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OPPTS 870.3100/ DACO 4.3.1/ OECD 408 

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#: 1,003,285 

Signature: ~~~ ~ 

Date 6~/tJt&t3 ~I 

Signatu~ 

Date 0~ /of/VJ I j 

DATA EVALUATION RECORD 

STUDY TYPE: 90-Day Oral Toxicity (gavage, rats); OPPTS 870.3100 [' 82-1a] (rodent); 

OECD 408. 

PC CODE: 072599 DP BARCODE: D411279 

TEST MATERIAL (PURITY): silver nanoparticles at least 99.98% pure 

CITATION: Kim, Y.S., Song, M.Y., Park, J.D., et al. (2010) Subchronic oral toxicity of silver 

nanoparticles. Particle and Fiber Toxicology, 7:20. Published online 2010 

August 6. doi: 10.1186/1743-8977-7-20 MRID 49052006. 

http:/ /www.ncbi.nlm.nih.gov/pmc/articles/PMC2928176/pdf/1743-8977 -7-20.pdf 

EXECUTIVE SUMMARY: 

In a 90-day oral toxicity study (MRID 49052006), silver nanoparticles (purity at least 99.98%, 

median diameter is 56 nm [ carboxymethylcellulose-coated] and geometric standard deviation is 

1.46 nm) were administered to ten (Fisher 344) rats/sex/dose by gavage at dose levels of0.5% 

carboxymethylcellulose (vehicle control), 30 mg/kg/day (low-dose), 125 mg/kg/day (middledose), and 500 mg/kg/day (high-dose) for 13 weeks. Bodyweight was measured weekly. 

Clinical chemistry and hematology was measured at the end of the study. Organ weight and 

histopathology were determined at study termination along with analysis of selected tissue for 

silver (Ag) concentration. 

Food consumption was not affected by treatment. There was a significant decrease in dose 

related body weight at the mid dose level in rats at 10 weeks and at the high dose level in rats at 

4, 5, 7 and 13 weeks of exposure. Similarly, there were no significant or biological relevant 

organ weight changes. There was a significant increase (P < 0.01) in alkaline phosphatase (ALP) 

for female rats in the high-dose groups, although it did not reach statistically significant in the 

mid- and high-dose males. A significant increase (P < 0.01) in cholesterol in the mid- and highdose males and the high-dose females and a significant increase (P < 0.05) in total bilirubin in 

the mid-dose males were observed; whereas, a significant decrease in magnesium, protein, and 

inorganic phosphorus was noted in the mid- and high-dose females. A significant increase (P < 

0.05) in monocytes was reported in the high-dose female rats. The reticulocyte count for the 

low-dose female rats decreased significantly (p < 0.05) when compared with the control group. 
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Treatment-related bile-duct hyperplasia, with or without necrosis (focal, multifocal, or lobular), 

fibrosis, and/or pigmentation were identified in all treated animals, both males and females. 

Treatment-related minimum or mild renal unilateral or bilateral mineralization was observed in 

all treated female rats. A dose-dependent increase in pigmentation of villi in the intestines was 

observed in all treated male rats and a slight change in pigmentation was noted in high dose 

female rats. 

There was a statistically significant (P < 0.01) dose-dependent increase in the silver 

concentration of all tissues evaluated, with a two-fold higher accumulation of silver in the 

kidneys of female rats, compared with the male rats across all the dose groups, indicating a 

gender-dependent distribution. 

Based on histopathological evidences of liver toxicity (bile-duct hyperplasia with focal, 

multifocal, or lobular necrosis) in both males and females, a LOAEL is determined to be~ 30 

mglkglday and a NOAEL could not be established(< 30 mglkglday). 

The 90-day oral toxicity study is classified Acceptable/non-guideline and may be used for 

regulatory purposes; however, it does not satisfy the guideline requirement OPPTS 870.3100 for 

a subchronic oral study in the rat. 

