Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) I Page 1 of 13 

OPPTS 870.3100/ DACO 4.3.11 OECD 408 

Silver nanoparticles/ (PC Code 072599) 

EPA Reviewer: Jonathan Chen, Ph. D. 

Risk Assessment and Science Support Branch 

Antimicrobial Division 

EPA Secondary Reviewer: Jonathan Leshin, Ph. D. 

Risk Assessment and Science Support Branch 

Antimicrobial Division 

TXR#: 1,003,295 

Erythrocyte Micronucleus Assay (rat) 2007 

OPPTS 870.5395 

Signature: l(}t(t?Ykuc {)(k-\... 

"'""' Date _ __:O~~~.!....:h':rc~/~B=---

Signature:"'/~:;· ______ ~ _ 

Date Oi> / t) V ~o( 3 

II DATAEVALUATIONRECORD II 

STUDY TYPE: 1) Repeated Dose (28-Day) Oral Toxicity [gavage]- rats; OPPTS 870.3050; 

OECD TG 407 and 2) Erythrocyte Micronucleus Assay- rats; OPPTS 870.5395; OECD TG 474 

PC CODE: 072599 DP BARCODE: D411279 

TEST MATERIAL (PURITY): Nanosilver (52.7-70.9 run, average particle size: 60 run; 

99.98% pure) 

SYNONYMS: Not available 

CITATION: Kim, YS, Kim, JS and Cho, HS, et. al. (2008) Twenty-Eight-Day Oral Toxicity, 

Genotoxicity, and Gender-Related Tissue Distribution of Silver Nanoparticles in 

Sprague-Dawley Rats. Korea Environment and Merchandise Testing Institute, 

Incheon, Korea. Inhalation Toxicology. 20: 575-583. MRID 49052005. 

SPONSOR: National Institute of Toxicological Research, Korea Food and Drug 

Administration. 

EXECUTIVE SUMMARY: In a repeated dose oral toxicity study (MRID 49052005), silver 

nanoparticles (99.98% a.i.; 60 run average particle size; carboxymethylcellulose [CMC]-coated) 

were administered via gavage (10 ml/k:g dosing volumes) to 10 Sprague-Dawley rats/sex/dose at 

dose levels ofO (vehicle control, 0.5% carboxymethylcellulose [CMC]), 30 (low-dose), 300 

(medium-dose), or 1000 (high-dose) mg/kg bw/day for 28 days. Following sacrifice, selected 

organ weight and histopathological examinations were performed. Tissue concentrations of 

silver (Ag) in the testis, kidney, brain, lungs, stomach and blood were also determined. In 

addition, bone marrow was collected from the femurs of all animals and an in vivo micronucleus 

assay conducted for determination of cytotoxicity and genotoxicity. 

No dose-related changes were observed in body weight, food consumption, or organ weight in 

male and female rats compared to the control group, except for an increase in the weight of the 
Silver nanoparticles/ (PC Code 072599) 

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OPPTS 870.5395 

brain for the high-dose males (p<0.05) and the liver of the middle-dose females (data not 

shown). 

Effects on blood parameters were limited. There were significant increases in alkaline 

phosphatase (ALP) in mid- and high-dose (300 and 1000 mglkg/day group) male rats and in 

high-dose females (p<O.Ol) and significant increases in cholesterol in high-dose males (p<0.05) 

and in mid- and high-dose females (p<O.Ol); whereas, total protein was decreased significantly 

in high-dose males (p<O.Ol). There were also significant increases in hematology (red blood cell 

counts, hemoglobin, and hematocrit in mid- and high-dose females (p<O.Ol) and an increase in 

mean corpuscular volume (MCV) in high dose male rats. These data were accompanied by a 

significant decrease the coagulation time in terms of the active partial thromboplastin time 

(APTT) decreased significantly(p < .01) in the female rats at 1000 mglkg/day. 

It is noted that tissue distribution analysis revealed significant amount of Ag in the blood, brain 

and kidneys of males and females of all dose groups; in the male testes of all groups; and in the 

stomach of mid (male) and high doses (male and female). There was also a significant increase 

in silver in the mid (male and female) and high dose (female only) liver. The lungs showed an 

increase in all dose levels, except for low dose males. 

