NANOSIVLER (PC Code 072599) Subchronic (90-day) Oral Toxicity Study (Rodents) (2010)/Page I of 12 OPPTS 870.3100/ DACO 4.3.1/ OECD 408


### EPAReviewer: Jonathan Chen, Ph. D. 

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

### RiskAssessment and Science SupportBranch Antimicrobial Division

TXR#: 1,003,285 

### Signature:

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DATA EVALUATIONRECORD

STUDY TYPE: 90-Day Oral Toxicity (gavage, rats); OPPTS 870.3100 [' 82-1a] (rodent); OECD 408.


### PC CODE: 072599 

### DP BARCODE: D411279


### TESTMATERIAL (PURITY): silvernanoparticles at least 99.98% pure


### CITATION: Kim, Y.S., Song, M.Y., Park, J.D., et al. (2010) Subchronic oral toxicity ofsilver 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. Bodyweightwas measured weekly. Clinical chemistry and hematology was measured at the end ofthe study. Organ weight and histopathology were determined at study termination along with analysis ofselectedtissue 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 atthe high dose level in rats at 4, 5, 7 and 13 weeks ofexposure. Similarly, there were no significant orbiological relevant organ weight changes. There was a significant increase (P 

### < 0.01) in alkaline phosphatase (ALP) for female rats inthe high-dose groups, although it did not reach statistically significant in the mid- andhigh-dose males. A significant increase (P 

### < 0.01) in cholesterol inthe mid- and highdose males and the high-dose females and a significant increase (P 

### < 0.05) intotal 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 withthe control group.NANOSIVLER (PC Code 072599) Subchronic (90-day) Oral Toxicity Study (Rodents) (2010)/Page 2 of 12 OPPTS 870.3100/ DACO 4.3.1/ OECD 408
Treatment-related bile-ducthyperplasia, with or without necrosis (focal, multifocal, or lobular), fibrosis, and/orpigmentation were identified in all treated animals, both males and females. Treatment-relatedminimum ormildrenal unilateral orbilateral mineralization was observed in all treated female rats. A dose-dependent increase inpigmentation ofvilli inthe intestines was observed in all treated male rats and a slight change inpigmentation was noted inhigh dose female rats.
There was a statistically significant (P 

### < 0.01) dose-dependent increase inthe silver concentration ofall tissues evaluated, with a two-fold higher accumulation ofsilver inthe kidneys offemale rats, comparedwiththe male rats across all the dose groups, indicating a gender-dependent distribution.
Based onhistopathological evidences ofliver 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 maybe used for regulatory purposes; however, it does not satisfy the guideline requirement OPPTS 870.3100 for
a subchronic oral study inthe rat.


### COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were notprovided.NANOSIVLER (PC Code 072599) Subchronic (90-day) Oral Toxicity Study (Rodents) (2010)/Page 3 of12 OPPTS 870.3100/DACO 4.3.1/ OECD 408


### I. MATERIALSANDMETHODS:
A. MATERIALS:
1. 

### Test material: Description:

Lot/batch #: Purity: Compound stability: CAS # ofTGAI: Structure: Silver nanoparticles Silver nanoparticles, The median diameter and geometric standard deviation ofsilver nanoparticles in 0.5% aqueous carboxymethylcellulose 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) adlibitum 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: notprovided.
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 onthe 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. Dietpreparation and analysis: The rats were fed a rodent diet (Harlan Teklad, USA) and filtered water ad libitum. Information on dietpreparation, details on storage, stability, and concentration were not provided.
Results: Homogeneity analysis: notprovided Stability analysis: not providedNANOSIVLER (PC Code 072599)



### Concentration analysis: notprovided Subchronic (90-day) Oral Toxicity Study (Rodents) (2010)/Page 4 of 12 OPPTS 870.3100/ DACO 4.3.1/ OECD 408



### 5. Statistics: Statistical evaluation was performedby analysis oftwo-tailed student'st-test or analysis ofvariance (ANOVA) following multiple comparisontests with Duncan'smethod. The level ofstatistical 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 onthe frequency of animals beingtested for signs oftoxicity 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 ofweighingthe animals during the studyperiod. However, body weight changes were represented graphically (Figure 2 ofthe 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 inthe study, although were briefly mentioned inthe result section ofthe study report.


### 4. Ophthalmoscopic examination: No specific ophthalmological examinations were conducted; however, the eyes were examined for abnormalities, inflammation ofthe Harderian gland, and othermicroscopic findings for histopathology.


### 5. Hematology and clinical chemistry: Afterthe completion ofthe 13-weekexperiment, 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.NANOSIVLER (PC Code 072599)



### 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) Subchronic (90-day) Oral Toxicity Study (Rodents) (2010)/Page 5of 12 OPPTS 870.3100/DACO 4.3.11 OECD 408

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 inthis study.