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality 

statements were not provided. 
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I. MATERIALSANDMETHODS: 

A. MATERIALS: 

1. Test material: 

Description: 

Lot/batch #: 

Purity: 

Compound stability: 

CAS # of TGAI: 

Structure: 

Silver nanoparticles 

Silver nanoparticles, The median diameter and geometric standard deviation of silver 

nanoparticles in 0.5% aqueous carboxymethyl cellulose analyzed by electron microscopy is 

56 nm ± 1.46 nm, respectively. 

99.98% a.i. 

Not available 

2. Vehicle: Carboxymethylcellulose 0.5% (CMC, Sigma USA) 

3. Test animals: 

Species: 

Strain: 

Age/weight at study initiation: 

Source: 

Housing: 

Diet: 

Water: 

Environmental conditions: 

Acclimation period: 

B. STUDY DESIGN: 

Rats 

Fisher 344 

five weeks old 

Japan SLC Inc. 

polycarbonated cages with a maximum of3 rats per cage. 

fed a rodent diet (Harlan Teklad, USA) 

ad libitum 

Temperature: 22.2 ± 1.7 oc 

Humidity: 48.4 ± 6.0% 

Air changes: 

Photoperiod: 12 hrs light/dark 

For 7 days before starting the experiment 

1. In life dates: The study's Start: not provided; End: not provided. 

2. Animal assignment: Animals were assigned into 4 different groups with 10 rats/sex/dose 

group. The dose groups were 30 mg/kg/day (low-dose), 125 mg/kg/day (mid-dose), 500 

mg/kg/day (high-dose), and 0.5% carboxymethylcellulose (a vehicle control) 

3. Dose selection rationale: The dose levels selected for this 90-day oral toxicity study was 

based on the 28-day oral toxicity study (Kim et al., 2007) using Sprague-Dawley rats with a 

NOAEL and LOAEL of30 mg/kg and 300 mg/kg, respectively. 

4. Diet preparation and analysis: The rats were fed a rodent diet (Harlan Teklad, USA) and 

filtered water ad libitum. Information on diet preparation, details on storage, stability, and 

concentration were not provided. 

Results: 

Homogeneity analysis: not provided 

Stability analysis: not provided 
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Concentration analysis: not provided 

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5. Statistics: Statistical evaluation was performed by analysis oftwo-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. Relative organ weights, serum values, 

hematological values, reticulocyte counts, histopathology, and tissue silver distribution were 

analyzed for male and female rats. 

C. METHODS: 

1. Observations: 

la. Cageside observations: No specific information was provided on the frequency of 

animals being tested for signs of toxicity and mortality. 

lb. Clinical examinations: No specific information was provided on clinical examinations. 

lc. Neurological evaluations: The study did not conduct any neurological evaluations. 

2. Body weight: The study did not specifically describe the frequency of weighing the animals 

during the study period. However, body weight changes were represented graphically 

(Figure 2 of the study article) for each week during the study. 

3. Food consumption and compound intake: The food consumption along with the food 

efficiency, and compound intake were not specifically stated in the study, although were 

briefly mentioned in the result section of the study report. 

4. Ophthalmoscopic examination: No specific ophthalmological examinations were 

conducted; however, the eyes were examined for abnormalities, inflammation of the 

Harderian gland, and other microscopic findings for histopathology. 

5. Hematology and clinical chemistry: After the completion of the 13-week experiment, food 

was withheld for 24 hours before necropsy, and the rats were anesthetized with C02 gas. 

