Registration Dossier

Administrative data

Endpoint:
acute 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

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: ‘‘Technical Guideline for Acute Toxicity Test of chemical drugs’’ (SFDA, 2004 - PR China)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
GLP compliance:
yes
Remarks:
All operations were carried out under the Good Laboratory Practice (GLP) Regulations of the State Food and Drug Administration of China.
Test type:
fixed dose procedure
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 and environmental conditions:
TEST ANIMALS
- 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.
- Weight at study initiation: 158.2 – 167.4 g for males and 138.4 – 156.4 g for females.
- Fasting period before study: overnight.
- Housing: three rats of the same gender from the same group were held in the same plastic cage.
- Diet ad libitum.
- Water ad libitum.
- Acclimation period: 1 week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25
- Humidity (%): 55 ± 15
- Air changes (per hr): 12
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg bw.
Doses:
16 g/kg bw
No. of animals per sex per dose:
6
Control animals:
yes
Remarks:
intragastrical administration of sterile saline (10mL/kg).
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were thoroughly observed after test article administration immediately for the onset of any toxic signs and once daily thereafter for 14 days of observation period. Survival, feed intake (days 2 and 8), and body weight (days 0, 3, 7, 10, and 13) were monitored.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, other: hematology, biochemistry.
- Hematology: erythrocyte count (RBC), haemoglobin concentration (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocyte count (RET), white blood cell count (WBC) and differential, platelet count (PLT), prothrombin time (PT) and activated partial thromboplastin time (APTT)
- Clinical biochemistry: biochemical indexes includes serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), creatine kinase (CK), alkaline phosphatase (ALP), urea nitrogen (UREA), total serum protein (TP), albumin (ALB), albumin/globulin ratio (A/G), blood glucose (GLU), total bilirubin (TBIL), creatinine (Crea), total cholesterol (CHOL), triglycerides (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), sodium (Na), potassium (K), and chloride (Cl).
Statistics:
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 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.

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 16 000 mg/kg bw
Mortality:
No death was recorded in any of the groups during 14 days of the study.
Clinical signs:
No clinical signs related to the administration of naringin at 16 g/kg dose were observed.
Body weight:
Normal body weight gains and food intakes were observed in male and female of both groups.
Gross pathology:
The macroscopic examination of the organs revealed no changes in the necropsies of any of the animals.
Other findings:
- The hematology assessments, including RBC, HGB, HCT, MCV, MCH, MCHC, RET, WBC, white blood cell differential count, PLT and PT for rats treated with naringin, were not remarkably different compared to control rats. APTT of naringin treatment group is significantly longer than that of control group (p < 0.05), but stayed within the normal reference range.
- The biochemical analyses indicated that no significant differences between the control and naringin treated groups were detected for any of the parameters.

Any other information on results incl. tables

Table 1. Hematology data for SD rats given a single oral dose (16 g/kg) of naringin.

Parameters

Control group

Treatment group

WBC

(109/L)

3.616

±

1.230

4.181

±

1.061

NEU

(%)

10.538

±

3.906

8.356

±

2.685

LYM

(%)

85.15

±

4.81

86.18

±

2.96

MONO

(%)

2.384

±

1.200

3.188

±

0.829

EOS

(%)

1.500

±

0.589

1.756

±

0.715

BASO

(%)

0.4345

±

0.3763

0.5299

±

0.2767

RBC

(1012/L)

6.342

±

0.285

6.446

±

0.318

HGB

(g/L)

134.6

±

6.2

137.6

±

3.5

HCT

(%)

39.80

±

1.64

40.73

±

0.96

MCV

(fL)

62.79

±

1.55

63.29

±

2.31

MCH

(pg)

21.23

±

0.59

21.37

±

0.80

MCHC

(g/L)

338.0

±

2.7

337.4

±

4.7

RET

(%)

3.250

±

1.023

3.403

±

1.192

PLT

(109/L)

1014.8

±

114.1

1078.7

±

62.0

PT

(s)

7.75

±

0.75

7.56

±

0.51

APTT

(s)

14.58

±

1.04

15.72

±

0.81*

 

Table 2. Serum biochemical data for rats given a single oral dose (16 g/kg) of naringin.

Parameters

Control group

Treatment group

ALP

(U/L)

205.10

±

59.71

182.04

±

45.42

ALT

(U/L)

32.36

±

3.80

31.53

±

5.01

AST

(U/L)

111.14

±

16.23

108.61

±

18.58

CK

(U/L)

448.0

±

117.2

406.8

±

131.4

Urea

(mmol/L)

6.469

±

1132

6.033

±

1565

Crea

(lmol/L)

20.33

±

2.31

18.27

±

3.59

TP

(g/L)

52.33

±

2.10

53.17

±

1.47

ALB

(g/L)

38.57

±

1.76

39.41

±

1.48

A/G

 

2.813

±

0.171

2.880

±   0.26

GLU

(mmol/L)

6453

±

0.953

6441

±

0.640

TBIL

(µmol/L)

1.67

±

0.61

1.94

±

0.68

CHOL

(mmol/L)

1323

±

0.367

1316

±

0.316

TG

(mmol/L)

0.340

±

0.182

0.438

±

0.216

HDL-c

(mmol/L)

0.403

±

0.089

0.409

±

0.065

LDL-c

(mmol/L)

0.235

±

0.098

0.219

±

0.107

K+

(mmol/L)

3873

±

0.302

3880

±

0.198

Na+

(mmol/L)

144.59

±

1.06

144.60

±

2.04

Cl

(mmol/L)

105.72

±

1.67

105.03

±

1.44

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
EU criteria.
Conclusions:
The test item has an LD50 greater than 16g/kg in rats.
Executive summary:

To determine the acute oral toxicity of the test item in rats, a limit test was performed, according to the ‘‘Technical Guideline for Acute Toxicity Test of chemical drugs’’(SFDA, 2004, China), similar to OECD 420 (GLP study). The test item was orally administered to 6 male and 6 female Sprague-Dawley rats, at a single bolus dose of 16 g/kg bw, by gavage. All animals were thoroughly observed immediately after administration for the onset of any toxic signs and once daily thereafter for 14 days. Survival, feed intake, and body weight were monitored. There were no mortality, adverse clinical signs, abnormal changes in body weights or food consumption, toxicologically relevant changes in hematology, clinical biochemistry and macroscopic findings during 14 days of the acute toxicity study. Under test conditions, the test item was found to be non-toxic by oral route, with an LD50 > 16g/kg in rats.