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. 

### RiskAssessment and Science SupportBranch Antimicrobial Division EPA Secondary Reviewer: Jonathan Leshin, Ph. D. 

### RiskAssessment and Science SupportBranch AntimicrobialDivision 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 ofSilverNanoparticles in Sprague-Dawley Rats. KoreaEnvironment and Merchandise Testing Institute, Incheon, Korea. Inhalation Toxicology. 20: 575-583. MRID 49052005. 

### SPONSOR: National Institute ofToxicological 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 ofall animals and an in vivo 

### micronucleus assay conducted for determination ofcytotoxicity and genotoxicity. No dose-related changes were observed in body weight, food consumption, or organ weight in male and female rats comparedto the control group, except for an increase in the weight oftheSilver nanoparticles/ (PC Code 072599) Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) I Page2 of 13 OPPTS 870.3100/ DACO 4.3.1/ OECD 408 Erythrocyte Micronucleus Assay (rat) 2007 OPPTS 870.5395 brain for the high-dose males (p<0.05) and the liver ofthe middle-dose females (data not shown). Effects on bloodparameters were limited. There were significant increases in alkaline phosphatase (ALP) in mid- andhigh-dose (300 and 1000 mglkg/day group) male rats and in high-dose females (p<O.Ol) and significant increases in cholesterol inhigh-dose males (p<0.05) and in mid- and high-dose females (p<O.Ol); whereas, total protein was decreased significantly inhigh-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 coagulationtime interms ofthe active partial thromboplastin time (APTT) decreased significantly(p < .01) inthe female rats at 1000 mglkg/day. Itis noted that tissue distribution analysis revealed significant amount ofAg inthe blood, brain and kidneys ofmales and females ofall dose groups; inthe male testes ofall groups; and in the stomach ofmid (male) and high doses (male and female). There was also a significant increase in silver inthe mid (male and female) andhigh dose (female only) liver. The lungs showed an increase in all dose levels, except for low dose males. Histopathological examinationrevealedbile-duct hyperplasia around central vein, infiltration of inflammatory cells, including eosinophils, and dilation ofthe central vein inthe liver ofmale and female rats. Additionally, a dose-dependent significant increase (p<O.Ol) in nanosilver distribution in mosttissues (liver, kidneys, stomach, brain, lungs, testes, and blood) inboth males and females, with a two-fold higher accumulation inthe kidneys offemale rats, was observed, when compared with male rats across all dose groups. There were no statistically significant differences inthe micronucleated polychromatic erythrocytes (MN PCEs) orratio ofpolychromatic erythrocytes among the total erythrocytes after silver nanoparticle exposure. Based on significant increases in alkaline phosphatase (ALP) and cholesterol (CHO), significant changes inhematology, and accompaniedbyhistopathological evidences ofliver toxicity (bileduct hyperplasia around central vein, infiltration ofinflammatory cells, and dilation ofthe central vein), 

### the NOAEL is 

### 30 mglkglday and the LOAEL is 

### 300 mglkglday. This 28-dayrepeated 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 inrats 

### (Refer to deficiency sectionIILB). 

### COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Data Confidentiality statements are not provided.Silver nanoparticles/ <PC Code072599) Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) I Page 3 of 13 OPPTS 870.3100/DACO 4.3.11 OECD 408 Erythrocyte Micronucleus Assay (rat) 2007 OPPTS 870.5395 

### I. MATERIALS AND METHODS A. MATERIALS: 1. 

### TestMaterial: 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) adlibitum filtered water ad libitum Temperature: 23 ± 2°C Humidity: 55±7% Air changes: Notreported Photoperiod: 12 hrs dark/12 hrs light Two weeks



### 1. 

### In life dates

- notreported 

### 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 notpresented inthis reportSilver nanoparticles/ <PC Code 072599) Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) IPage 4 of 13 OPPTS 870.3100/DACO 4.3.11 OECD 408 Erythrocyte Micronucleus Assay (rat) 2007 OPPTS 870.5395


### 4. Treatmentpreparation, administration, and analysis: Dosing formulations were notpresented in this study report. 

### 5. Statistics The body weights, organ weights and the results for bloodbiochemistry 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 ofcageside observations for mortality ortoxicity were not provided in this report. 1 b. Clinical Examinations The frequency ofclinical examinations conducted was notprovided in this report. lc. Neurological Evaluations Neurological evaluations were not reported in this study. 