Histopathological examination revealed bile-duct hyperplasia around central vein, infiltration of 

inflammatory cells, including eosinophils, and dilation of the central vein in the liver of male and 

female rats. Additionally, a dose-dependent significant increase (p<O.Ol) in nanosilver 

distribution in most tissues (liver, kidneys, stomach, brain, lungs, testes, and blood) in both males 

and females, with a two-fold higher accumulation in the kidneys of female rats, was observed, 

when compared with male rats across all dose groups. 

There were no statistically significant differences in the micronucleated polychromatic 

erythrocytes (MN PCEs) or ratio of polychromatic erythrocytes among the total erythrocytes 

after silver nanoparticle exposure. 

Based on significant increases in alkaline phosphatase (ALP) and cholesterol (CHO), significant 

changes in hematology, and accompanied by histopathological evidences of liver toxicity (bileduct hyperplasia around central vein, infiltration of inflammatory cells, and dilation of the central 

vein), the NOAEL is 30 mglkglday and the LOAEL is 300 mglkglday. 

This 28-day repeated dose oral toxicity study is classified as Acceptable/non-guideline and may 

be used for regulatory purposes; however, it does not satisfy the Guideline requirements (OPPTS 

870.3050) for a 28-day oral toxicity study in rats (Refer to deficiency section IILB). 

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality 

statements are not provided. 
Silver nanoparticles/ <PC Code 072599) 

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I. MATERIALS AND METHODS 

A. MATERIALS: 

1. Test Material: 

Description: 

Lot/Batch #: 

Purity: 

Compound Stability: 

CAS#: 

Nanosilver 

Nanoparticles, (52.7-70.9 run, average; 60 nm) 

Not reported 

99.98 % a.i. 

Not reported 

Not available 

2. Vehicle and/or positive control: Aqueous carboxymethylcellulose, 0.5% 

3. Test animals: 

Species: 

Strain: 

Age/weight at study 

initiation: 

Source: 

Housing: 

Diet: 

Water: 

Environmental 

conditions: 

Acclimation period: 

B. STUDY DESIGN: 

Rat 

Sprague-Dawley 

Six -week-old/283 g(males)/192 g(females) 

OrientBio (Korea) 

Polycarbonate cages (maximum 3 rats/cage) 

Rodent diet, Harlan Teklab, Plaster International Co., Korea) ad libitum 

filtered water ad libitum 

Temperature: 23 ± 2°C 

Humidity: 55± 7% 

Air changes: Not reported 

Photoperiod: 12 hrs dark/12 hrs light 

Two weeks 

1. In life dates - not reported 

2. Animal assignment: Animals were assigned to the test groups noted in Table 1. 

TABLE 1: Study design 

Test Group Nominal Dose Actual Dose #Male #Female 

(mglkg bw/d) (mglkg bw/d) 

Control 0 0 10 10 

Low 30 30 10 10 

Mid 300 300 10 10 

High 1000 1000 10 10 

3. Dose selection rationale 

The dose selection rationale not presented in this report 
Silver nanoparticles/ <PC Code 072599) 

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4. Treatment preparation, administration, and analysis: 

Dosing formulations were not presented in this study report. 

5. Statistics 

The body weights, organ weights and the results for blood biochemistry and hematology were 

compared using a multiple variance analysis and Duncan's multiple range tests. These analyses 

were used for the three experimental groups and the fresh-air control rats. 

C. METHODS: 

1. Observations: 

la. Cageside Observations 

The frequency of cageside observations for mortality or toxicity were not provided in this report. 

1 b. Clinical Examinations 

The frequency of clinical examinations conducted was not provided in this report. 

lc. Neurological Evaluations 

Neurological evaluations were not reported in this study. 

2. Body weight 

Animal body weight changes were observed throughout of the study according to the dose of 

silver nanoparticles during the 28-day experiment. The frequency of observation data was not 

reported. 

3. Food consumption 

During the 28-day exposure period, there were no significant differences in food consumption. 

4. Ophthalmoscopic examination: 

No ophthalmoscopic data provided in this report. 

