm, USA) and a TL9000 Chromatographic Station. The mobile phase was prepared by a 45/55 (v/v) mixture of methanol/water and the pH was adjusted to 3.0 with acetic acid. The injection volume was 20 ll. The UV detector was set at a wavelength of 283 nm. The HPLC purity of naringin was determined to be >98.3% by external standard method.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Male and female Sprague-Dawley (SD) rats, certified specific pathogen-free, were purchased from Slack Shanghai Laboratory Animal Co., Ltd. (Shanghai, China) under the license number SCXK(HU) 2007-0005.
- Age at study initiation: 6 weeks
- Weight at study initiation: 158.2 – 167.4 g for males and 138.4 – 156.4 g for females.
- Housing: Animals were housed in suspended plastic cages with feed and water available ad libitum. 4 animals per cage.
- Acclimation period: 1 week.
- Age: One hundred and seventy-six six-week-old rats.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25ºC
- Humidity (%): 55+-15%
- Air changes (per hr):
- Photoperiod: 12 hrs dark / 12 hrs light.
- Air change: 10-12 cycles/h.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

10mL/kg bw.

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Naringin was dissolved in sterile water for injection and orally administered at a gavage volume of 10 ml/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The purity analysis was performed on a Shimadzu (HPLC) LC-6A instrument (Shimadzu Corp., Kyoto, Japan) with a Dionex C18 column (5 µm, 4.6 mm 250 mm, USA) and a TL9000 Chromatographic Station. The mobile phase was prepared by a 45/55 (v/v) mixture of methanol/water and the pH was adjusted to 3.0 with acetic acid. The injection volume was 20 ll. The UV detector was set at a wavelength of 283 nm. The HPLC purity of naringin was determined to be >98.3% by external standard method.

Duration of treatment / exposure:

13 weeks.

Frequency of treatment:

6 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 males and 6 females per dose for the 13-weeks study; plus 4 males and 4 females kept for the recovery study, 8 males and 8 females kept for a 6 months study, and 4 males and 4 females kept for the 6-month recovery study.

Control animals:

yes, concurrent vehicle

Details on study design:

3 test groups (44 rats/group, m/f 1:1), subdivided: 12, 8, 16, and 8 animals for 13-week subchronic toxicity, subchronic toxicity recovery, 6-month chronic toxicity and chronic toxicity recovery, respectively.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (first at the time of dose administration and approximately 1–2 h following dose administration)
- Observations included, but were not limited to, changes in skin, fur, eyes, appearance, genital organ, glandular secretion (salivary gland secretions), oral mucosa, faecal characteristics, respiration, circulation and behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week in the first 4 weeks and weekly thereafter.

FOOD EFFICIENCY:
- Mean daily food consumption was calculated once a week by subtracting the weight of the remaining food from the weight of the supplied food. The 24 h feed intake pattern was measured on rats weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: not specified.
- Dose groups that were examined: all.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during euthanasia procedure.
- Anaesthetic used for blood collection: phenobarbital sodium (60 mg/kg bw.)
- Animals fasted: Yes (night fasting)
- How many animals: 12
- Parameters: WBC (white blood cell count and differential), NEU, LYM, MONO, EOS, BASO, RBC (erythrocyte count), HGB (haemoglobin concentration), HCT (haematocrit), MCV (mean corpuscular volume), MCH (mean corpuscular haemoglobin), MCHC (mean corpuscular haemoglobin concentration), RET (reticulocyte count), PLT (platelet count), PT (prothrombin time), APTT (activated partial thromboplastin time).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during euthanasia procedure.
- Animals fasted: No data
- How many animals: 12
- Parameters: ALP (alkaline phosphatase), ALT (serum alanine aminotransferase), AST (serum aspartate aminotransferase), CK (creatine kinase), Urea (urea nitrogen), Crea (creatinine), TP (total serum protein), ALB (albumin), A/G (albumin/globulin ratio), GLU (blood glucose), TBIL (total bilirubin), CHOL (total cholesterol), TG (triglycerides), HDL-c (high-density lipoprotein cholesterol), LDL-c (low density lipoprotein-cholesterol), K+ (potassium), Na+ (sodium), Cl- (chloride).

