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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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.

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.

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

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

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.

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.

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.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 250 mg/kg bw/day (nominal)

Based on:

dissolved

Sex:

male/female

Basis for effect level:

other: No toxicologically significant effects observed at the highest dose level.

Key result

Critical effects observed:

no

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

Number of animals with lesion		Control		Naringin (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

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.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2014

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:

6-month study, animals dosed 6 days/week.

GLP compliance:

yes

Remarks:

No certificate included in publication.

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:

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

Test material form:

solid

Specific details on test material used for the study:

Naringin was extracted from Citrus grandis ‘Tomentosa’ by water and purified by recrystallization as described previously (Li et al., 2013c). The HPLC purity of naringin was determined to be >98.3% by external standard method.
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:

TEST ANIMALS
- Source: male and female SD rats, certified specific pathogen-free, were purchased in good health from Slack Shanghai Laboratory Animal Co., Ltd.(Shanghai, China) under the license number SCXK (HU) 2007-0005.
- Age at study initiation: 4 to 6-week-old rats.
- Weight at study initiation: At initial of dosing, the animal weights ranged from 109.6 to 142.2 grams (mean, 127.5 ± 6.9) for males and 116.3 to 158.9 grams (mean, 140.2 ± 9.9) for females.
- Fasting period before study: overnight
- Housing: Animals were housed in labelled polypropylene cages (four rats per cage) with feed and water available ad libitum.
- Acclimation period: 1 week.

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:

Since naringin is a flavonoid with slight solubility (1.1 g/L) in water (Lauro et al., 2007), suspension of naringin in sterile water was made up fresh each day just before it was administrated to the animals by using oral gavage at a dosing volume of 10 ml/kg body weight. During administration, suspension of naringin was always shaken well with suspended magnetic stir, thereby ensuring its homogeneity and stability.

PREPARATION OF DOSING SOLUTIONS: suspension of naringin in sterile water was made up fresh each day just before it was administrated to the animals.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Amount of vehicle (if gavage): 10 ml/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The HPLC purity of naringin was determined to be >98.3% by external standard method. Dosing solutions were prepared fresh every day.

Duration of treatment / exposure:

6 months.

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:

8 males and 8 females for the 6-month study, plus 4 males and 4 females for the recovery period (1 month).

Control animals:

yes

Details on study design:

One hundred and seventy-six rats were randomly assigned to control and three test groups, each consisting of 22 males and 22 females. 8 animals were assigned to the recovery subgroup; recovery period was designated as one month. Control animals received sterile water alone.
- Rationale for selecting satellite groups: one group was kept to observe the recovery after administration.
- Post-exposure recovery period in satellite groups: 1 month

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily (morning and afternoon; first at the time of dose administration and approximately 1–2 h following dose administration) for signs of clinical toxicity and mortality.
- 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
- Body weight was recorded twice per week in the first 4 weeks after dosing and then once a week thereafter.

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

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). PT and APTT were measured using blood plasma, while other haematology parameters used EDTA-anticoagulated whole blood. Serum from non-anticoagulated whole blood collected in separator tubes was analysed for changes in biochemistry parameters.

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). Serum sex hormone levels, including double hydrogen testosterone (DHT, a male sex hormone) and estradiol (E2, the primary ovarian estrogen), were determined by radioimmune analysis using commercially available kits obtained from Diagnostics Systems Laboratories, Inc., Corporate Headquarters, USA and Union Medical & Pharmaceutical Technology Co Ltd., Tianjin, China, respectively.

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. In addition, all tissues from rats dying or killed during the study at unscheduled times and all tissues showing macroscopic abnormalities were also examined by histopathologic examination.

Statistics:

The values are expressed as the mean ± SD. Body weight, organ weight (both absolute and relative weights), food consumption, hematology, serum biochemistry and serum sex hormone analyses were tested by conducting One-Way Analysis of Variance (ANOVA) using SPSS 13.0 statistical software. If 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 clinical signs related to the administration of naringin were observed. From the second month to the sixth month after administration, hair loss was noted on some skin areas including the necks in three female rats from control group, the necks in one female and one male rats from 50 mg/kg group, the necks, ears and/or around the eyes in four female and one male rats from 250 mg/kg of naringin group, the backs, necks, chests, abdomens and femoral regions in three female and two male rats from 1250 mg/kg group. Histopathological examination showed that no any histological changes occurred. Microorganism examination showed that no any pathogenic microorganisms were found on these skin areas. After cessation of naringin administration, hair loss gradually returned to normal and completely disappeared at the end of recovery period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality and clinical signs related to the administration of naringin were observed except that one female rat in 50 mg/kg group and one female rat in 1250 mg/kg group died at days 76 and 24 after dosing, respectively, due to improper intragastric administration on basis of macroscopic and histopathologic examination.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Statistical analysis of mean weekly bw showed consecutive and/or isolated periods of significant decrease in some treatment groups (p < 0.05). However, mean weekly body weights of female rats in 50 mg/kg group on weeks 28–30 were significantly increased (p < 0.05) compared with the corresponding control values.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At weeks 5, 13, 15–16, 21–22, 24, food consumption in 1250 mg/kg group females was less than the corresponding control values (p < 0.05), while more than the control value at week 18.