5. Hematology & Clinical Chemistry: 

Blood was collected from the abdominal aorta and collected in heparinized vacutainers and 

analyzed for hematology and clinical chemistry from all surviving animals. The CHECKED (X) 

parameters were examined. 
Silver nanoparticlesl (PC Code 072599) 

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a. Hematology 

X Hematocrit (HCT)* X Leukocyte differential count* 

X Hemoglobin (HGB)* X Mean corpuscular HGB (MCH)* 

X Leukocyte count (WBC)* X Mean corpusc. HGB conc.(MCHC)* 

X Erythrocyte count (RBC)* X Mean corpuscular volume (MCV)* 

X Platelet count* X Reticulocyte count 

X Blood clotting measurements* 

X (Thromboplastin time) 

X (Clotting time) 

X (Prothrombin time) 

* Recommended for 28-day oral rodent studies based on Gu1dehne 870.3050 

b. Clinical Chemistry 

ELECTROLYTES OTHER 

X Calcium X Albumin* 

X Chloride X Creatinine* 

X Magnesium X Urea nitrogen* 

X Phosphorus X Total Cholesterol* 

X Potassium* (K) Globulins 

X Sodium* (NA) X Glucose* 

ENZYMES (more than 2 hepatic enzymes, eg., *) X Total bilirubin 

X Alkaline phosphatase (AP)* X Total protein (TP)* 

Cholinesterase (ChE) X Triglycerides 

X Creatine phosphokinase Serum protein electrophoresis 

X Lactic acid dehydrogenase (LDH) 

X Alanine aminotransferase (ALT/also SGPT)* 

X Aspartate aminotransferase (AST/also SGOT)* 

X Gamma glutamyl transferase (GGT)* 

Glutamate dehydrogenase 

'---- Sorbitol dehydrogenase* 

* Recommended for 28-day oral rodent studies based on Gu1delme 870.3050 

6. Urinalysis* 

No urinalysis data provided in this report. The CHECKED (X) parameters were examined. 

Appearance* Glucose* 

Volume* Ketones 

Specific gravity I osmolality* Bilirubin 

pH* Blood I blood cells* 

Sediment (microscopic) Nitrate 

Protein* Urobilinogen 

* OptiOnal for 28-day oral rodent studies based on Gu1delme 870.3050 
Silver nanoparticles/ <PC Code 072599) 

7. Sacrifice and Pathology 

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All animals were sacrificed by cervical dislocation on schedule and subjected to gross 

pathological examination. The CHECKED (X) tissues were collected for histological 

examination. The (XX) organs, in addition, were weighed. 

DIGESTIVE SYSTEM CARDIOVASC./HEMA T. 

XX Tongue Aorta, thoracic* XX 

Salivary glands* XX Heart*+ 

XX Esophagus* Bone marrow* 

XX Stomach* Lymph nodes* 

Duodenum* XX Spleen*+ 

Jejunum* XX Thymus*+ 

Ileum* XX 

Cecum* UROGENITAL 

Colon* XX Kidneys*+ 

Rectum* XX Urinary bladder* XX 

XX Liver*+ XX Testes*+ 

Gall bladder* (not rat) XX Epididymides*+ 

Bile duct* (rat) XX Prostate* 

XX Pancreas* XX Seminal vesicles* 

RESPIRATORY XX Ovaries*+ 

XX Trachea* XX Uterus*+ 

XX Lung* Mammary gland* 

XX Nose* 

XX Pharynx* 

XX Larynx* 

* Recommended for 28-day oral rodent studies based on Gu1delme 870.3050 

+ Organ weights required for rodent studies. 

II. RESULTS 

A. OBSERVATIONS: 

NEUROLOGIC 

Brain*+ 

Peripheral nerve* 

Spinal cord (3 levels)* 

Pituitary* 

Eyes (optic nerve )* 

GLANDULAR 

Adrenal gland*+ 

Lacrimal gland 

Parathyroid* 

Thyroid* 

OTHER 

Bone (sternum and/or femur) 

Skeletal muscle 

Skin* (treated & untreated areas) 

All gross lesions and masses* 

1. Clinical signs of toxicity: Twenty-eight days of repeated oral doses of silver nanoparticles did 

not show any significant changes in body weight for male and female rats. 

2. Mortality: This study did not mention any mortality that occurred on study. 

3. Neurological Evaluations: This study mentioned that there was increase in the weight of the 

brain for the high-dose (1 000 mg/kg) male rats. 

B. BODY WEIGHT AND WEIGHT GAIN: It was reported that the male and female rats did 

not show any significant changes in body weight according to the dose of silver nanoparticles 

during the 28-day experiment. The data tables for body weight and weight gain were not 

included in this report. 