Blood was then drawn from the abdominal aorta, and collected in heparinized vacutainers, 

and analyzed for hematology and clinical chemistry. The CHECKED (X) parameters were 

examined. 
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a. Hematology: 

X Hematocrit (HCT)* 

Hemoglobin (HGB)* 

Leukocyte count (WBC)* 

Erythrocyte count (RBC)* 

Platelet count* 

Blood clotting measurements* 

(Thromboplastin time) 

(Clotting time) 

X (Prothrombin time) 

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Leukocyte differential count* 

Mean corpuscular HGB (MCH)* 

Mean corpusc. HGB conc.(MCHC)* 

Mean corpusc. volume (MCV)* 

Reticulocyte count 

* Recommended for 90-day oral rodent studies based on Guideline 870.3100 

b. Clinical chemistry: 

X ELECTROLYTES X OTHER 

X Calcium X Albumin* 

Chloride Creatinine* 

Magnesium Urea nitrogen* 

Phosphorus Total Cholesterol* 

Potassium* Globulins 

Sodium* Glucose* ==~~~~~~~~======~=9~~~ ENZYMES (more than 2 hepatic enzymes eg., *) Total bilirubin 

~~==========~================9~~~ Alkaline phosphatase (ALK)* Total protein (TP)* 

Cholinesterase (ChE) Triglycerides 

Creatine phosphokinase Serum protein electrophoresis 

Lactic acid dehydrogenase (LDH) 

Alanine aminotransferase (ALT/also SGPT)* 

Aspartate aminotransferase (AST/also SOOT)* 

Sorbitol dehydrogenase* 

Gamma glutamyl transferase (GGT)* 

Glutamate dehydrogenase 

* Recommended for 90-day oral rodent studies based on Guideline 870.3100 

6. Urinalysis: Urinalysis was not conducted in this study. 

7. Sacrifice and pathology: After the rats were killed by cervical dislocation, the CHECKED 

(X) organs were collected, weighed, and fixed with 10% formalin solution containing neutral 

phosphate-buffered saline; subsequently, the organs were embedded in paraffin, stained with 

hematoxylin and eosin, for histological examination. In addition, organs like the liver, 

kidneys, lungs, blood, brain, and testes were examined for determining the tissue 

concentration of silver. In order to determine the silver concentration, the tissues were 

digested with cone. nitric acid by using microwave digestion system (MARS 230/60, CEM) 

and analyzed with flameless method using atomic absorption spectrophotometer equipped 

with a Zeeman graphite furnace (Perkin Elmer 51 OOZL, Zeeman Furnace Module, USA) 

based on the NIOSH 7300 method. 
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DIGESTIVE SYSTEM X CARDIOV ASC./HEMA T. 

X Tongue Aorta* 

Salivary glands* X Heart*+ 

X Esophagus* Bone marrow* 

Stomach* Lymph nodes* 

Duodenum* X Spleen*+ 

Jejunum* X Thymus*+ 

Ileum* 

Cecum* X UROGENITAL 

Colon* X Kidneys*+ 

Rectum* X Urinary bladder* 

X Liver*+ X Testes*+ 

Gall bladder (not rat)* X Epididymides*+ 

Bile duct (rat) X Prostate* 

X Pancreas* X Seminal vesicles* 

X RESPIRATORY X Ovaries*+ 

X Trachea* X Uterus*+ 

X Lung* Mammary gland* 

X Nose* 

Pharynx* 

Larynx* 

*Recommended for 90-day oral rodent studies based on Gu1delme 870.3100 

+ Organ weights required for rodent studies. 

II. RESULTS: 

A. OBSERVATIONS: 

X NEUROLOGIC 

X Brain*+ r--- Peripheral nerve* r--- Spinal cord (3 levels)* 

r---x Pituitary* r--- Eyes (optic nerve )* 

X GLANDULAR 

X Adrenal gland*+ 

Lacrimal gland 

Parathyroid* 

r--x- Thyroid* 

X OTHER 

Bone (sternum and/or femur) r--- Skeletal muscle r--- Skin* 

r---

'--- All gross lesions and masses* 

-

-

-

-

1. Clinical sims of toxicitv: No specific cageside observations along with the details on 

observing the clinical examinations during the study were reported. 