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 identified in the eyes of any study animals over the course of the study.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few significant changes were noted higher HGB level in 50 mg/kg female group, lower NEU percentage in 1250 mg/kg male group, higher RBC, HGB and HCT levels in 250 and 1250 mg/kg male groups at the end of 6-month treatment period.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

After 6 months, the levels of Crea, TP, ALB and TBIL of female rats and the levels of Urea, GLU and Cl of male rats from 1250 mg/kg group were significantly decreased compared with corresponding control values (p < 0.05).

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Liver and spleen-to-body weight ratios were significantly elevated in 250 mg/kg female group (p < 0.05) after 6 months.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of recovery period, some effects were noted in individuals from various dose groups. All of those pathological changes were sporadically detected in control and naringin treatment groups. There existed no pathological lesions in any other tissues. These results indicated that there were no pathological changes in the organs or tissues that could be attributed to naringin treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Various lesions, both after 6 months treatment and after recovery period, in minimal or slight severity; but also found in control group. There were no pathological changes in the organs or tissues that could be attributed to naringin treatment.
After 6 months, the lesions mainly included accumulation of foam cells in pulmonary alveoli and local pneumopleuritis, vacuolar degeneration and focal necrosis of liver cells, chronic progressive nephropathy, renal tubular and/or interstitial calcification, focal myocardial degeneration necrosis and inflammatory cell infiltration in heart. At the end of recovery period, the lesions mainly included accumulation of foam cells in pulmonary alveoli, chronic progressive, focal myocardial degeneration necrosis and inflammatory cell infiltration, and cystic dilatation of gastric glands. The severities of all of these lesions are minimal and were sporadically detected both in control and naringin treatment groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

In present toxicity study, no naringin-related deaths or abnormalities in clinical signs were noted except that reversible and non-pathological hair loss was identified.
In conclusion, daily doses of 50, 250, and 1250 mg/kg of naringin for six months were well tolerated and did not cause either lethality or toxic clinical symptoms and changes in both sexes of rats except for slight body weight decrease and non-pathological and reversible hair loss, which returned to normal after 4 weeks recovery. The NOAEL of naringin is proposed to be greater than 1250 mg/kg/day following daily oral administrations to SD rats for six months. Using the body surface area normalization method, a dose of 1250 mg/kg naringin in rat corresponds to 200 mg/kg in humans, or 12 g for a 60 kg human.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 250 mg/kg bw/day (nominal)

Based on:

dissolved

Sex:

male/female

Basis for effect level:

other: No toxicologically significant effects observed at the highest dose tested.

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1. Haematological values of rats treated orally with test item (0, 50, 250, 1250 mg/kg bw) for 6 months.