C. FOOD CONSUMPTION: 
Silver nanoparticles/ <PC Code 072599) 

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1. Food consumption: It was reported that there were no significant differences in food 

consumption between male and female rats and the control group. 

D. OPHTHALMOSCOPIC EXAMINATION: This study did not mention any 

ophthalmoscopic examination data. 

E. BLOOD ANALYSIS: Before blood collection the rats were withheld food for 24 hours and 

anesthetized with ether. The blood was drawn from the abdominal aorta, collected in 

heparinized vacutainers, and analyzed 

1. Clinical Chemistry: The data showed a statistically significant decrease in total protein for 

high-dose (1 000 mg/kg/day) male rats following a 28-day oral exposure to silver nanoparticles 

(Table 1A). The ALP in mid-dose (p < 0.05) and high-dose (p < 0.01) male rats and high-dose 

(p < 0.05) female rats increased significantly compared to that in the control. There were 

significant increases in cholesterol in both the high-dose (p < 0.05) male rats and the mid- and 

high-dose (p< 0.01) female rats (see Tables 1A and 1B below). 

TABLE lA 

Blood biochemistry data for male rats (n = I 0, mean ± SO) 

Component Control Low Middle High 

ALB 2.26± 0.12 2.23±0.08 2.30±0.07 2.28 ± 0.12 

ALP 427.20 ± 70.61 475.40 ± 98.54 613.70 ± 128.6IQ 837.70 ± 221.53Q 

Ca 10.01 ± 0.43 9.75 ± 0.30 9.73 ± 0.32 9.65 ±0.42 

CHO 69.00± 8.23 73.10 ± 16.03 82.10 ± 21.68 87.50 ± 13.05b 

CRE 0.72 ±0.09 0.68±0.04 0.71 ±0.07 0.68 ±0.14 

GLU 120.20 ± 14.07 106.80 ± 16.77 124.00 ± 24.76 121.20± 31.44 

AST 130.10 ± 35.67 129.80 ± 35.35 133.20 ± 45.42 131.40 ± 30.28 

ALT 39.10 ± 5.61 41.70± 5.98 43.10 ± 5.53 47.10± 10.27 

TP 5.79±0.21 5.61 ±0.25 5.77±0.24 5.53 ± 0.20" 

BUN 19.87 ± 3.89 19.87 ± 3.89 20.19± 3.45 19.91 ± 3.93 

TBIL 0.006 ± 0.011 0.012 ± 0.012 0.008 ± 0.010 0.002 ± 0.004 

IP 9.89±0.60 9.72 ± 0.56 9.72 ±0.61 9.90±0.82 

TG 31.90 ± 11.20 27.20 ± 15.40 38.10± 17.56 25.50± 13.09 

CPK 841.90 ± 427.80 718.20 ± 358.60 873.20 ± 468.96 770.50 ± 376.63 

Na 145.10 ± 1.85 145.10±2.23 144.30 ± 2.45 144.90 ± 1.20 

K 6.53 ± 2.06 6.39± 1.9 7.06 ± 2.74 6.04 ± 1.53 

C1 102.10±0.88 102.10 ± 1.29 102.00 ± 1.15 101.90 ± 1.20 

Note. ALB (g/dl), albumin; ALP(IU/1.), alkaline phosphlllliSC; Ca (mg/dl), calcium; CHO (mg/dl), total cholesterol; CRE (mg/dl), creatinine; 

GLU (mg/dl), glucose; AST (IU/1.), asputate; aminocransferase; ALT (IU/1.), alanine aminottansfc:rase; TP(g/dl), tOial pi'Oiein; BUN (mg/dl), 

blood un:a nitrogen; T-Bll. (mg/dL), tOial bilirubin; IP (mg/dl). inorganic phosphorus; TG (mg/dl), triglyceride; CPK (U/1.), creatine phosphokinase; Na (mmoi/L), sodium; K (mmoi/L), potassium; Cl (mmoi/L), chloride. 

