Silver nanoparticles/ (PC Code072599)


### EPA Reviewer: Jonathan Chen, Ph. D. 

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

### RiskAssessment and Science SupportBranch Antimicrobial Division
TXR: 1,003,206 28-Day Oral Toxicity in (Mice) (2010) IPage 1 of 14 OPPTS 870.3050 I OECD 407 

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STUDY TYPE: Repeated-Dose (28-Day) Oral Toxicity [diet]- mice; OPPTS 870.3050; OECD 407. 

### PC CODE: 072599 

### DP BARCODE: D411279 

### TEST MATERIAL (PURITY): Silver nanoparticles 

### SYNONYMS: Nanosilverparticles 

### CITATION: Park, E., E. Bae, J. Yi, et al. (2010) Repeated-dose toxicity and inflammatory responses inmice by oral administration ofsilver nanoparticles. Environmental Toxicology and Pharmacology. 30:162-168. MRID 48385201. 

### SPONSOR: Not applicable 

### EXECUTIVE SUMMARY:
There are two independent studies reported inthis paper: 1. A 28-daymice oral study

- to studythe potential toxicological effects associated with oral exposure to nano-sized silver; and 2. A 14-day mice size difference toxicity test- To studythe effects ofsize on tissue distribution ofdosed nano-silver. 

### 28-day mice oral study: In the repeated-dose oral toxicity study, silvernanoparticles of42 nm diameters in deionized water were orally administered in mice, ICR strain, 6 per group at dose levels of0.25, 0.5, and 1.0 mg/kg/day for 28 days. Following sacrifice, selected organ weights were measured. Histopathology was conducted only onthe liver, kidney and intestine at the highest dose (1.0 mg/kg/day) group. Tissue concentrations ofsilver (Ag) inthe testis, kidney, brain, lungs,Silver nanoparticles/ (PC Code 072599) 28-Day Oral Toxicity in (Mice) (2010) I Page 2 of 14 OPPTS 870.3050 I OECD 407
stomach and blood were also determined. In addition, blood chemistry and serum cytokine levels were measured.
Atthe dose level of 1.0 mg/kg/day, significant increases in serum enzyme level ofalkaline phosphatase (p <0.01) and aspartate transaminase (p <0.05) were observed inboth males and females. The females group displayed a significant increase in alanine transaminase level (p <0.01). Such increased in serum enzyme levels indicated the liverto be one ofthe target organs ofconcern. In addition, a dose-dependent, statistically-significant increase inthe concentration ofpro-inflammatory cytokines (IL-l, IL-6), Thl-type cytokines (IL-12), Th2-type cytokines (IL4, IL-10), and**TGF-~** in serum, and IgE was observed.
A slightly increase inthe proportion ofB cells was observed in all treatment groups. Atthe 1.0 mg/kg/day dose level, NK, NKT, B, and T cell distribution were all increased compared to the control group. The ratio ofCD4+/CD8+ T cells was decreased indicating an increase ofCD8+T cell distribution.
Inthe 28-day repeated-dose oral toxicity studyinmice with silver nanoparticles of42 nm diameter, statistically significant changes in clinical chemistry, including increase in serum liver enzymes in males (alkaline phosphatase and aspartate transaminase) and females (alkaline phosphatase, aspartate transaminase, and alanine transaminase). A slight cell infiltration inthe cortex ofthe kidneys was observed at 1.0 mg/kg/day dose level inbothmales and females. The toxicological significance ofthese changes is questionable. Therefore, 

### No NOAEL orLOAEL values can be determined from this 28-day repeated-dose toxicity study.
The data ofthe 28-dayrepeated dose oral toxicity study in mice are 

