Silver

Repeated dose toxicity: oral

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-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Silver

EC Number:

231-131-3

EC Name:

Silver

Cas Number:

7440-22-4

Molecular formula:

Ag

IUPAC Name:

Silver

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 or test system 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 / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
30 mg/kg/day (low dose)
Basis:
other: actual administered

Doses / concentrations 2

Remarks:

Doses / Concentrations:
125 mg/kg/day (middle dose)
Basis:
other: actual administered

Doses / concentrations 3

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

1

Dose descriptor:

other:

Basis for effect level:

other: see 'Remark'

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

2

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

3

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.