Sacrifice and pathology:

GROSS PATHOLOGY: Necropsies included examination of the visceral organs, external surface, all orifices, as well as the cranial, thoracic, abdominal and pelvic cavities including viscera. Gross lesions were examined from all animals in all groups. The vital organs from each rat, such as brain, pituitary, heart, liver, spleen, lungs, kidneys, adrenal glands, thymus, testis, epididymides, bladder, ovaries and uterus, were removed and weighed. Paired organs were weighed together. Organ-to-final-body-weight and organ-to-brain-weight ratios were calculated.

HISTOPATHOLOGY: At the time of necropsy, the following tissues and organs were collected: brain, spinal cord (cervical, thoracic and lumbar), gastrointestinal tract (oesophagus, submaxillary gland, stomach, duodenum, jejunum, ileum, cecum, colon and rectum), pancreas, heart, liver, spleen, lungs, kidneys, ureter, prostate, seminal vesicle, epididymis, testis, ovaries, uterus, mammary gland, sciatic nerve, bladder, pituitary gland, adrenal glands, thyroids, parathyroid glands, thymus, trachea, aorta, bone marrow, lymph node (mesenteric lymphoid node, submandibular lymph node). Epididymis and testis were fixed in modified Davidson’s fluid, while others in 10% neutral buffered formalin. After fixation, the collected tissue samples were processed into paraffin blocks. The labelled paraffin blocks were sectioned at 4–8 µm and the paraffin ribbons of the sectioned tissue were placed on clean glass microscope slides. Upon completion of staining with hematoxylin and eosin, microscopic examinations were first performed on all tissues from all animals in the control and 1250 mg/kg naringin treatment groups euthanized at the scheduled necropsy. If treatment-related changes were noted in a particular organ or tissue in 1250 mg/kg naringin treatment group, extended examination was conducted on the corresponding organs or tissues from lower dose treated groups.

Statistics:

One-Way ANOVA using SPSS 13.0 statistical software. When statistically significant differences were indicated, the least significant difference (LSD) test was employed for comparisons between groups. Levene’s test was used to assess the homogeneity of variances in data. If the variance was not homogeneous, the Kruskal–Wallis test was applied. When statistically significant differences were indicated, the Mann–Whitney U test was employed for inter-group comparison. Statistically significant differences between groups were defined as p < 0.05.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No mortality or abnormal clinical signs related to the administration of naringin were observed.

Mortality:

no mortality observed

Description (incidence):

No mortality or abnormal clinical signs related to the administration of naringin were observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights in female and male rats at high dose were less than control values after weeks 6 and 11. Compared with control group, the decrease in mean body weight in females were significant (p < 0.05) at weeks 7, 8, 12, 13. The ability of the test item to regulate fatty acid, cholesterol and glucose metabolism may be considered as a possible explanation of these alterations. The body weight loss was not associated with other clinical signs, and no related indication of pathological abnormality was observed.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Any changes observed were neither continuous nor dose-related.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Any changes observed were neither continuous nor dose-related.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmological abnormalities were observed in any of naringin treatment or control animals over the course of the study.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Lymphocytes(%) increased at high dose, other parameters unchanged. The alterations in hematology and clinical biochemistry analyses were assumed to be toxicologically irrelevant because they were within normal physiological ranges and were not dose-related or reflected by any changes in other related parameters.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Level of urea decreased at high dose, TBIL levels of all groups were significantly decreased; the other parameters remained unchanged. The alterations in hematology and clinical biochemistry analyses were assumed to be toxicologically irrelevant because they were within normal physiological ranges and were not dose-related or reflected by any changes in other related parameters.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional behavioral results of the naringin treatment groups of male and female rats were considered comparable to control groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute heart and lung weights were significantly decreased in females at high dose, ovary weight and ovary-to-bw ratio significantly increased in female at 250 mg/kg. Heart-to-brain and lung-to-brain weight ratios in the female 1250 mg/kg decreased. However, these changes were not sex or dose-related, were within the normal laboratory ranges, and/or were not supported by any other consistent or toxicologically significant changes in hematological and blood biochemistry analyses and histopathological examination. Therefore, these differences were considered incidental in nature without toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Dirty red spots of 0.1–0.2 cm in diameter scattered irregularly on the surface of liver lobes and isabeling particles of 0.2 cm in diameter were observed at the juncture of left and right middle lobes in one male rat (250mg/kg). No similar signs were observed in any other animal.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

see table 1. Various lesions (minimal severity) for the high dose group. In the histopathological examination, the lesions in the lungs, liver, spleen, kidneys, duodenum, heart and stomach were noted. However, all of those pathological changes were sporadically detected in controls and the rats administrated with 1250 mg/kg of naringin, and no consistent histopathological changes were observed in either sex. Therefore, these lesions could be considered to be spontaneous and/or incidental in nature but not relevant to the treatment