### 2. Bodyweight Animal body weight changes were observed throughout ofthe study according to the dose of silver nanoparticles during the 28-day experiment. The frequency ofobservation 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: Bloodwas collected from the abdominal aorta and collected inheparinized vacutainers and analyzed for hematology and clinical chemistry from all surviving animals. The CHECKED (X) parameters were examined.Silver nanoparticlesl (PC Code 072599) Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) IPage 5 of 13 OPPTS 870.3100/DACO 4.3.1/ OECD 408 Erythrocyte Micronucleus Assay (rat) 2007 OPPTS 870.5395


### 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 Ureanitrogen* X Phosphorus X Total Cholesterol* X Potassium* (K) Globulins X Sodium* (NA) X Glucose* ENZYMES (more than 2 hepatic enzymes, eg., *) X Totalbilirubin X Alkaline phosphatase (AP)* X Totalprotein (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 dataprovided inthis 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 870.3050Silver nanoparticles/ <PC Code072599) 

### 7. Sacrifice and Pathology Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) IPage 6 of 13 OPPTS 870.3100/DACO 4.3.1/ OECD 408 Erythrocyte Micronucleus Assay (rat) 2007 OPPTS 870.5395


### All animals were sacrificed by cervical dislocation on schedule and subjectedto 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 oftoxicity: Twenty-eight days ofrepeated oral doses ofsilver 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 inthe weight ofthe brain for the high-dose (1

### 000 mg/kg) male rats. 

### B. BODYWEIGHTAND WEIGHT GAIN: It

### was reported that the male and female rats did not show any significant changes inbody weight according to the dose ofsilver nanoparticles during the 28-day experiment. The data tables for body weight and weight gain were not included inthis report. 

### C. FOOD CONSUMPTION:Silver nanoparticles/ <PC Code 072599) Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) IPage 7 of 13 OPPTS 870.3100/DACO 4.3.1/ OECD 408 Erythrocyte Micronucleus Assay (rat) 2007 OPPTS 870.5395 

### 1. Food consumption: It

### was reported that there were no significant differences infood consumptionbetween 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 intotal protein for high-dose (1000 mg/kg/day) male rats following a 28-day oral exposure to silver nanoparticles (Table 1A). The ALP inmid-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 inthe control. There were significant increases incholesterol inboththe 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 = I0, 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:anitrogen; T-Bll.(mg/dL), tOial bilirubin; IP(mg/dl). inorganic phosphorus;TG (mg/dl), triglyceride; CPK (U/1.), creatinephosphokinase; Na (mmoi/L), sodium; K (mmoi/L), potassium; Cl(mmoi/L), chloride.

•Significant difference vs. control, p < .0I. bSignificant difference vs. control, p < .OS.Silver nanoparticles/ (PC Code 072599) Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) IPage 8 of 13 OPPTS 870.3100/DACO 4.3.11 OECD 408 ErythrocyteMicronucleus Assay (rat)2007 OPPTS 870.5395
TABLE lB Bloodbiochemistrydata for femalerm (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 ± 17.74 1 117.50 ± 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

"Significantdilfcn:nc:e vs.conlrOI, p < .01.


### 2. Hematology: The MCV (p 

### < 0.05) inthe high-dose male rats andthe RBC (p 

### < 0.05 

### top < 0.01), Hb (p 

### < 0.01), and HCT (p 

### < 0.01) inboththe mid- and high-dose female rats increased significantly compared to that inthe controls. Hematological values for male and female rats are given in Tables 2A and 2B below. In

### addition, the coagulationtime in terms ofthe 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) IPage 9 of 13 OPPTS 870.3100/DACO 4.3.1/ OECD 408 Erythrocyte Micronucleus Assay (rat)2007 Silver nanoparticles/ <PC Code 072599) OPPTS 870.5395 TABLE2B Hematological data forfemale 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
.71 11 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

•significantdifference 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. "Significantdifference vs. control. p < .05. 10.96 ± 2
.89 10.28 ± 3.67 8.81 ± 2.48"Silver nanoparticles/ <PC Code 072599) Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) I Page 10 of 13 OPPTS 870.3100/DACO 4.3.1/ OECD 408 Erythrocyte Micronucleus Assay (rat) 2007 OPPTS 870.5395 F. URINALYSIS: No data onurinalysis reported. G. SACRIFICE AND PATHOLOGY: Atthe end ofthe 28 day oral test, the rats were necropsied after 24 hours. Silver distribution intissue was examined. This examinationrevealed that all the groups exposed to silver nanoparticles for 28 days showed dose-dependent increases inthe silver concentration withinthe testes, kidneys, liver, brain, lungs, stomach andblood of male rats andthe kidneys, liver, brain, lungs, stomach andblood offemale rats. The kidneys also showed a sex-dependent accumulation ofsilver, with a two-fold higher accumulation inthe female rats when compared with the male rats across all dose groups (Table 4)
. Table 4. Tissue contentofsilver 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

. •Sianificantdiffeml(:e vs.c:onuot. p < .OS; ••sianificantdiffe"'nc:e vs.control. p < .01. 1. Organweight: The report states that there were no significant organ-weight changes observed in either male orfemale rats after 28 days ofsilver nanoparticle administration, with exception in the weight ofthe brain and liver. There was a weight increase inthe brain for the high-dose male rats (p 