		Administration period								Recovery period
		0 mg/kg		50mg/kg		250mg/kg		1250mg/kg		0 mg/kg		50mg/kg		250mg/kg		1250mg/kg
Female		(n = 8)		(n = 8)		(n = 8)		(n = 8)		(n = 3)		(n = 4)		(n = 3)		(n = 4)
WBC	(10^9/L)	2.40	± 0.82	2.88	± 0.84	1.89	± 0.65	2.59	± 0.61	2.47	± 0.55	1.93	± 0.63	2.45	± 0.20	2.90	± 1.67
NEU	(10^9/L)	0.468	± 0.236	0.486	± 0.210	0.350	± 0.140	0.427	± 0.261	0.437	± 0.292	0.414	± 0.167	0.323	± 0.045	1.202	± 1.047
LYM	(10^9/L)	1.82	± 0.75	2.24	± 0.62	1.42	± 0.58	2.08	± 0.72	1.90	± 0.27	1.41	± 0.44	1.96	± 0.14	1.46	± 0.67
MONO	(10^9/L)	0.050	± 0.044	0.082	± 0.035	0.065	± 0.048	0.026	± 0.023	0.055	± 0.021	0.053	± 0.043	0.098	± 0.017	0.146	± 0.092
EOS	(10^9/L)	0.055	± 0.009	0.068	± 0.020	0.047	± 0.015	0.058	± 0.023	0.072	± 0.010	0.045	± 0.020	0.058	± 0.005	0.072	± 0.014
BASO	(10^9/L)	0.003	± 0.003	0.005	± 0.004	0.003	± 0.003	0.003	± 0.006	0.007	± 0.002	0.009	± 0.012	0.012	± 0.007	0.017	± 0.013
NEU	(%)	20.0	± 10.6	16.4	± 3.7	19.1	± 7.8	18.2	± 15.5	16.6	± 7.4	21.2	± 5.2	13.1	± 1.1	35.2	± 17.8
LYM	(%)	75.0	± 11.6	78.0	± 2.9	74.6	10.5	78.5	± 15.5	77.8	± 5.9	73.5	± 3.4	8± 0.0	± 1.0	55.2	± 17.8*
MONO	(%)	2.43	± 2.37	2.94	± 1.36	3.61	± 2.76	0.89	± 0.59	2.39	± 1.31	2.7	± 1.91	3.96	± 0.40	5.77	± 4.94
EOS	(%)	2.45	± 0.71	2.40	± 0.40	2.57	± 0.65	2.31	± 1.01	2.95	± 0.46	2.27	± 0.62	2.39	± 0.24	3.09	± 1.58
BASO	(%)	0.142	± 0.152	0.169	± 0.125	0.167	± 0.182	0.107	± 0.171	0.266	± 0.050	0.422	± 0.416	0.501	± 0.253	0.810	± 1.013
RBC	(10^12/L)	7.33	± 0.29	7.57	± 0.25	7.33	± 0.38	7.57	± 0.24	7.62	± 0.39	7.20	± 0.55	7.42	± 0.24	7.35	± 0.21
HGB	(g/L)	139	± 4	145	± 5*	141	±6	143	± 4	145	± 6	137	± 8	144	± 3	139	± 3
HCT	(%)	41.9	± 1.3	43.3	± 1.4	42.6	1.8	43.1	±1.3	43.5	± 1.8	4± 0.8	± 2.4	43.1	± 0.4	41.8	± 1.1
MCV	(fL)	57.2	± 0.8	57.3	± 1.6	58.3	1.9	57.0	± 1.1	57.1	± 3.3	56.8	± 1.2	58.1	± 2.3	56.9	± 1.7
MCH	(pg)	19.0	± 0.3	19.1	± 0.5	19.3	± 0.6	18.9	± 0.5	19.0	± 1.1	19.0	± 0.4	19.4	± 1.0	18.9	± 0.6
MCHC	(g/L)	332	± 2	334	± 2	331	± 5	332	± 4	333	± 1	335	± 0	334	± 4	333	± 1
RET	(%)	2.03	± 0.39	2.12	± 0.24	1.98	± 0.32	1.97	± 0.23	2.47	± 0.26	2.03	± 0.32	2.88	± 0.44	2.33	± 0.67
PLT	(10^9/L)	871	78	859	± 60	849	± 65	828	± 129	897	± 89	818	± 37	891	± 73	858	± 106
PT	(s)	7.0	± 0.4	7.1	± 0.3	7.0	± 0.5	7.2	± 0.5	7.8	± 0.6	7.8	± 0.9	7.8	± 0.3	7.5	± 0.2
APTT	(s)	16.4	± 0.8	17.3	± 2.6	17.0	± 1.3	17.0	± 1.6	16.0	± 1.0	15.3	± 3.2	16.0	± 2.2	14.6	± 1.1

Applicant's summary and conclusion

Conclusions:

The test item has a NOAEL > 1250 mg/kg bw/d in rats, after 6 months oral administration.

Executive summary:

The subchronic toxicity of naringin was studied on rats for 6 months, by a method according to "Technical Guideline for Long Term Toxicity Test of chemical drugs" (SFDA, 2005), similar to OECD TG 408, with GLP (no certificate available). 8 male and 8 female Sprague Dawley rats were administered the test substance 6 days a week by gavage at doses of 0 (control), 50, 250, or 1250 mg/kg for the 6 -months study, plus 4 males and 4 females that were kept for a 1 -month recovery period (same treatment). During the 6-month treatment period and one month recovery period, no mortality and toxicologically significant changes in clinical signs, opthalmoscopic examination, hematology, clinical biochemistry, serum sex hormone, macroscopic findings, organ weights and histopathological examination were noted and attributed to naringin administration. Although consecutive and/or isolated periods of significant body weights and food consumption decreases were relevant to naringin administration, they were not considered toxicologically significant. In addition, slight, non-pathological and reversible hair loss was noted during the 6-month treatment period and considered as a kind of change possibly relevant to naringin administration; however, it was not considered adverse change and to be of toxicological significance. Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) of naringin in rats is greater than 1250 mg/kg/day when administered orally for 6 consecutive months.