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

No other pathological lesions were found in brain, spinal cord (cervical, thoracic and lumbar cords), gastrointestinal tract (oesophagus, submaxillary gland, jejunum, ileum, cecum, colon and rectum), pancreas, ureter, prostate, seminal vesicle, epididymis, testis (males only), ovary and uterus (females only), mammary gland, sciatic nerve, bladder, pituitary gland, adrenal glands, thyroids, parathyroid glands, thymus, trachea, aorta, bone marrow and lymph node (mesenteric lymphoid node and submandibular lymph node). These results indicated that there were no pathological changes in the organs or tissues that could be attributed to naringin treatment.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effects observed

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1. Histopathological findings in rats treated orally with test item for 13 weeks.

Number of animals with lesion		Control		Test item (1250 mg/kg)
Organs	Lesions	Female (n = 6)	Male (n = 6)	Female (n = 6)	Male (n = 6)
Lungs	Haemorrhage	0	0	1	0
	Interstitial chronic inflammatory infiltrate	1	1	1	1
	Interstitial inflammation around thickened wall blood vessels	0	1	0	0
Liver	Haemorrhage	0	0	0	1
	Vacuolar degeneration of liver cells	2	5	1	0
	Wegener	1	1	1	2
Spleen	Focal necrosis accompanied with haemorrhage	0	1	0	0
Kidneys	Chronic progressive nephropathy	2	5	0	2
	Renal tubular and/or interstitial calcification	1	0	0	0
	Renal tubular cystic disease	2	2	1	0
Duodenum	Focal chronic inflammatory cell infiltration of intestinal villus	0	0	0	1
Stomach	Cystic dilatation of gastric glands	1	0	0	0
	Focal haemorrhage of gastric mucosa	0	1	0	0
Heart	Focal myocardial degradation and necrosis accompanied with or without fibrous tissue hyperplasia and inflammatory cell infiltration	1	1	0	3

 

 

Table 2. Absolute and relative weights of organs of female rats treated with test item for 13 weeks.

 

Parameters	Dose (mg/kg/day)
	0		50		250		1250
Body weight (g)	315.5±30.0		307.4±21.1		286.3±38.2		271.6±9.9*
Brain	1.92±0.12		1.98±0.03		1.98±0.08		1.89±0.11
Heart	1.01±0.11		1.09±0.08		0.92±0.09		0.87±0.11*
Liver	8.21±0.42		7.88±0.55		7.81±1.09		7.50±0.46
Spleen	0.65±0.09		0.65±0.04		0.66±0.16		0.61±0.09
Lungs	1.42±0.19		1.38±0.07		1.34±0.10		1.18±0.04*^a
Thymus	0.27±0.07		0.24±0.09		0.24±0.06		0.23±0.07
Kidneys	1.93±0.19		1.94±0.17		1.92±0.34		1.74±0.15
Adrenals	0.064±0.006		0.058±0.007		0.068±0.015		0.055±0.006
Ovaries	0.083±0.012		0.109±0.033		0.115±0.018*		0.087±0.018
Uterus	0.74±0.18		0.65±0.27		0.85±0.20		0.67±0.29
Pituitary	0.015±0.004		0.015±0.002		0.014±0.003		0.015±0.003
Organ-to-body weight ratio (%)
Brain	0.61±0.06		0.64±0.03		0.70±0.07*		0.70±0.05*
Heart	0.32±0.02		0.36±0.04		0.32±0.03		0.32±0.03
Liver	2.62±0.23		2.56±0.09		2.73±0.13		2.76±0.13
Spleen	0.21±0.02		0.21±0.03		0.23±0.05		0.22±0.03
Lungs	0.45±0.05		0.45±0.02		0.47±0.03		0.44±0.02
Thymus	0.08±0.02		0.08±0.03		0.09±0.03		0.09±0.02
Kidneys	0.61±0.04		0.63±0.03		0.67±0.04		0.64±0.04
Adrenals	0.021±0.003		0.019±0.003		0.024±0.005		0.020±0.003
Ovaries	0.026±0.002		0.036±0.012		0.041±0.010*		0.032±0.007
Uterus	0.24±0.07		0.21±0.10		0.30±0.09		0.24±0.10
Pituitary	0.0047±0.0016		0.0047±0.0008		0.0050±0.0004		0.0054±0.0009
Organ-to-brain weight ratio (g/g)
Heart	0.53±0.07		0.55±0.04		0.47±0.04		0.46±0.05*
Liver	4.29±0.16		3.99±0.23		3.94±0.48		3.97±0.31
Spleen	0.34±0.03		0.33±0.03		0.33±0.08		0.32±0.03
Lungs	0.74±0.07		0.70±0.03		0.68±0.04*		0.63±0.02*
Thymus	0.14±0.04		0.12±0.04		0.12±0.03		0.12±0.04
Kidneys	1.01±0.06		0.98±0.07		0.97±0.15		0.92±0.08
Adrenals	0.034±0.004		0.029±0.004		0.035±0.008		0.029±0.003
Ovaries	0.043±0.006		0.055±0.017		0.059±0.011		0.046±0.010
Uterus	0.39±0.09		0.33±0.14		0.43±0.12		0.35±0.15
Pituitary	0.0077±0.0021		0.0073±0.0012		0.0072±0.0011		0.0078±0.0014

