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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented publication.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: silver nanoparticles (60nm), not further characterised; source: Namatech (Korea)
Details on test material:
- Name of test material (as cited in study report): silver nanoparticles
- Molecular formula (if other than submission substance): Ag
- Molecular weight (if other than submission substance): 107.87
- Physical state: solid
- Analytical purity: at least 99.98 % pure
- Other: silver nano particles purchased form NAMATECH Co., Ltd. (Korea)
- Count median diameter and geometric standard deviation in 0.5% aqueous carboxymethylcellulose analysed by transmission electron microscopy were 56 nm and 1.46, respectively.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: specific-pathogen free (SPF) Fisher 344 rats were purchased from Japan SLC Inc. (Japan)
- Age at study initiation: 4 weeks
- Housing: during the acclimation and experimental periods, rats were housed in polycarbonate cages (maximum of 3 rats per cage).
- Diet: ad libitum; rodent diet (Harlan Teklad, USA)
- Water: ad libitum; filtered water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 +/- 1.7
- Humidity (%): 48.4 +/- 6.0
- Photoperiod: 12 hours dark/light cycle
No further details are given.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
aqueous, 0.5 % solution (from Sigma, USA)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: no details

VEHICLE
- Justification for use and choice of vehicle (if other than water): CMC is a standard vehicle in toxicity studies
- Amount of vehicle (if gavage): dosing volumes were 10 mL/kg
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
30 mg/kg/day (low dose)
Basis:
other: actual administered
Remarks:
Doses / Concentrations:
125 mg/kg/day (middle dose)
Basis:
other: actual administered
Remarks:
Doses / Concentrations:
500 mg/kg/day (high dose)
Basis:
other: actual administered
No. of animals per sex per dose:
The rats were divided into 4 groups of 10 rats:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected based on previous observations in a 28-day oral toxicity study by Kim et al. (2008).
No further details are given.
Positive control:
No positive control substance was tested.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: no data

FOOD CONSUMPTION: Yes

FOOD EFFICIENCY: No data

WATER CONSUMPTION: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before necropsy after 13 weeks of treatment
- Anaesthetic used for blood collection: CO2
- Animals fasted: for 24 hours
- How many animals: all animals
- Parameters checked: WBC (white blood cell count), RBC (red blood cell count), Hb (haemoglobin concentration), HTC (hematocrits), MCV (mean corpuscular volume), MCH (mean corpuscular haemoglobin), MCHC (mean corpuscular haemoglobin concentration), RDW (red cell distribution width), PLT (platelet count), MPV (mean platelet volume), NE# (number of neutrophils), NE% (percent of neutrophils), LY# (number of lymphocytes), LY% percent of lymphocytes), MO# (number of monocytes), MO% (percent of monocytes), EO# (number of eosinophils), EO% (percent of eosinophils), BA# (number of basophils), and BA% (percent of basophils) using a blood cell counter.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before necropsy after 13 weeks of treatment
- Animals fasted: for 24 hours
- How many animals: all animals
- Parameters checked: ALB (albumin), ALP (alkaline phosphatase), Ca (calcium), CHO (cholesterol), CRE (creatinine), gamma-GT (gamma-glutamyl transpeptidase), GLU (glucose), GOT (glutamic oxaloacetic transaminase), GPT (glutamic pyruvic transminase), IP (inorganic phosphorus), LDH (lactate dehydrogenase), MG (magnesium), TP (total protein), UA (uric acid), BUN (blood urea nitrogen), TBIL (total bilirubin), CK (creatine phosphokinase), Na (sodium), K (potassium), Cl (chloride), TG (triglyceride), and A/G (ratio of albumin to globulin) using a biochemical blood analyser.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Before necropsy, food was withheld for 24 h and the rats were anesthetised with CO2 gas. The rats were killed by cervical dislocation.

GROSS PATHOLOGY: Yes
- Adrenal glands, bladder, testes, ovaries, uterus, epididymis, seminal vesicle, heart, thymus, thyroid gland, trachea, esophagus, tongue, prostate, lungs, nasal cavity, kidneys, spleen, liver, pancreas, and brain were removed carefully and weighed.

HISTOPATHOLOGY: Yes:
- The organs were fixed in a 10% formalin solution containing neutral phosphate-buffered saline.
- Thereafter, the organs were embedded in paraffin, stained with hematoxylin and eosin, and examined under light microscopy.
Other examinations:
Silver distribution study:
- Tissues were digested with conc. nitric acid by using a microwave digestion system.
- The concentration of silver was analysed with a flameless method using an atomic absorption spectrophotometer equipped with a Zeeman graphite furnace.
- The concentration of silver in tissues was expressed as μg/g wet weight.
Statistics:
Statistical analysis was performed with SPSS (Version 12). Statistical evaluation was performed by analysis of two-tailed Student’s t-test or analysis of variance (ANOVA) following multiple comparison tests with Duncan’s method. The level of statistical significance was set at p < 0.05.

Results and discussion

Results of examinations

Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
BODY WEIGHT AND WEIGHT GAIN
- No significant dose-related changes were found in females.
- Body weight of high dose males (500 mg/kg bw/d) was significantly (p<0.05) decreased at weeks 4, 5 and 7 and at study termination.
- Body weights of mid dose males (125 mg/kg bw/d) was significantly (p<0.05) lower at week 10.