### categorized as "qualitative" and are not appropriatefor quantitative use (i.e., dose selection). The data can be discussed inthe risk characterization as additional lines ofevidence to supportrisk conclusions regarding metabolism, adverse effects ofconcern, life stage susceptibility, and mode oftoxic action. It does not satisfythe guideline requirement for a 28-day oral rodent studybased on Guideline 870.3050. The information onthe purity ofsilvernanoparticles, data on statistical significance oncytokine production, B-cell distribution, inflammatory cell infiltrates are not provided. Histopathology was conducted only onthe liver, kidney and intestine atthe highest dose (1.0 mg/kg/day) group. Thereasoning for observing an increase inthe CD8+cell distribution while analyzing the CD4+/CD8+ ratio were not adequately addressed. Others, including food and water consumptions, neurological evaluations, hematology and ophthalmological examinations, and clinical biochemistry determinations along with a detailed gross necropsy, are also required.


### 14-daymice daily single dose study: A 14-dayoral toxicity study with various sizes (22 nm, 42 nm, 71 nm, and 323 nm) ofsilver nanoparticles in deionized waterwas conducted at a single dose level of 1.0 mg/kg/day in mice (5/group). Statistically significant (p <0.01) amounts ofsilver nanoparticles were accumulated in all the tested organs (brain, lungs, liver, kidneys, and testes) in mice being administered orallySilver nanoparticles/ (PC Code072599) 28-Day Oral Toxicity in (Mice) (2010) IPage3 of 14 OPPTS 870.3050 I OECD 407
with 22 run, 42 run, and 71 run silvernanoparticles, with the greatest accumulation seen in the 22 run treatment group. This greatest accumulation at 22 run diameter size ofsilver nanoparticles raises significant concerns aboutpenetration, retention, assimilation, and elimination within the organs ofsilvernanoparticles at smaller size compared to larger size, such as 323 run. The results are summarized in Table 2 below. In addition, the level ofTGF-P was significantly increased (p<0.01) in mice treated with smallersizednanosilver particles, such as 22 run, 42 run and 71 run, but not with larger-sized nanosilver particles (e.g., 323 run), when compared with the control group. 

### The study indicating the particle size is importantin determiningthe tissue distribution after oralexposure ofthe nano-sized material.
COMPLIANCE: Signed and dated GLP, Quality Assurance, andNo Data Confidentiality statements are not required.Silver nanoparticles/ (PC Code 072599) 28-Day Oral Toxicity in (Mice) (2010) I Page4 of 14 OPPTS 870.3050 I OECD 407


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

### TestMaterial: Silvernanoparticles (Cat. No. 484059, Sigma-Aldrich, USA) Description: Lot/Batch #: Purity: Compound Stability: CAS#:
2. 

### Vehicle and/orpositive control: Tetrahydrofurane, Fluka, 99.8% pure 

### 3. Test animals:
Species: Mice Strain: ICR Age/weight at study 6-weeks initiation: Source: Orient-Bio Animal Company (Seongnam, Gyunggi-do, Korea) Housing: Not provided. The author stated using the principles from "Guide for the Care and Use of Laboratory Animals issued by the Animals Care and Committee ofNational Veterinary Research and Quarantine Service" for caring ofthe animals. Diet: Not provided. The author stated using the principles from "Guide for the Care and Use of Laboratory Animals issued by the Animals Care and Committee ofNational Veterinary Research and Quarantine Service" for caring ofthe animals. Water: Not provided. The author stated using the principles from "Guide for the Care and Use of Laboratory Animals issued by the Animals Care and Committee ofNational Veterinary Research and Quarantine Service" for caring ofthe animals. Environmental Temperature: 23 ± l°C conditions: Humidity: 55±5% Air changes: Not provided. Photoperiod: 12 hrs dark! light cycle Acclimation period: 1-weekpriorto the initiation ofthe study



### B. STUDY DESIGN: 1. 

### In life dates

- Start: not applicable End: not applicable
2. 