• Significant difference vs. control, p < .0 I. 

bSignificant difference vs. control, p < .OS. 
Silver nanoparticles/ (PC Code 072599) 

Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) I Page 8 of 13 

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TABLE lB 

Blood biochemistry data for female rm (n = 10, mean± SD) 

Control Low Middle Higb 

ALB 2.44±0.16 2.42±0.09 2.39±0.10 2.39 ± 0.10 

AlP 367.60 ± 125.67 335.40 ± 78.98 403.30 ± 75.93 499.00 ± 107.34« 

Ca 9.79±0.28 9.55 ±0.38 9.61 ±0.41 9.79±0.53 

CHO 89.60±9.07 85.60± 12.19 102.90 ± 17.741 117.50 ± 14.08« 

CRE 0.68±0.06 0.67 ±0.08 0.68±0.08 0.75±0.07 

GLU 12S.OO ± 15.58 121.30 ± 12.94 115.40± 16.70 122.80 ± 14.20 

AST 143.70 ± 33.06 152.50 ± 26.33 145.70 ± 26.73 137.30 ± 35.41 

ALT 40.50 ± 10.54 44.10±8.47 42.80±6.41 45.30± 7.97 

TP 5.79±0.22 5.83± 0.22 5.74±0.23 5.79±0.24 

BUN 19.85 ± 1.77 21.13 ±4.08 21.96 ± 3.32 23.76 ± 6.19 

TBIL 0.006±0.011 0.002 ± 0.004 0.007 ± 0.009 0±0 

IP 8.53 ± 1.08 8.17±0.68 8.14 ± 1.04 8.85 ± 1.28 

TG 16.00± 8.04 12.50± 3.98 11.50± 5.60 13.60 ±4.65 

CPK 872.20 ± 375.37 940.40 ± 343.86 844.30 ± 213.28 170.80 ± 281.56 

Na 142.80 ± 1.23 143.10± 1.91 143.60 ± 1.65 143.10 ± 1.45 

K 4.79±0.62 4.50±0.49 4.38±0.38 5.01 ±0.81 

Cl 101.10± 1.91 102.30 ± 1.25 101.80 ± 1.55 101.60 ± 1.43 

"Significant dilfcn:nc:e vs. conlrOI, p < .01. 

2. Hematology: The MCV (p < 0.05) in the high-dose male rats and the RBC (p < 0.05 top < 

0.01), Hb (p < 0.01), and HCT (p < 0.01) in both the mid- and high-dose female rats increased 

significantly compared to that in the controls. Hematological values for male and female rats are 

given in Tables 2A and 2B below. In addition, the coagulation time in terms of the active partial 

thromboplastin time (APTT) decreased significantly (p < 0.01) in the female rats compared to 

that for the control group (Tables 3A and 3B). 

TABLE2A 

Hematological data for male rats 

Control Low Middle High 

WBC 11.89±4.28 12.45± 3.22 12.68 ±2.85 11.20 ± 2.61 

RBC 9.05 ± 1.81 9.34±0.38 9.66±0.68 9.65 ±0.59 

Hb 14.84± 3.14 15.37 ± 0.44 15.60± 1.01 16.03 ± 0.93 

HCT 65.20 ± 13.59 66.66±2.33 67.83±4.76 71.20 ± 3.23 

MCV 71.96 ± 3.63 70.19±3.34 70.30± 3.18 73.88 ± 2.61a 

MCH 16.36±0.75 16.29±0.51 16.17± 0.47 16.60± 0.47 

MCHC 22.74±0.85 23.23 ±0.82 23.02 ± 0.77 22.51 ± 0.83 

RDW 17.04 ± 1.05 17.41 ± 2.66 18.04± 3.57 18.91 ± 2.95 

PLT 681.60 ± 211.80 709.40 ± 79.52 730.40 ± 75.14 666.30 ± 152.05 

MPV 5.93 ±0.22 5.92 ±0.32 5.94±0.32 5.93 ± 0.25 

NEU 2.56±0.89 2.33±0.91 2.32±0.43 2.93 ±0.93 

LYO 95.45 ± 1.79 95.79 ± 1.64 95.71 ± 0.74 95.09 ± 1.57 

MONO 1.55 ± 0.38 1.48 ± 0.60 1.81 ± 0.35 1.63 ± 0.47 

EOS 0.32 ±0.48 0.19±0.46 0.13±0.08 0.27 ±0.38 

BASO 0.13 ±0.26 0.09±0.22 0.03±0.02 0.08 ± 0.12 

Note. WBC(K!uL). White blood cells; RBC(M/uL), Red blood cells; Hb (g/dL). Hemoglobin; HCf(%), Hematocrits; MCV (II), Mean 

corpuscular volume; MCH (pg), Mean COIJluscular hemoglobin concentration; ROW(%), Red cell distribution width; PLT (K/uL), Platelets; 

MPV (II), Mean platelet volume; NEU (%), Neutrophils; LYO (%),Lymphocytes; MONO(%). Monocytes: EOS (%), Eosinophils; BASO (%), 

Basophils. 