2. Mortality: No assessment on mortalities and morbidities were reported during the study. 

3. Neurological evaluations: No neurological evaluations were conducted. 

B. BODY WEIGHT AND WEIGHT GAIN: There were no significant body weight 

related changes in female rats; however, there were significant decreases in the body weights of 

male rats in middle-dose group at 10 weeks, and high-dose at 4, 5, 7, and 13 weeks of exposure. 

C. FOOD CONSUMPTION AND COMPOUND INTAKE: 

1. Food consumption: There were no significant differences in food consumption and water 

intake between treated animals with the control group. 

2. Compound consumption: No information on compound consumption in terms of time 

weighted averages was reported. 

3. Food efficiency: No information on food efficiency related to changes in body weight was 

reported. 
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D. OPHTHALMOSCOPIC EXAMINATION: No specific ophthalmological findings 

were reported; but the histopathological findings in the males and females exhibited minimal eye 

abnormality and inflammation of the Harderian gland. 

E. BLOOD ANALYSES: 

1. Hematology: In females, the reticulocyte count in the low-dose groups decreased (p < 0.05), 

and monocytes in high-dose groups increased (p <0.05) significantly. The blood clotting 

measurements like active partial thromboplastin time, and prothrombin time when compared 

with the controls did not show any significant changes. 

2. Clinical chemistry: The alkaline phosphatase significantly (p <0.01) increased in females at 

high-dose groups. Similarly, but not significantly, male rats in the middle and high-dose 

groups showed an increase in levels of alkaline phosphatase. The total cholesterol level was 

significantly (p <0.01) higher in the middle and high-dose groups male rats, and high-dose 

group female rats. The total bilirubin remained unchanged in females, but was significantly 

(p <0.05) higher in the middle-dose group male rats. Only the female rats showed a 

significant (p < 0.05) decrease in inorganic phosphorous, total protein, and magnesium (p 

<0.01) levels in middle and high-dose groups. Although not statistically significant, the 

amount of lactate dehydrogenase reduced in treated animals by half in middle and high-dose 

groups. Likewise, not statistically significant, creatine phosphokinase in female rats 

increased threefold in the lowest dose group. 

F. URINALYSIS: Urinalysis was not conducted for this 90-day oral study. 

G. SACRIFICE AND PATHOLOGY: 

1. Organ weight: No significant organ-weight changes were observed in either male or female 

rats, except an increase (p < 0.05) in the weight of the left testis for the high-dose males, and 

a decrease in the weight of right kidney in the low and middle-dose females. Although not 

statistically significant in changes, the right side ovary and the uterus weight reduced 

considerably in all dose groups when compared with the controls. 

2. Gross pathology: Not statistically significant, a majority of the changes observed in the 

histopathological findings were in the liver, kidneys, and intestines of male and female rats 

(Table 1 ). Also, an increase in minimal unilateral tubular mineralization in all the treated 

female rats was observed when compared with controls. 
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Table 1: Histopathological findings for male and female rats after 90-day oral administration of 

s1 "I ver pa rti c I es 

Organs Histopathological Rats Control Low-dose Middle-dose High-dose 

findings (N = 10) (N= 10) (N = 10) {N= 10) 

Liver Minimal duct Male 4 7 8 6 

hyperplasia Female 3 7 8 7 

Focal necrosis Male 0 4 3 4 

Female 0 2 2 1 

Kidneys Abnormality Male 0 0 1 4 

Female 5 8 7 9 

Intestines Abnormality Male 0 0 8 8 

Female 0 0 0 5 

Yell ow pigmentation Male 0 0 1 8 

of villi Female 0 0 0 0 

Faint yellow Male 0 0 7 0 

pigmentation of villi Female 0 0 0 5 

3. Silver distribution in the tissues: There was a statistically significant (p <0.01) dosedependent increase in the silver concentration in all the treated animals. In general, females 

showed an increase in distribution of silver in their lungs, liver, kidney, brain and lungs. A~ 

2-fold Ag content in the kidney at all dosages when compared to male rats. 