### 7. Sacrifice and pathology: Afterthe rats were killedby 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 inparaffin, stained with hematoxylin and eosin, for histological examination. Inaddition, organs like the liver, kidneys, lungs, blood, brain, andtestes were examined for determining the tissue concentration ofsilver. In orderto determine the silver concentration, the tissues were digested with cone. nitric acid byusing microwave digestion system (MARS 230/60, CEM) and analyzed with flameless methodusing atomic absorption spectrophotometer equipped with a Zeeman graphite furnace (PerkinElmer 51OOZL, Zeeman Furnace Module, USA) based onthe NIOSH 7300 method.NANOSIVLER (PC Code 072599) Subchronic (90-day) Oral Toxicity Study (Rodents) (2010)/Page 6 of 12 OPPTS 870.3100/ DACO 4.3.1/ OECD 408

DIGESTIVE SYSTEM X CARDIOV ASC./HEMAT. 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 oftoxicitv: No specific cageside observations along with the details on observing the clinical examinations during the study were reported.
2. 

### Mortality: No assessment onmortalities and morbidities were reported during the study.
3. 

### Neurological evaluations: No neurological evaluations were conducted.
B. BODYWEIGHT AND WEIGHT GAIN: There were no significantbody weight related changes in female rats; however, there were significant decreases in the body weights of male rats inmiddle-dose group at 10 weeks, and high-dose at4, 5, 7, and 13 weeks ofexposure.
C. FOOD CONSUMPTIONAND COMPOUND INTAKE:
1. 

### Food consumption: There were no significant differences in food consumption and water intake betweentreated animals withthe control group.
2. 

### Compound consumption: No information on compound consumption interms oftime weighted averages was reported.
3. 

### Food efficiency: No information onfood efficiency related to changes inbody weight was reported.NANOSIVLER (PC Code 072599) Subchronic (90-day) Oral Toxicity Study (Rodents) (2010)/Page 7 of 12 OPPTS 870.3100/DACO 4.3.1/ OECD 408



### D. OPHTHALMOSCOPIC EXAMINATION: No specific ophthalmological findings
were reported; butthe histopathological findings inthe males and females exhibitedminimal eye abnormality and inflammation ofthe Harderian gland.


### E. BLOOD ANALYSES:
1. Hematology: In females, the reticulocyte count inthe low-dose groups decreased 

### (p < 0.05), and monocytes inhigh-dose groups increased 

### (p <0.05) significantly. The blood clotting measurements like active partial thromboplastintime, andprothrombintime when compared withthe 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, butnot significantly, male rats inthe middle andhigh-dose groups showed an increase in levels ofalkaline phosphatase. The total cholesterol level was significantly 

### (p <0.01) higher inthe middle and high-dose groups male rats, and high-dose group female rats. The total bilirubinremained unchanged in females, but was significantly 

### (p <0.05) higher inthe middle-dose group male rats. Onlythe 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 oflactate dehydrogenase reduced intreated animals byhalfinmiddle and high-dose groups. Likewise, not statistically significant, creatine phosphokinase in female rats increasedthreefold inthe 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) inthe weight ofthe left testis for the high-dose males, and
a decrease inthe weight ofrightkidney inthe low and middle-dose females. Although not statistically significant in changes, the right side ovary andthe uterus weightreduced considerably in all dose groups when comparedwiththe controls.
2. 

### Gross pathology: Not statistically significant, a majority ofthe changes observed inthe histopathological findings were in the liver, kidneys, and intestines ofmale and female rats (Table 1 ). Also, an increase inminimal unilateral tubular mineralization in all the treated female rats was observed when compared with controls.NANOSIVLER (PC Code 072599) Subchronic (90-day) Oral Toxicity Study (Rodents) (2010)/Page 8 of 12 OPPTS 870.3100/ DACO 4.3.1/ OECD 408

Table 1: Histopathological findings for male and female rats after 90-day oral administration of "I rti I s1 ver pa c 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 Yellow pigmentation Male 0 0 1 8 ofvilli Female 0 0 0 0 Faintyellow Male 0 0 7 0 pigmentation ofvilli Female 0 0 0 5



### 3. Silverdistribution in

### the tissues: There was a statistically significant (p 

### <0.01) dosedependent increase inthe silver concentration in all the treated animals. In general, females showed an increase in distribution ofsilver intheir lungs, liver, kidney, brain and lungs.**A~** 2-fold Ag content inthe kidney at all dosages when comparedto male rats.