"Significant diffe~nce vs. control, p< .05. 
Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) I Page 9 of 13 

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TABLE2B 

Hematological data for female rats 

Control Low Middle High 

WBC 9.81 ± 2.59 8.00 ± 3.71 9.40±2.74 9.74 ±2.54 

RBC 7.84±0.58 1.15 ± 1.01 8.48 ±0.55b 8.53 ± 0.4541 

Hb 13.36 ± 0.93 13.11 ± 1.87 14.43 ± 0.784 14.74 ± 0.6841 

HCT 65.18 ± .5.32 64.94 ± 9.27 71.69 ± 4.7111 73.42 ± 4.3541 

MCV 83.09 ±2.39 83.67 ± 1.89 84.66± 3.42 86.08 ± 3.81 

MCH 17.04 ±0.46 16.91 ± 0.81 17.04± 0.62 17.30 ± 0.70 

MCHC 20.55 ± 0.98 20.22 ± 1.29 20.17 ± 0.97 20.11 ± 0.83 

RDW 17.79 ± 1.02 18.18 ± 1.46 18.18 ± 0.93 18.29±0.86 

PLT 428.80 ± 96.77 297.80 ± 181.56 354.00 ± 139.86 359.80 ± 97.66 

MPV 5.86± 0.35 5.77 ± 0.37 5.98±0.27 5.79 ± 0.30 

NEU 3.69± 3.08 2.49± 1.05 2.88± 1.20 3.17 ±0.67 

LYO 93.67 ± 5.42 95.58 ± 1.66 95.23 ± 2.03 94.99 ± 1.05 

MONO 1.80 ± 0.71 1.56± 0.36 1.48±0.56 1.64 ± 0.51 

EOS 0.61 ± 1.31 0.28 ±0.29 0.27±0.30 0.14 ± 0.15 

BASO 0.23 ± 0.58 0.09±0.09 0.13 ± 0.19 0.06±0.06 

•significant difference vs. control, p < .01. 

6Significant difference vs. control, p < .OS. 

Table 3A 

Coagulation time for male rats (sec, mean ± SD) 

Groups PT AP'IT 

Control (n = 1 0) 15.11 ±0.96 9.26 ± 2.14 

Low (n = 10) 14.80±0.96 9.90 ± 2.19 

Middle (n = 10) 14.95±o.n 10.87 ±2.33 

High(n =9) 15.01 ±0.91 11.27 ± 3.19 

Note. AP1T: active panial thromboplastin time, PT: prothrombin time. 

Table 3B 

Coagulation time for female rats (sec. mean ± SO) 

Group PT Group AP'IT 

Dose Dose 

Control (n = 9) 15.39 ± 0.73 Control (n = 9) 12.23 ± 2. 13 

Low (n=9) 14.78 ± 1.20 Low(n =9) 

Middle (n = 9) 14.46± 0.83 Middle (n = 8) 

High (n = 9) 14.80 ±0.93 High (n = 7) 

Note. PT: prothrombin time: AP'IT: active panial thromboplastin time. 

"Significant difference vs. control. p < .05. 

10.96 ± 2.89 

10.28 ± 3.67 

8.81 ± 2.48" 
Silver nanoparticles/ <PC Code 072599) 

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F. URINALYSIS: No data on urinalysis reported. 

G. SACRIFICE AND PATHOLOGY: At the end ofthe 28 day oral test, the rats were 

necropsied after 24 hours. Silver distribution in tissue was examined. This examination revealed 

that all the groups exposed to silver nanoparticles for 28 days showed dose-dependent increases 

in the silver concentration within the testes, kidneys, liver, brain, lungs, stomach and blood of 

male rats and the kidneys, liver, brain, lungs, stomach and blood of female rats. The kidneys 

also showed a sex-dependent accumulation of silver, with a two-fold higher accumulation in the 

female rats when compared with the male rats across all dose groups (Table 4). 

Table 4. 