Table 2: Tissue silver content aft~r 90-day oral administrati~n of silver nanoparticles 

Silver (llg/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 • 0.10 ± 0.08 0.001 ± 0.000 

Female O.Ql ± 0.01 0.03 ± 0.01 0.01 ± 0.01 • 0.05 ± 0.02 0.002 ± 0.002 

~ mg/Kg 

Male 6.56 ± 0.33*" 4.20 ± 1 .57** • 1.4~ ± 0.37*" b 0.47 ± 0.18*" 1 .94 ± 0.64** b 0.111 ± 0.016*" 

Female 8.56 ± 3.22** • 7.98 ± 0.~1** b 0.38 ± 0.05*" 4.~7 ± 0.00 b 0.087 ± 0017*" 

125 mg/1<9 

Male 11 .84 ± 1.62"* 10.1~ ± 2.0~ b 8.82 ± 21 3** b 0.6~ ± 0.06*" 10.~7 ± 3.81*" 0.1 ~1 ± 0.037*" b 

Female 2~.13 ± ~.74*" b 37.09 ± 17.44** b 0.77 ± 0.11*" 17.64 ± ~.06*" 0.122 ± O.QlO*" b 

500 mg/Kg 

Male 23.75 ± ~.13** 68.65 ± 335~ ~.1 ~ ± 32.82** b 3.54 ± 1.44*" 56.Q4 ± 51.14 0.41 ~ ± 0.083*" 

Female ~.75 ± 31.65*" 226.88 ± 55.64*" b 3.70 ± 1.17*" 45.83 ± 1 1.43** 0.303 ± 0.()~~ 

-Significant diff~r~c~ V5. control, p < 0.01. (ANOVA) 

• Significant dlffer~nc~ vs. dl5tlnctlon cf ~x. p < 0.05. (T-t~st) 

b Significant dlfferenc~ vs. distinction of s~x, p < O.o1. (T-te3t) 

III. DISCUSSION AND CONCLUSIONS: 

A. INVESTIGATORS' CONCLUSIONS: The study described the liver and the kidneys 

as the potential target organs being affected by silver nanoparticles, with twice as likely of 
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observing silver deposition in kidneys of female rats. In addition, the study found an increase in 

the silver concentrations in the intestinal villi, and in blood, which indicated the absorption of 

silver from nanoparticles into the circulatory system and other organs. Moreover, the target 

organs of toxicity with silver nanoparticles seem to be similar to silver ions exposure via 

inhalation and oral route of exposures (Kim et al, 2008, Sung et al, 2009). With the evidence 

from previous studies on varying silver nanoparticles sizes, routes of exposure, and treatmentrelated endpoints, the authors suggested, that the deposition of silver is neither dependent on the 

particle size (tested for earlier inhalation studies at 15 to 19 nm or oral study at 56 to 60 nm) nor 

on the route of exposure. However, in order to determine "the relationship between particle 

effect and effect from ionized silver a quantitative examination oftarget tissues for the presence 

ofnanoparticles with a concurrent measurement oftissue ionized silver as opposed to measuring 

total tissue silver alone must be conducted." The study reported the NOAEL = 30 mg/kg, and 

LOAEL = 125 mg/kg/day, based on the results described in the study. 

B. REVIEWER COMMENTS: All the observed effects are summarized in Table 3. 

Reviewer is in consensus with the author's suggestion on evaluating the effect of silver 

nanoparticles and silver ions. From the study results, both liver and kidneys seem to be the 

target organs of silver nanoparticles deposition, with females exhibiting higher distribution of 

silver nanoparticles in kidneys. 