### Animal assignment: Animals were randomly assigned to both 14-day and 28-day for repeated-dose toxicity testto silvernanoparticles. In the 14-day study, various sizes such as 22 nm, 42 nm, 71 nm, and 323 nm ofsilver nanoparticles were tested at one single dose of 1.0 mg/kg/day with 5 mice per group. In 28-day repeated-dose toxicitytest, only one size, 42 nm, silver nanoparticles atvarious doses of0.25 mg/kg/day, 0.5 mg/kg/day, and 1.0 mg/kg/day were tested with 6 mice per group. The control group was treated withde-ionized water.Silver nanoparticles/ (PC Code 072599) 28-Day Oral Toxicity in (Mice) (2010) IPage 5 of 14 OPPTS 870.3050 I OECD 407



### 3. Dose selection rationale: No rationale was provided for studyingthe various silver nanoparticle sizes inthe 14-day study orthe dose selection rationale with 42 run diameter size of silver nanoparticles 

### in the 28-day study. 

### 4. Treatmentpreparation, administration, and analysis: Silver nanoparticles were suspended with sonication withtetrahydrofurane (99.8%, Fluka)whichwas evaporated by stirringthe suspension. The evaporatedtetrahydrofurane volume was compensated withthe addition of deionized waterto the suspension for a day or two to finally replace tetrahydrofurane. Thereafter, the silvernanoparticles suspension was filtered through varying pore sizes ofpolycarbonated isopore filters to yield different sizes ofsilver nanoparticles.
For the 14-day study, the different sizes ofsilver nanoparticles in diameters were 22 run, 42 run, 71 run, and 323 run, whichwere administered orally in a single dose of 1.0 mg/k.g/day. Forthe 28-day repeated-dose toxicity study, only the 42 run size silver nanoparticle was administered orally at 3 different doses, whichwere 0.25 mg/k.g/day, 0.5 mg/k.g/day, and 1.0 mg/k.g/day. The control group was prepared withthe deionized water. 

### Results Homogeneity(% coefficient ofvariation) Notprovided 

### Stability (range of% ofinitial concentration) Notprovided 

### Concentration (range of% ofnominal concentration) Notprovided



### 5. Statistics

- The results ofthe treated group were compared using the student'st-test and one way ANOVA analysis withthe control group for analyzing several parameters like accumulation ofsilver in the organs, serum level ofTGF-p, ratio oforganto body weight exposed to varying size ofsilver nanoparticles, and blood biochemical parameters. 

### C. METHODS: 1. 

### Observations:
la. Cageside Observations- No specific data or information was provided onthe frequency of mortality and toxicity.
lb. Clinical Examinations- No specific data or information was provided onthe frequency of clinical examinations
lc. Neurological Evaluations- No neurological evaluations were conducted for this study.Silver nanoparticles/ (PC Code072599) 28-Day Oral Toxicity in (Mice) (2010) I Page 6 of14 OPPTS 870.3050 I OECD 407

2. Body weight: Bodyweight was measured onday 0, 7, 14, 21, and 28. 3. Food consumption: No information or data were provided on food consumption. 4. Ophthalmoscopic examination: Eyes were not examined inthis study. 5. Hematology & Clinical Chemistry: No specific data orinformation was provided on hematology; however, for biochemical analysis, 1ml ofblood was collected from the retroorbital venous plexus from eachmouse using heparinized capillary tubes. Fromthe withdrawn 1 ml blood, 100 Ill was used for cell phenotype analysis, and the rest was centrifuged at 3000 rpm for 10 minfor preparing the serum. The serum was stored inthe -80 °C freezer priorto blood biochemical analyses, whichwere measured using an auto-analyzer (Hitachi 7180, Hitachi, Japan). Inthe repeated oral toxicity test, blood was obtained from 3 mice ofeach group for cell phenotype and blood biochemistry analysis, and the other 3 were used for blood withdrawing for cytokine assay.