Tissue content of silver 

Silver (mg/g wet weight) 

Sex Testes Kidneys Liver Brain Lungs Stomach Blood 

Control 

M 0.1 ±0.1 0.02 ± O.ol 0.02±0.0 0.03±0.0 0.04±0.0 0.2±0.1 0.01 ±0.01 

F 0.02 ±0.00 0.01 ±0.0 0.02±0.0 0.02±0.0 0.2±0.1 0.00±0.00 

Low 

M 1.2 ± 0.4" 1.3 ± 0.6" 0.5 ±0.3 o.3 ± o.o-• 0.2 ± 0.1 10.7±8.8 o.t8 ± o.o-· F 2.8±0.9 .. 0.5±0.3 0.2 ± 0.1** 0.1 ±o.o• 6.9±3.6 o.t6 ±o.o·• 

Middle 

M 3.6±0.6*• 6.0± 2.4* 8.7 ± 3.7• 0.8 ± O.t•• 4.3 ± 0.9"0 56± 25° 0.43 ±o.1•• 

F 16.8±2.800 9.6 ± 3.6" 0.6 ± 0.1"* 6.1±2S 37± 19 0.44± 0.1*" 

High 

M 7.4 ± 1.7** 24± tt• 71 ±56 2.0±0.3** 17±4• 228 ± 79• 0.80±0.200 

F 69 ± 22*" 65 ± 32" 1.8 ± 0.3** 27±8 .. 176± 44*" 0.70±0.3 .. 

Nott. M, male: F. Cemale. 

• Sianificant diffeml(:e vs. c:onuot. p < .OS; ••sianificant diffe"'nc:e vs. control. p < .01. 

1. Organ weight: The report states that there were no significant organ-weight changes observed 

in either male or female rats after 28 days of silver nanoparticle administration, with exception in 

the weight of the brain and liver. There was a weight increase in the brain for the high-dose male 

rats (p < 0.05) and the liver for the mid-dose female rats. The data for these findings were not 

presented in this report. 

2. Gross pathology: The study report did not state whether gross pathological changes were 

observed. 

3. Microscopic pathology: Histopathological findings for rat liver are (a) bile duct hyperplasia 

around central vein to hepatic lobule; (b) infiltration of inflammatory cells, including 

eosinophils, in hepatic lobule and portal tract; (c) infiltration of inflammatory cells in and 

beneath the central vein; and (d) dilated central vein filled with bluish amorphous material and 

blood cells. Interestingly, there appeared to be an increased accumulation of silver via 

microscopic examination in the kidneys of female rats when compared with male rats. No 

significant microscopic fmdings were reported in adrenal glands, urinary bladder, testes, 

epididymus, seminal vesicle, spleen, pancreas, thymus, thyroid gland, trachea, esophagus, 

tongue, lungs, heart, brain, ovaries, uterus, or nasal cavity. 
Silver nanoparticles/ (PC Code 072599) 

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OPPTS 870.5395 

H. ADDITIONAL ANALYSIS: A rat bone marrow micronucleus assay test was conducted at 

the end of the 28-day oral study for the detection of cytogenetic damage. The study author 

examined the number ofmicronucleated polychromatic erythrocytes (MN-PCEs) among 2000 

polychromatic erythrocytes (PCEs) per animal (bone marrow) using a fluorescent microscope. 

To evaluate bone marrow toxicity, the PCE/(PCE + NCE) ratio was calculated. 

The frequency ofmicronucleated polychromatic erythrocytes (MN PCEs) in 2000 PCEs for the 

male rats was 6.00, 6.60, and 7.40 for the groups exposed to concentrations of 30, 300, and 1000 

mglkg/day respectively, while that for the control was S.20. Plus, the frequency ofMN PCEs in 

2000 PCEs for the female rats was 3.50, 2.40, and 3.40 for the groups exposed to concentrations 

of 30, 300, and 1000 mg/kg/day. Respectively, while that for the control was 2.SO. Thus, 

although a dose-related increase was found in the number of micronucleated polychromatic 

erythrocytes (MN PCEs) in the male rats, no significant treatment-related increase ofMN PCEs 

was detected in the male and female rats when compared to the corresponding negative controls 

(Tables SA and SB). No statistically significant difference in the PCE/(PCE+ NCE) ratio, 

representing the absence of bone marrow cytotoxicity, was observed in the male and female rats 

after silver nanoparticle exposure when compared with the control (Tables SA and SB). 