Values are mean ± SD for 6 rats in each group.
^a Data from 5 rats.
^* Statistically significant compared to control (p < 0.05).

 

 

Table 3. Haematological values of rats treated orally with test item for 13 weeks.

 

Parameters	Dosage (mg/kg /day)
	0	50	250	1250
WBC (10^9/L)	4.21 ± 1.96	3.17 ± 2.05	3.26 ± 1.87	3.70 ± 1.95
NEU (10^9/L)	1.075 ± 1.148	0.453 ± 0.291	0.450 ± 0.256	0.491 ± 0.334
LYM (10^9/L)	2.86 ± 1.58	2.53 ± 1.69	2.65 ± 1.61	3.05 ± 1.61
MONO (10^9/L)	0.173 ± 0.177	0.109 ± 0.073	0.086 ± 0.034	0.080 ± 0.072
EOS (10^9/L)	0.087 ± 0.033	0.060 ± 0.031	0.062 ± 0.032	0.071 ± 0.034
BASO (10^9/L)	0.014 ± 0.010	0.012 ± 0.008	0.009 ± 0.006	0.011 ± 0.010
NEU% (%)	24.1 ± 15.2	14.6 ± 4.5	14.4 ± 4.1	12.9 ± 4.5
LYM% (%)	69.5 ± 16.9	79.4 ± 4.6	79.8 ± 5.4	82.7 ± 4.8^*
MONO% (%)	3.81 ± 2.60	3.49 ± 0.75	3.32 ± 1.77	2.05 ± 1.20
EOS% (%)	2.22 ± 0.68	2.11 ± 0.73	2.20 ± 0.86	2.04 ± 0.53
BASO% (%)	0.356 ± 0.260	0.410 ± 0.243	0.284 ± 0.194	0.288 ± 0.216
RBC (10^12/L)	8.08 ± 0.60	7.83 ± 0.48	8.04 ± 0.51	7.98 ± 0.40
HGB (g/L)	145 ± 8	145 ± 6	145 ± 6	146 ± 6
HCT (%)	43.2 ± 2.5	43.0 ± 1.6	43.3 ± 1.9	43.5 ± 1.5
MCV (fL)	53.6 ± 1.7	55.0 ± 1.9	54.0 ± 2.1	54.5 ± 1.6
MCH (pg)	18.0 ± 0.7	18.5 ± 0.6	18.0 ± 0.7	18.3 ± 0.6
MCHC (g/L)	336 ± 4	336 ± 3	335 ± 3	336 ± 3
RET (%)	2.01 ± 0.39	2.03 ± 0.36	2.09 ± 0.24	1.84 ± 0.26
PLT (10^9/L)	1035 ± 200	940 ± 127	1010 ± 99	960 ± 90
PT (s)	7.2 ± 0.9	7.0 ± 0.8	7.2 ± 0.9	7.5 ± 1.0
APTT (s)	15.6 ± 2.4	15.6 ± 1.9	16.0 ± 1.2	16.0 ± 2.1

Values are mean ± SD for 12 rats in each group except 11 for the control group. Blood sample from one rat in 1250 mg/kg naringin treatment group coagulated. The hematological data were not determined.
^* Statistically significant compared to control group (p < 0.05).

