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Administrative data

Description of key information

Acute toxicity: oral. Key study. Method according to the ‘‘Technical Guideline for Acute Toxicity Test of chemical drugs’’(SFDA, 2004, China), similar to OECD 420, GLP study (no certificate available). The test item has an oral LD50 > 16g/kg for rats.

Acute toxicity: dermal. Key study. Method according to OECD 402, GLP study. The test item has a dermal route LD50 > 2000 mg/kg bw in rats.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
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
Reference:
Composition 1
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 information:
Composition 1
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 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
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.
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.

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

 

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
16 000 mg/kg bw
Quality of whole database:
The study has a Klimisch score of 2.

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 15th to November 29th, 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Remarks:
(SPF Caw)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage JANVIER LABS (53940 Le Genest St Isle – France)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: the males were 7 weeks old and the females 8 weeks old.
- Weight at study initiation: the mean weight of male rats was 273.8 g, and the mean weight of female rats 217.2 g.
- Housing: during the treatment, animals were kept in individual cages. They were solid-bottomed clear polycarbonate cages with a stainless steel mesh lid, containing dust free weed shavings (changed at least 2 times per week).
- Diet (e.g. ad libitum): foodstuff (ENVIGO 2016) ad libitum.
- Water (e.g. ad libitum): tap-water from public distribution system ad libitum.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 15/11/2016 To:29/11/2016.
Type of coverage:
other: non-occlusive
Vehicle:
DMSO
Details on dermal exposure:
TEST SITE
- Area of exposure: at least 10% of the body surface area
- % coverage: at least 10% of the body surface area
- Type of wrap if used: non-occlusive porous gauze dressing (50x50 mm2 non-woven swab, 4-layer patch, MEDISTOCK) secured in position with a strip of surgical adhesive tape (50 mm wides hypoallergenic micropore™ adhesive tape from 3M).

REMOVAL OF TEST SUBSTANCE
- Washing: the gauze dressings were removed and the treated area was rinsed with distilled water.
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mL/kg bw
- Concentration (if solution): 0.2 g/ml
- Constant volume or concentration used: yes
- For solids, paste formed: no

VEHICLE
- Amount(s) applied (volume or weight with unit): 10 mL
Duration of exposure:
24 h
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes
Remarks:
control group referred to historical values.
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: daily observations, the animals were weighed on day 0 (just before administering the test item) then on day 2, day 7, and day 14.
- Necropsy of survivors performed: yes. Observed organs: oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, spleen, liver, thymus, trachea, lungs, heart, kidneys, urinary bladder, testicles, adrenals, pancreas. As no abnormalities were observed, no microscopic examinations were performed.
- Other examinations performed: clinical signs, body weight. Histopathological examination was not performed because no macroscopic anomalies were observed in any organ.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality occurred during the study.
Clinical signs:
No systemic clinical signs related to the administration of the test item were observed. Erythema was noted in all animals (10/10) at 24h post-dose; it was totally reversible at day 3. Dryness of the skin was noted in females at day 2 and in all animals at day 3. The skin recovered a normal aspect on day 4. Yellow coloration was noted in all animals at 24h post-dose.
Body weight:
The body weight evolution of the animals remained normal throughout the study.
Gross pathology:
The macroscopic examinations of the animals at the end of the study did not reveal treatment-related changes.
Interpretation of results:
GHS criteria not met
Remarks:
EU criteria.
Conclusions:
The dermal LD50 of the test item in rats is greater than 2000 mg/kg bw.
Executive summary:

To determine the acute dermal toxicity of the test item in rats, a limit test was performed, according to OECD 402 (GLP study). The test item was applied onto the intact skin of 10 Sprague Dawley rats (5 males, 5 females) at a dose of 2000 mg/kg bw. All animals were observed once daily for 14 days, survival and body weight were monitored. There were no mortality, adverse clinical signs, abnormal changes in body weights or macroscopic findings. Therefore, the test item was found to be non-toxic by dermal route, with an LD50 > 2000 mg/kg bw in rats.

Endpoint conclusion
Endpoint conclusion:
no study available
Dose descriptor:
LD50
2 000 mg/kg bw
Quality of whole database:
The study has a Klimisch score of 1.

Additional information

Acute toxicity: oral. 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, and observed for 14 days. 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. Therefore, with an LD50 > 16g/kg in rats.

Acute toxicity: dermal. To determine the acute dermal toxicity of the test item in rats, a limit test was performed, according to OECD 402 (GLP study). The test item was applied onto the intact skin of 10 Sprague Dawley rats (5 males, 5 females) at a dose of 2000 mg/kg bw. All animals were observed once daily for 14 days, survival and body weight were monitored. There were no mortality, adverse clinical signs, abnormal changes in body weights or macroscopic findings. Therefore, the test item was found to be non-toxic by dermal route, with an LD50 > 2000 mg/kg bw in rats.

Acute toxicity: other routes. Supporting study. The test item was administered intraperitoneally at doses of 2570, 1977, 1521, 1170, and 900 mg/kg test item to 5 ICR mice. The intraperitoneal LD50 of the test item in mice was 1650 mg/kg bw.

Justification for classification or non-classification

Based on available information (oral LD50 > 16 g/kg in rats, dermal LD50 > 2000 mg/kg bw in rats), the substance is not classified for acute toxicity according to CLP Regulation (EC) No. 1272/2008.