Table SA 

Frequency of MNPCEs and PCE/(PCE + NCE) ratio in bone maiTOW of male rats treated orally with indicated doses of silver 

nanoparticles for 28 days 

Dose Number of rats Frequency of MN PCEs PCEI(PCE + NCE) 

(mglkg/day) (male) in 2000 PCEs (mean ± SE) (mean±SE) 

0 10 5.20± 0.63 0.30±0.024 

30 10 6.00±0.93 0.25 ±0.019 

300 10 6.60±0.64 0.28±0.036 

1000 10 7.40±0.54 0.26±0.023 

Note. MN PCE. micronucleated polychromatic erythrocytes; PCE, polychromatic erythrocytes; NCE. nonnochromatic erythrocytes. 

Table SB 

Frequency of MNPCEs and PCE/(PCE + NCE) ratio in bone marrow of female rats treated orally with md1cated doses ot sliver 

nanoparticles for 28 days 

Dose Number of rats Frequency of MN PCEs PCE/(PCE + NCE) 

(mg/kg/day) (female) in 2000 PCEs (mean ± SE) (mean±SE) 

0 10 2.50±0.43 0.37±0.022 

30 10 3.50±0.50 0.32±0.019 

300 10 2.40±0.52 0.34±0.023 

1000 10 3.40±0.64 0.30 ±0.019 

Note. MN PCE. micronucleated polychromatic erythrocytes; PCE. polychromatic erythrocytes; NCE. normochromatic erythrocytes. 
Silver nanoparticles/ (PC Code 072599) 

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III. INVESTIGATORS' DISCUSSION AND CONCLUSIONS: The investigators concluded 

that exposure to over 300 mg/kg/day of silver nanoparticles may result in slight liver damage. 

Liver toxicity evaluated by histopathology included dilation of the central vein, bile-duct 

hyperplasia, and increased foci. It was also stated that a gender-related difference in the 

accumulation of silver was noted in the kidneys, with a two-fold increase in the female kidneys 

when compared with silver accumulation in male kidneys. Further, the study author stated that 

there was an increase in blood coagulation time along with dose-dependent increases of silver 

concentrations in the blood, indicating that the orally absorbed silver nanoparticles were able to 

enter the blood circulation and affect peripheral blood coagulation. 

A. REVIEWER COMMENTS: The reviewer concurs that oral exposure to silver nanoparticles 

induced dose-dependent changes in clinical chemistry, hematology, and nanosilver accumulation 

in major tissues/organs evaluated, at dose level equal or greater than 300 mg/kg/day, following 

repeat exposure of this test substance over a 28-day time period. Although the dose level was 

not clearly stated in the content of the article for the observed histopathology changes in the 

liver, the author indicated in the abstract section that" ...... some significant dose-dependent 

changes were found in the alkaline phosphatase and cholesterol values in either the male or 

female rats, seeming to indicate that exposure to over more than 300 mg of silver nanoparticles 

may result in slight liver damage ....... ". The reviewer believes the histopathology changes in 

the liver happened at 300 mg/kg/day and higher. 

The NOAEL is 30 mglkglday and the LOAEL is 300 mglkglday, Based on significant 

increases in alkaline phosphatase (ALP) and cholesterol (CHO), significant changes in 

hematology, histopathological fmdings in the liver (bile-duct hyperplasia around central vein, 

infiltration of inflammatory cells, and dilation of the central vein), and dose-dependent 

significant accumulation of nanosilver in tissues, including gender-dependent accumulation in 

the kidneys. 

B. STUDY DEFICIENCIES: 

Major deficiencies (individual animal data not provided in published study) 

• Clinical observations (including sensory reactivity and functional observational battery) 

• Body weight 

• Gross necropsy and microscopic examination 

• Food consumption 

Minor deficiencies (data/information not provided in published study) 

• Dose selection rationale 

• Dose of 7 days/week 

• Urinalysis data 

• Justification of vehicle used 

• The dose level(s) at which the histopathology changes in the liver were observed 
Silver nanoparticles/ (PC Code 072599) 

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C. STUDY CLASSIFICATION: This 28-day repeated dose oral toxicity study is classified as 

Acceptable/non-guideline and may be used for regulatory purposes; however, it does not 

satisfy the Guideline requirements (OPPTS 870.3050) for a 28-day oral toxicity study in rats.