 

Table 4. Serum biochemistry of rats treated orally with test item for 13 weeks

 

Parameters	Dosage (mg/kg /day)
	0	50	250	1250
ALP (U/L)	56.4 ± 22.7	52.9 ± 20.8	58.9 ± 20.9	58.8 ± 16.4
ALT (U/L)	29.5 ± 6.8	31.0 ± 9.6	31.1 ± 10.3	26.1 ± 7.1
AST (U/L)	99.2 ± 27.2	97.2 ± 23.9	97.1 ± 17.7	90.6 ± 27.8
CK (U/L)	313 ± 134	324 ± 144	305 ± 93	333 ± 179
LDH (U/L)	958 ± 525	871 ± 486	927 ± 321	918 ± 584
Urea (mmol/L)	7.54 ± 1.42	7.57 ± 1.18	6.66 ± 1.30	5.77 ± 0.80*
Crea (µmol/L)	58.2 ± 13.6	53.1 ± 9.5	51.4 ± 8.1	47.4 ± 8.2
TP (g/L)	67.2 ± 5.1	66.5 ± 5.3	68.2 ± 5.9	65.5 ± 2.1
A/G	42.9 ± 6.9	43.8 ± 5.2	44.1 ± 5.5	43.7 ± 3.1
ALB (g/L)	1.83 ± 0.42	1.95 ± 0.30	1.83 ± 0.28	2.02 ± 0.32
GLU(mmol/L)	6.28 ± 1.03	6.41 ± 0.69	6.48 ± 0.88	5.83 ± 0.69
TBIL (µmol/L)	3.2 ± 0.7	2.4 ± 0.6*	2.5 ± 0.7*	2.2 ± 0.6*
CHOL (mmol/L)	1.57 ± 0.47	1.40 ± 0.26	1.47 ± 0.45	1.52 ± 0.38
TG (mmol/L)	0.94 ± 0.33	0.86 ± 0.49	1.10 ± 0.53	0.82 ± 0.34
HDL-c (mmol/L)	1.48 ± 0.42	1.38 ± 0.22	1.37 ± 0.37	1.49 ± 0.35
LDL-c (mmol/L)	0.12 ± 0.06	0.09 ± 0.04	0.10 ± 0.05	0.14 ± 0.09
K^+(mmol/L)	3.99 ± 0.37	3.91 ± 0.21	3.93 ± 0.25	3.75 ± 0.33
Na^+(mmol/L)	142.1 ± 1.2	142.0 ± 1.6	142.8 ± 1.3	143.0 ± 1.5
Cl^- (mmol/L)	104.0 ± 1.8	103.7 ± 0.8	104.5 ± 1.2	103.3 ± 0.9

Values are mean ± SD for 12 rats in each group.
^* Statistically significant compared to control (p < 0.05).

Applicant's summary and conclusion

Conclusions:

The test item has a NOAEL > 1250 mg/kg bw/d in rats, after 90-day oral administration.

Executive summary:

The 90d repeated oral dose toxicity was studied on Sprague-Dawley rats according to the ‘‘Technical Guideline for Acute Toxicity Test of chemical drugs’’(SFDA, 2004, China), similar to OECD 408 (GLP study, certificate not available). The test item was orally administered to 22 male and 22 female Sprague-Dawley rats, at doses of 0 (control), 50, 250 or 1250 mg/kg bw/d. All animals were thoroughly observed immediately after administration for the onset of any toxic signs and once daily thereafter. Survival, feed intake, and body weight were monitored. During the subchronic oral toxicity study, no mortality and toxicologically significant changes in clinical signs, food consumption, opthalmoscopic examination, hematology, clinical biochemistry, serum sex hormone, macroscopic findings, organ weights and histopathological examination except for slight body weight decrease were noted and attributed to naringin administration. Under test conditions, the test item was found to have a NOAEL of 1250 mg/kg in rats.