a. Clinical Chemistry

ELECTROLYTES OTHER Calcium X Albumin* Chloride X Creatinine* Magnesium X Urea nitrogen* Phosphorus Total Cholesterol* Potassium* (K) Globulins Sodium* (NA) Glucose* ENZYMES (more than 2 hepatic enzymes, eg., *) Total bilirubin X Alkaline phosphatase (AP)* X Total protein (TP)* Cholinesterase (ChE) Triglycerides Creatine phosphokinase Serumprotein electrophoresis Lactic acid dehydrogenase (LDH) X Alanine aminotransferase (ALT/also SGPT)* X Aspartate aminotransferase (AST/also SGOT)* Gamma glutamyl transferase (GGT)* Glutamate dehydrogenase Sorbitol dehydrogenase*



### *Recommended for 28-day oral rodent studies based on Guideline 870.305
6. Urinalysis: Although optional for 28-day repeated dose toxicity study, no information or data was provided onurinalysis. 7. Sacrifice and Pathology: Althoughthe author did not specifically state about the histological techniques used 

### inthis study, the kidneys, livers, and small intestines ofthe control andtreated groups were fixed with 10% neutral buffered formalin and processedusing routine histological techniques. Afterthe paraffin embedding, which was not explained inthe article, 3 llm sectionsSilver nanoparticles/ (PC Code072599) 28-Day Oral Toxicity in (Mice) (2010) I Page 7 of 14 OPPTS 870.3050 I OECD 407

were cut and stained with hematoxylin and eosin for histopathologic evaluation. Injuries, cellular changes, and inflammations were examinedmicroscopically.
All animals after blood drawing were sacrificed and subjectedto grosspathological examination check. The CHECKED (X) tissues were collected for histological examination.

X X X

X DIGESTIVE SYSTEM CARDIOV ASC.IHEMAT. Tongue Aorta, thoracic* X Salivary glands* Heart*+ Esophagus* Bone marrow* Stomach* Lymph nodes* Duodenum* Spleen*+ Jejunum* Thymus*+ Ileum* Cecum* UROGENITAL Colon* X Kidneys*+ Rectum* Urinary bladder* Liver*+ Testes*+ Gall bladder* (not rat) Epididymides*+ Bile duct* (rat) Prostate* Pancreas* Seminal vesicles* RESPIRATORY Ovaries*+ Trachea* Uterus*+ Lung* Mammary gland* Nose* Pharynx* Larynx* * Recommended for 28-day oral rodent studies based on Gmdehne 870.3050 + Organ weights required for rodent studies. 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*

8. 

### Inflammatory responses: The author further evaluatedthe inflammatory responses by measuring the amount ofcytokine and lgEproduction, and the ratio between CD4+to CD8+ cells.

a. Measurement ofcytokines- Enzyme-Linked Immunosorbent Assay (ELISA) kit was used for determining the concentrations ofeach cytokine inthe serum. About 1 00 Ill ofcapture antibody was used for coating eachwell ofa 96-well plate and incubated overnight at4 °C. The serum or standard was then added to each individual well after washing andblockingthe plate withthe assay dilution and maintained at room temperature for 2 hours. After another wash ofthe plate, a biotin-conjugate detecting mouse antibody was added to each well and incubated atroom temperature for 1hour. In addition, before detecting with TMB solution, the plate was washed again and further incubated with avidin-HRP for 30 min. By adding 1 M H3P04 reactions were stopped, and at 450 nm the absorbance was measured with an ELISA reader. Finally, the cytokine was evaluated from the linearportion ofthe generated standard curve.Silver nanoparticles/(PC Code072599) 28-Day Oral Toxicity in (Mice) (2010) IPage 8 of 14 OPPTS 870.3050 I OECD 407 b. Measurement ofImmunoglobulin E (IgE)

- Similarto the measurement ofthe cytokines, the ELISAkit was used for determining the concentration oflgE in the serum. Each ofthe 96 well plates were coated with the coating antibody, incubated overnight at 4 °C, then washed and blocked for 1hour at room temperature. The serum and lgE standard were added to all the wells and left for an hour at room temperature. Following another wash ofthe plate, detection antibodies were added to each well and incubated for anhour at room temperature. Finally with another washthe plate was further incubated with color development solution, and the reaction was terminated bythe addition of2M H2S04. The absorbance was measured with anELISAreader at 450 nm and IgE was calculated from the linearportion ofthe standard curve.