FOOD CONSUMPTION
- No significant differences were seen between control and treated groups (data not presented).

WATER CONSUMPTION
- No significant differences were seen between control and treated groups (data not presented).

HAEMATOLOGY
- No treatment-related changes were observed, except a significant increase in monocytes (p<0.05) in high dose females (500 mg/kg bw/d).

CLINICAL CHEMISTRY
- ALP appeared to be incresaed (not significant) in mid and high dose males (125 and 500 mg/kg bw/d).
- ALP was significantly (p<0.01) increased in high dose females (500 mg/kg bw/d).
- Cholesterol was significantly (p<0.01) increased in mid and high dose males (125 and 500 mg/kg bw/d) and high dose females.
- Total bilirubin was significantly (p<0.05) increased in mid dose males (125 mg/kg bw/d), without any dose-related trend.
- In high dose females (500 mg/kg bw/d), magnesium (p<0.01), total protein and inorganic phosporus (p<0.05) were significantly decreased.

ORGAN WEIGHTS
- Left testes weights were significantly (p<0.05) increased in high dose males (500 mg/kg bw/d), possibly reflecting lower terminal body weights.
- Right kidney weights were significantly (p<0.05) decreased in low and mid dose females (125 and 500 mg/kg bw/d), without dose-relation.

HISTOPATHOLOGY: NON-NEOPLASTIC (Incidence in the control, 30, 125, 500 mg/kg bw/d groups)
- Bile duct hyperplasia was observed in 4/10, 7/10, 8/10 and 6/10 males and 3/10, 7/10, 8/10 and 7/10 females, respectively.
- Focal, multifocal or lobular necrosis was noted in 0/10, 4/10, 5/10 and 4/10 males and 0/10, 2/10, 2/10 and 2/19 females, respectively.
- Bile duct hyperplasia with or without necrosis/fibrosis was regarded as minimal and due to silver nanoparticles.
- Minimal or mild renal unilateral or bilateral mineralisation was observed in 5/10, 8/10, 7/10 and 9/10 females, respectively.
- In the intestines, pigmentation of villi was observed in 0/10, 0/10, 8/10 and 8/10 males, respectively.
- Only slight treatment-related pigmentation was seen in high dose females (5/10).

OTHER FINDINGS: SILVER DISTRIBUTION
- There was a statitically significant (p<0.01) dose-related increase of silver deposition in testes, liver, kidneys, brain, lungs and blood of treated rats.
- A two-fold higher accumulation of silver was seen in kidneys of females compared to males.

Effect levels

open allclose all
Dose descriptor:
other:
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: incidence of liver damage by increased levels of alkaline phosphatase and cholesterol
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: incidence of liver damage by increased levels of alkaline phosphatase and cholesterol

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table: Silver concentration in tissue and blood (μg/g wet weight)

Dose/Sex

Testes

Liver

Kidneys

Brain

Lungs

Blood

0 mg/Kg

 

 

Male

0.04 ± 0.02

0.02 ± 0.01

0.04 ± 0.02

0.02 ± 0.01 a

0.10 ± 0.08

0.001 ± 0.000

Female

 

0.01 ± 0.01

0.03 ± 0.01

0.01 ± 0.01 a

0.05 ± 0.02

0.002 ± 0.002

30 mg/Kg

 

 

Male 

6.56 ± 0.33**

4.20 ± 1.57** a

1.49 ± 0.37** b

0.47 ± 0.18**

1.94 ± 0.64** b

0.111 ± 0.016**

Female 

 

8.56 ± 3.22** a

7.98 ± 0.91** b

0.38 ± 0.05**

4.97 ± 0.90 b

0.087 ± 0.017**

125 mg/Kg

 

 

Male 

11.84 ± 1.62**

10.19 ± 2.09** b

8.82 ± 2.13** b

0.69 ± 0.06**

10.97 ± 3.81**

0.191 ± 0.037** b

Female

 

29.13 ± 9.74** b

37.09 ± 17.44** b

0.77 ± 0.11**

17.64 ± 9.06**

0.122 ± 0.010** b

500 mg/Kg

 

 

Male

23.75 ± 9.13**

68.65 ± 33.59**

99.19 ± 32.82** b

3.54 ± 1.44**

56.04 ± 51.14

0.419 ± 0.083**

Female

 

98.75 ± 31.65**

226.88 ± 55.64** b

3.70 ± 1.17**

45.83 ± 11.43**

0.303 ± 0.099**

 

**Significant difference vs. control, p < 0.01. (ANOVA)

a Significant difference vs. distinction of sex, p < 0.05. (T-test)

b Significant difference vs. distinction of sex, p < 0.01. (T-test)

Applicant's summary and conclusion

Conclusions:
The target organ for the silver nanoparticles was found to be the liver in both the male and female rats after 90-day of exposure to silver nanoparticles. Significant dose-related changes were found in alkaline phosphatase and cholesterol levels of male and female rats at and above 125 mg/kg bw/d, indicating slight liver damage. Histopathology revealed slightly higher incidences of bile-duct hyperplasia with or without necrosis, fibrosis and/or pigmentation in treated animals together with a dose-dependent accumulation of silver in all tissues examined.
A NOAEL of 30 mg/kg and LOAEL of 125 mg/kg was established in the present study.