### Table 2: Tissue silver content**aft~r** 

### 90-day oral**administrati~n** 

### ofsilver 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 ± 11.43** 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, withtwice as likely ofNANOSIVLER (PC Code 072599) Subchronic (90-day) Oral Toxicity Study (Rodents) (2010)/Page 9 of 12 OPPTS 870.3100/ DACO 4.3.1/ OECD 408 observing silver deposition inkidneys offemale rats. In addition, the study found anincrease in the silver concentrations 

### inthe intestinal villi, and inblood, which indicatedthe absorption of silver from nanoparticles into the circulatory system and other organs. Moreover, the target organs oftoxicity with silver nanoparticles seem to be similarto silver ions exposure via inhalation and oral route ofexposures (Kim et al, 2008, Sung et al, 2009). Withthe evidence from previous studies onvarying silver nanoparticles sizes, routes ofexposure, and treatmentrelated endpoints, the authors suggested, that the deposition ofsilver is neither dependent onthe particle size (tested for earlier inhalation studies at 15 to 19 nm or oral study at 56 to 60 nm) nor onthe route ofexposure. However, inorder to determine "the relationship betweenparticle effect and effect from ionized silver a quantitative examination oftargettissues for the presence ofnanoparticles with a concurrent measurement oftissue ionized silver as opposed to measuring total tissue silver alone mustbe conducted." The study reported the NOAEL = 30 mg/kg, and LOAEL 

### = 125 mg/kg/day, based onthe results described inthe study.



### B. REVIEWER COMMENTS: All the observed effects are summarized in Table 3. Reviewer is in consensus with the author's suggestion onevaluating the effect ofsilver nanoparticles and silver ions. From the study results, both liver and kidneys seem to be the target organs ofsilver nanoparticles deposition, with females exhibiting higher distribution of silver nanoparticles inkidneys.
Based on significant increases in alkaline phosphatase (ALP) and cholesterol (CHO), significant changes inhematology, andhistopathological findings inthe liver (bile-duct hyperplasia with focal, multifocal, lobular necrosis), reviewerbelieves liver is the primarytarget organ. Similar kinds ofeffects were noticed inthe 28-day oral study (Kim et al, 2008). With all these consideration, reviewerbelieves the liver effects noticed inthe low-dose group, including bileduct hyperplasia and necrosis are real effects. Therefore, based on histopathological evidences of liver toxicity (bile-ducthyperplasia with focal, multifocal, or lobular necrosis) in bothmales 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 notreport any detailed information on the food, compound intake, and food efficiency, along withhomogeneity, 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 mustbe provided onthe same. Also, anintestinal distribution ofsilver nanoparticles would be helpful to correlate it withthe intestinal histopathological findings. 5. There was a dramatic increase in creatine phosphokinase at 30 mg/kg/day infemale rats, an explanation mustbeprovided.
Minor deficiencies: 1. The start and the end dates ofthe study were notprovided. 2. The consumption analysis must be provided as a time-weighted average.NANOSIVLER (PC Code 072599) Subchronic (90-day) Oral Toxicity Study (Rodents) (2010)/Page 10 of 12 OPPTS 870.3100/DACO 4.3.1/ OECD 408
3. The study did notprovide detailed data onmicroscopic histopathological findings. 4. The Table 1 ofthe 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 inthe method's section ofthe study article. 5. The albumin/globulin ratio was notreported. 6. The Tables 8 and 9 onhistopathological findings did notprovide any detailed information onthe kind ofabnormalities, and the microscopic findings that were being examined for this study.NANOSIVLER (PC Code 072599) REFERENCE: Subchronic (90-day) Oral Toxicity Study (Rodents) (2010)/Page II ofI2 OPPTS 870.3I00/DACO 4.3.11 OECD 408

Kim, J.S., E. Kuk, K. N. Yu et al. (2007). Antimicrobial effects ofsilver 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 ofSilverNanoparticles in SpragueDawley Rats. KoreaEnvironment 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 inhalationtoxicity ofsilver nanoparticles. Toxicol Sci. 1 08(2):452-61.NANOSIVLER (PC Code 072599) Subchronic (90-day) Oral Toxicity Study (Rodents) (2010)/Page 12 of 12 OPPTS 870.3100/ DACO 4.3.1/ OECD 408

Table 3: Summary ofResults from MRID 49052006; Kim, YS, et al2010. 90-d oraltoxicity in rats (OECD TG 408) 0 (n=10) 30mg/kJl'd (n=10) 125 mg/kgld (n=10) 500 mglkgld (n=10) 

# a**~** 

# a**~** 

# a**~** 

# a**~** bw 

### ! wk 10 (p<0.05) 

### ! wk4, 5, 7 (p<0.05) Lefttestis 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 orbilateral 5 8 7 9 mineralization Pigmentation ofvilli 0 0 8 8 (the intestine) Silver cone. ofall the tissue samples Sig. dose-dependent

### t (p < 0.01) with a two-fold higher accumulation ofnAg in the kidneys offemale rats.