c. Immunophenotyping

- All the T cells, B cells, NK cells, CD4+ T cells, and CD8+ T cells were identified using conjugated anti-mouse antibodies. To reduce the non-specific binding ofthe antibodies, blood lymphocytes were blocked with Fc-block. In addition, cells were incubated inthe dark with 10 J..Ll ofthe appropriate flurochrome-conjugated antibody for 20 min at 4 °C, and were washed with a 500 J..Ll Fluroscence Activated Cell Sorter (FACS). The bloodwas lysed and rewashed with FACS atroom temperature. Thereafter, each ofthe samples was fixed with 1% paraformaldehyde until further analysis. The flow cytometry analysis was performed onthe FACSCalibur system and matched for each flurochrome with the control samples, and the data were analyzed using cellQuest software.
II. RESULTS
A. OBSERVATIONS: 

### 1. Clinical signs oftoxicity- No cageside observations and clinical examinations were provided for this study.
2. Mortality- No mortalities were reported in this study. 

### 3. NeurologicalEvaluations- No neurological evaluations were conducted inthis study.
B. BODYWEIGHT AND WEIGHT GAIN: Body weight changes were not provided for the 28-day study period with varying doses of42 nm size ofsilver nanoparticles. However, the body weight changeprovided for the 14-day study with varying sizes ofsilver nanoparticles, did not indicate any significant changes. Overall, the average body weight did increase by 5-6 gm both in controls and the treated groups during the 14-day study to 35.15 g. C. FOOD CONSUMPTION AND EFFICIENCY: 

### 1. Food consumption- No information or data was provided on food consumption.
2. Food efficiency- No information or data was provided onfood consumption.Silver nanoparticles/ (PC Code 072599) 28-Day Oral Toxicity in (Mice) (2010) IPage 9 of 14 OPPTS 870.3050 I OECD 407
D. OPHTHALMOSCOPIC EXAMINATION: No ophthalmoscopic examinations were conducted during the study.
E. BLOOD ANALYSES: 1. 

### Hematology- No information or data was provided on hematology 

### Clinical Chemistry

- The changes in clinical chemistry and serum cytokine level measurements are summarized in Table 1below. In the 28-day repeated dose toxicity test, the levels ofaspartate transaminase and alkaline phosphatase were statistically significantly higher in males dosed at 1.0 mg/kg/day when compared with the controls. In females, aspartate transaminase, alanine transaminase, and alkaline phosphatase were significantly (p <0.01) higher than controls. 

### Inflammatory responses- In orderto assess the inflammatory responses, the concentration ofpro-inflammatory cytokines (IL-l, IL-6, TNF-a), Th-1 type cytokines (IL-12 and IFN-y), Th-2 type cytokines (IL-4, IL-5, IL-10) and**TGF-~** in serum, and IgE were measured. In the 28-day repeated-dose toxicity study, an increase in all the above indicators was observed in mice treated with 1.0 mg/kg/day silver nanoparticles when compared with controls. In the 14-day study, the level ofTGF-**~in** serum was significantly higher in mice treated with 22 nm, 42 nm, and 71 nm silver nanoparticles.
In addition, not significantbut an increase in distribution ofNKcells and B cells were reported inboth 14-day study with 22 nm silvernanoparticles and 28-day study at 1.0 mg/kg/day dose level. When compared to controls with the CD4+/CD8+ ratio of3.99 inthe 14-day study, the lowest CD4+/CD8+ ratio of2.88 was reported with exposure to 22 nm size silver nanoparticles among all the other silver nanoparticles tested. Likewise, inthe 28-day study, at 1.0 mg/kg/day dose, the CD4+/CD8+ ratio was 2.76 versus 3.80 ofcontrols.Silver nanoparticles/ (PC Code072599) 28-Day Oral Toxicity in (Mice) (2010) I Page 10 of 14 OPPTS 870.3050 I OECD 407