### < 0.05) and the liver for the mid-dose female rats. The datafor 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 veinto hepatic lobule; (b) infiltration ofinflammatory cells, including eosinophils, inhepatic lobule andportal tract; (c) infiltration ofinflammatory cells in and beneaththe central vein; and (d) dilated central vein filled withbluish amorphous material and blood cells. Interestingly, there appeared to be an increased accumulation ofsilver via microscopic examination inthe kidneys offemale 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, ornasal cavity.Silver nanoparticles/ (PC Code 072599) Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) IPage 11 of13 OPPTS 870.3100/DACO 4.3.11 OECD 408 Erythrocyte Micronucleus Assay (rat) 2007 OPPTS 870.5395 H. ADDITIONAL ANALYSIS: A ratbone marrowmicronucleus assay test was conducted at the end ofthe 28-day oral study for the detection ofcytogenetic damage. The study author examinedthe number ofmicronucleatedpolychromatic erythrocytes (MN-PCEs) among 2000 polychromatic erythrocytes (PCEs) per animal (bone marrow) using a fluorescent microscope. To evaluate bone marrowtoxicity, the PCE/(PCE + NCE) ratio was calculated. The frequency ofmicronucleatedpolychromatic erythrocytes (MN PCEs) in 2000 PCEs for the male rats was 6.00, 6.60, and 7.40 for the groups exposed to concentrations of30, 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 of30, 300, and 1000 mg/kg/day. Respectively, while thatfor the control was 2.SO. Thus, although a dose-related increase was found inthe number ofmicronucleated polychromatic erythrocytes (MN PCEs) inthe male rats, no significanttreatment-related increase ofMNPCEs was detected inthe male and female rats when compared to the corresponding negative controls (Tables SA and SB). No statistically significant difference inthe PCE/(PCE+ NCE) ratio, representing the absence ofbone marrowcytotoxicity, was observed inthe male and female rats after silver nanoparticle exposure when compared withthe control (Tables SA and SB). Table SA Frequency ofMNPCEs and PCE/(PCE +NCE) ratio in bone maiTOW ofmale rats treated orally with indicated doses ofsilver nanoparticles for 28 days Dose Numberofrats Frequency ofMN 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 polychromaticerythrocytes; PCE, polychromatic erythrocytes; NCE. nonnochromatic erythrocytes.

Table SB Frequency ofMNPCEs and PCE/(PCE 

### +NCE) ratio in bone marrow offemale rats treated orally with md1cateddoses ot sliver nanoparticles for 28 days Dose Numberofrats Frequency ofMN 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. polychromaticerythrocytes; NCE. normochromatic erythrocytes.Silver nanoparticles/ (PC Code 072599) Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) I Page 12 of 13 OPPTS 870.3100/DACO 4.3.11 OECD 408 ErythrocyteMicronucleus Assay (rat) 2007 OPPTS 870.5395 

### III. INVESTIGATORS' DISCUSSION AND CONCLUSIONS: The investigators concluded that exposure to over 300 mg/kg/day ofsilver nanoparticles mayresult in slight liver damage. Liver toxicity evaluated byhistopathology included dilation ofthe central vein, bile-duct hyperplasia, and increased foci. It

### was also stated that a gender-related difference in the accumulation ofsilver was noted in the kidneys, with a two-fold increase inthe female kidneys when compared with silver accumulation in male kidneys. Further, the study author stated that there was an increase in blood coagulationtime along with dose-dependent increases ofsilver concentrations in the blood, indicating thatthe orally absorbed silver nanoparticles were able to enterthe blood circulation and affect peripheral blood coagulation. 

### A. REVIEWERCOMMENTS: The reviewer concurs that oral exposure to silver nanoparticles induced dose-dependent changes in clinical chemistry, hematology, and nanosilver accumulation inmajor tissues/organs evaluated, at dose level equal or greater than 300 mg/kg/day, following repeat exposure ofthis test substance over a 28-day time period. Although the dose level was not clearly stated in the content ofthe article for the observedhistopathology changes in the liver, the author indicated inthe abstract section that"

......some significant dose-dependent changes were found inthe alkaline phosphatase and cholesterol values in either the male or female rats, seeming to indicate that exposure to over more than 300 mg ofsilvernanoparticles mayresult in slight liver damage

.......". The reviewer believes the histopathology changes in the liver happened at 300 mg/kg/day and higher. 

### TheNOAEL is 30 mglkglday and theLOAEL 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 ofinflammatory cells, and dilation ofthe central vein), and dose-dependent significant accumulation ofnanosilver in tissues, including gender-dependent accumulation in the kidneys. 

### B. STUDY DEFICIENCIES: Majordeficiencies (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 

### Minordeficiencies (data/information not provided in published study) • Dose selection rationale • Dose of7 days/week • Urinalysis data • Justification ofvehicle used • The dose level(s) at whichthe histopathology changes in the liver were observedSilver nanoparticles/ (PC Code072599) Repeated Dose (28-Day) Oral Toxicity in (rat) 2007) IPage 13 of13 OPPTS 870.3100/DACO 4.3.1/ OECD 408 Erythrocyte Micronucleus Assay (rat) 2007 OPPTS 870.5395 

### 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 

### inrats.