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

changes in hematology, and histopathological findings in the liver (bile-duct hyperplasia with 

focal, multifocal, lobular necrosis), reviewer believes liver is the primary target organ. Similar 

kinds of effects were noticed in the 28-day oral study (Kim et al, 2008). With all these 

consideration, reviewer believes the liver effects noticed in the low-dose group, including bileduct hyperplasia and necrosis are real effects. Therefore, based on histopathological evidences of 

liver toxicity (bile-duct hyperplasia with focal, multifocal, or lobular necrosis) in both males and 

females, a LOAEL is determined to be~ 30 mglkglday and a NOAEL could not be 

established(< 30 mglkglday). 

C. STUDY DEFICIENCIES: 

Major deficiencies: 

1. The study did not report any detailed information on the food, compound intake, and food 

efficiency, along with homogeneity, stability, and concentration analysis. 

2. No cageside observations or clinical examinations were reported. 

3. The study did not conduct specific ophthalmological, urinalysis, and neurological tests. 

4. The silver nanoparticles distribution was not examined in ovaries, a brief justification 

must be provided on the same. Also, an intestinal distribution of silver nanoparticles 

would be helpful to correlate it with the intestinal histopathological findings. 

5. There was a dramatic increase in creatine phosphokinase at 30 mg/kg/day in female rats, 

an explanation must be provided. 

Minor deficiencies: 

1. The start and the end dates of the study were not provided. 

2. The consumption analysis must be provided as a time-weighted average. 
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3. The study did not provide detailed data on microscopic histopathological findings. 

4. The Table 1 of the study report lacked data on relative organ weights, for instance 

tongue, oesophagus, intestine, gall bladder, bile duct, pancreas, trachea, nasal cavity, 

urinary bladder, and thyroid gland even though they were listed in the method's section 

of the study article. 

5. The albumin/globulin ratio was not reported. 

6. The Tables 8 and 9 on histopathological findings did not provide any detailed 

information on the kind of abnormalities, and the microscopic findings that were being 

examined for this study. 
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REFERENCE: 

Subchronic (90-day) Oral Toxicity Study (Rodents) (201 0)/Page II of I2 

OPPTS 870.3I 00/ DACO 4.3.11 OECD 408 

Kim, J.S., E. Kuk, K. N. Yu et al. (2007). Antimicrobial effects of silver nanoparticles. 

Nanomedicine, 3:95-101. 

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 SpragueDawley Rats. Korea Environment and Merchandise Testing Institute, Incheon, Korea. 

Inhalation Toxicology. 20: 575-583. 

Sung, J.H., Ji, J,H., and Park, J.D et. al. (2009). Subchronic inhalation toxicity of silver 

nanoparticles. Toxicol Sci. 1 08(2):452-61. 
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Table 3: Summary of Results from MRID 49052006; Kim, YS, et al2010. 90-d oral toxicity in rats (OECD TG 408) 

0 (n=10) 30 mg/kJl'd (n=10) 125 mg/kgld (n=10) 500 mglkgld (n=10) 

a ~ a ~ a ~ a ~ bw ! wk 10 (p<0.05) ! wk 4, 5, 7 (p<0.05) 

Left testis weight t (p<0.05) 

Right kidney weight ! (p<0.05) ! (p<0.05) 

ALP t 

(P < 0.01) 

Cholesterol t (P < 0.01) t (P < 0.01) t 

(P < 0.01) 

Total bilirubin t (P < 0.05) 

Magnesium, protein, and inorganic ! (p<0.05?) ! (p<0.05) 

phosphorus 

Monocytes t (P < 0.05) 

Reticulocyte count ! (p<0.05) 

Minimal bile-duct hyperplasia 4 3 7 7 8 8 6 7 

Focal, multifocal, lobular necrosis 0 0 4 2 5 2 4 2 

MinimaVmild renal unilateral or bilateral 5 8 7 9 

mineralization 

Pigmentation of villi 0 0 8 8 

(the intestine) 

Silver cone. of all the tissue samples Sig. dose-dependent t (p < 0.01) with a two-fold higher accumulation of nAg in the kidneys offemale rats.