Table 1. Comparison ofClinical Chemistry and Inflammatory Responses atDose Level of 1.0 mglkglday TreatmentGroupwith Controls in a 28-Day Repeated-dose Study Levels (units) At dose level of 1.0 mglkglday Control AST (IU/1) 157.33 ± 47.98* 79.00± 14.18 Males ALT (lUll) 103.67 ± 77.31 48.00 ± 4.58 ALP (IU/1) 124.33 ± 37.98** 80.67 ± 3.79 AST (IU/1) 211.67±57.18** 79.67 ± 5.69 Females ALT (IU/1) 150.67 ± 13.05** 36.33 ± 5.13 ALP (IU/1) 181.00 ± 51.51** 87.67 ± 23.46 IL-l (pg/ml) 8.8 ± 07.0 NDa Pro-inflammatory IL-6 (pg/ml) ** 1.44 13.75 ± 0.57 cytokines TNF-a (pg/ml) 3.41 ± 0.06 1.21 IL-12 (pg/ml) ** 76.86 ± 5.20 35.5 Th-1 type cytokines IFN-y (pg/ml) 0.52 ± 0.04 ND IL-4 (pg/ml) 2.7 ± 0.07* ND Th-2 type cytokines IL-5 (pg/ml) 1.34 ± 0.01 ND IL-10 (pg/ml) 29.02 ± 1.70** ND TGF-P (pg/ml) 6.73 ± 0.52** ND IgE (ng/ml) 6.04 ± 0.74* 3.28 aNot detected. * ** Significantly different from control group: p<0.05; p<O.Ol (n=3, mean± SD).
F. URINALYSIS: Urinalysis was not conducted.
G. Sacrifice and Pathology: 1. Organ weight- No information was provided for the 28-day repeated toxicity study. In the 14-day study, the author statedthat the organ weights ofliver, kidney, testes, brain, and lungs were not affected in the treated groups. However, although it was not statistically significant, the liver and testes weights were higher than controls in mice administered 22 nm (and 42 nm for liver weight) silver nanoparticles, as the Fig.I showed onthe ratio oforgan/body weight.Silver nanoparticles/ (PC Code 072599) 

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### Liver Kidney Testis28-Day Oral Toxicity in (Mice) (2010) I Page 11 of 14 OPPTS 870.3050 I OECD 407



### Brain c::::J Control**~** 22run**~** 42run -71nm**~** 323nm



### Lung Fig 1: The ratio oforgan/body weight after administration ofdifferent sized silvernanoparticles in a 14-day oral toxicity study.



### 2. Tissue deposition ofthe Silver- The brain seemedto be the major site ofsilver deposition when the mice were orally exposed to 22 nm, 42 nm, and 71 nm diameter size silver nanoparticles. A significant (p <0.01) amount ofsilver was present in all the tested organs: brain, lungs, liver, kidneys, and testes when exposed to 22 nm. At42 nm size silver nanoparticles, lungs, kidneys, and testes had significant (p < 0.01) silver accumulation, andthe liver and kidneys at 71 nm silver nanoparticles size had significant (p <0.01) amount ofsilver, when comparedto controls. However, at 323 nm size ofsilver nanoparticles, no deposition was observed in any ofthe organs tested (see Table 2 below).
The organs inthe 28-dayrepeated oral toxicity study were nottested for silver accumulation.Silver nanoparticles/ (PC Code 072599) 28-Day Oral Toxicity in (Mice) (2010) IPage 12 of 14 OPPTS 870.3050 I OECD 407



### Table2. Accumulation ofSilver in the Organs in a 14-day Oral Study ofVarious Sized NanosilverParticles in Mice at a Single Dose of 1.0 mglkg/day Organs 22om 42nm 71nm 323nm Control Brain 255.56 

### ± 3.13** 138.80 

### ± 1.85** 51.32 

### ± 3.56** NDa ND 

### Lung 208.11 

### ± 7.65** 171.11 

### ± 9.05** 13.00 

### ± 22.50 ND ND 

### Liver 265.41 

### ± 1.55** 86.67 

### ± 150.11 220.95 

### ± 7.48** ND ND 

### Kidney 345.76 

### ± 10.75** 47.92 

### ± 16.40** 53.21 

### ± 1.53** ND ND 

### Testis 316.00 

### ± 5.40** 172.63 

### ± 8.75** ND ND ND

aNot detected. Significantly different from control group: •• p<O.Ol (n=5, mean± SD). 

### 3. Microscopic pathology- Inthe 28-dayrepeated-dose study, oral administration ofsilver nanoparticles at 1.0 mg/kg/day, a slight cell infiltration was observed inthe kidney's cortex region inmales and females. No other significant histopathological changes were reported in the treated groups ofthe 28-day study.


### III. INVESTIGATORS' DISCUSSIONAND CONCLUSIONS: Inthe 14-day study, the author described thatthe oral administration of22 nm size silvernanoparticles were easily translocated into the blood stream and accumulated significantly (p < 0.01) withinthe organs. Thebrain andkidneys had significant amount ofsilver nanoparticles deposition when exposed to 22 nm, 42, and 71 nm silver nanoparticles. The authors indicated the size dependent penetration along withthe brain, inthe lungs andtestes.
Inthe 28-dayrepeated-dose toxicitytest, statistically significant increases in liver enzymes (aspartate transaminase, alkaline phosphastase, and alanine transaminase) were reported at 1.0 mg/kg/day dose level. The author indicatedthatthis increase was solely due to the silver nanoparticles administered without any vehicle. In addition, the author suggestedthat since the study did not find anyhistopathological changes in the liver despite the increase inthe enzymes secretion, further studies are required onthe same. Also, the concentration ofpro-inflammatory cytokines (IL-l, IL-6, TNF-a), Th-1 type cytokines (IL-12 and IFN-y), Th-2 type cytokines (IL4, IL-5, IL-10),**TGF-~** in serum, IgE, B-cell and CD8+ distribution were higher inthe 28-day repeated-dose study at 1.0 mg/kg/day dose compared to controls. 

### The author did not determine anyNOAEL orLOAEL values for eitherthe 14-day or28-day repeated-dose toxicity study.
A. REVIEWER COMMENTS: The agency agrees thatthe size ofthe silvernanoparticles along withthe route ofexposure renders its translocation into the blood stream and deposition withinthe organs. Inthis case, the smallest, 22 nm diameter size silvernanoparticles demonstrated the greatest accumulation in all the tested organs: the brain, lungs, kidneys, testes, and livercompared to the other sizes tested, andthe accumulation was significantly (p < 0.01)Silver nanoparticles/ (PC Code 072599) 28-Day Oral Toxicity in (Mice) (2010) I Page 13 of 14 OPPTS 870.3050 I OECD 407 higher than controls. The largest silver nanoparticle tested was 323 run in diameter, which did not accumulate in any ofthe tested organs. All the relatively smaller sizes (22, 42, 71 run) to 323 run had significantly (p 

### < 0.01) accumulated inthe brain and kidneys, butthe accumulation inthe brainreduced with increase in silver nanoparticles size. This penetration as indicated bythe author raises concerns aboutthe retention and elimination ofthe nanoparticles after it has accumulated withinthe body. Additional investigations must adhere to understanding the biotransformation ofsilver nanoparticles at small sizes and low-doses. Also, clarification on observing a sudden decrease in silver nanoparticles accumulation inthe liver and kidneys at 42 run mustbe provided.
Inthe 28-day repeated-dose toxicity study, as suggested bythe author, further analysis ofthe liver is required to comprehend the statistically significant increase inthe liver enzyme despite not observing any histopathological changes/damages to the liver. Due to the lack ofdata on the significant levels ofthe increase in cytokines (Th-1 type cytokines and Th-2 type cytokines), Bcells, and allergic response (IgE), the author is required to provide additional data onthe same. The reduced ratio ofCD4+/CD8+ in boththe 14-day and 28-day repeated dose toxicity study as mentioned by the author is an "increase in the CD8+ cell distribution". This increase inthe CD8+ T cells means that the suppressor-cytotoxic T cells are more in numbers, which indirectly signifies thatthe silver nanoparticles affected the CD4+ T cells byreducing their numbers. Thus, the increase in CD8+ T cell distribution as specified bythe author is due to what? Moreover, for the production ofCD8+T cells, which are predominantly activated by a set ofreactions from the CD4+ T cells, specifically T-helper cells are necessary. Thus, the author mustprovide substantial reasoning to explicitly describe this increase in CD8+ cell distribution inthe study by either providing an initial count ofboth CD4+ and CD8+ cells before commencingthe experiment on their ratio to lucidly understand the actual effect ofsilver nanoparticles onthe same. Inthe 28-day repeated-dose oral toxicity study in mice with silver nanoparticles of42 run diameter, statistically significant changes in clinical chemistry, including increase in serum liver enzymes in males (alkaline phosphatase and aspartate transaminase) and females (alkaline phosphatase, aspartate transaminase, and alanine transaminase). These changes were not accompanied by any observed histopathological effect onthe liver. A slight cell infiltration in the cortex ofthe kidneys was observed at 1.0 mg/kg/day dose level in both males and females. The toxicological significance ofthese changes is questionable. Therefore, No NOAEL or LOAELvalues can be determined from this 28-day repeated-dose toxicity study.

B. STUDYDEFICIENCIES: Major deficiencies for the 28-dayrepeated-dose toxicity test: 

### 1. The purity ofsilvernanoparticles was notprovided 2. Additional data on statistical significance level ofcytokine production, B-cell distribution, and inflammatory cell infiltrates in kidney at 1.0 mg/kg/day dose level. 3. No information onneurological evaluations and hematology were providedSilver nanoparticles/ (PC Code 072599) 28-Day Oral Toxicity in (Mice) (2010) IPage 14 of 14 OPPTS 870.3050 I OECD 407
4. Increase in cell distribution ofCD8+ as statedbythe author from analyzing the CD4+/CD8+ratio in 28-day repeated-dose study is required. 5. Several other organsthat are mandatory for sacrifice and pathology mustbe studied for this 28-day repeated-dose study. Histopathology was conducted only onthe liver, kidney and intestine atthe highest dose (1.0 mg/kg/day) group. 6. Datamust be provided onurinalysis, hematology, and detailed gross necropsy. 7. Need for studying the inflammatory responses in mice inthis study was not elucidated in the introduction section ofthe article. The author must provide supporting evidence on the same. Minor deficiencies:
8. Information or data is required ontest substance homogeneity, food consumption, food efficiency, ophthalmological examination, cage-side observation, and mortalities. 

### C. STUDY CLASSIFICATION:
The data ofthe 28-day repeated dose oral toxicity study in mice are 

### categorized as "qualitative" are not appropriatefor quantitative use (i.e., dose selection), andthe data can be discussed inthe risk characterization as an additional lines ofevidence to supportrisk conclusions regarding metabolism, adverse effects ofconcern, life stage susceptibility, and mode oftoxic action. It does not satisfy the guideline requirement for a 28-day oral rodent studybased on Guideline 870.3050. The information onthe purity ofsilver nanoparticles, data on statistical significance on cytokine production, B-cell distribution, inflammatory cell infiltrates are not provided. Histopathology was conducted only onthe liver, kidney and intestine at the highest dose (1.0 mg/kg/day) group. The reasoning for observing an increase inthe CD8+ cell distribution while analyzing the CD4+/CD8+ratio were not adequately addressed. Others, including food and water consumptions, neurological evaluations, hematology and ophthalmological examinations, and clinical biochemistry determinations along with a detailed gross necropsy, are also required. 

### The 14-daymice studyidentifies the effects ofsize on tissue distribution ofdosed nanosilver.