Silver nanoparticles/ (PC Code 072599) 

EPA Reviewer: Jonathan Chen, Ph. D. 

Risk Assessment and Science Support Branch 

Antimicrobial Division 

EPA Secondary Reviewer: Jonathan Leshin, Ph. D. 

Risk Assessment and Science Support Branch 

Antimicrobial Division 

TXR: 1,003,206 

28-Day Oral Toxicity in (Mice) (2010) I Page 1 of 14 

OPPTS 870.3050 I OECD 407 

Signature: ~tfla« ~ 

Date o~/o!Uo/3 ~; . 

Signature: )iY ~ 

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II DATAEVALATIONRECORD II 

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: Nanosilver particles 

CITATION: Park, E., E. Bae, J. Yi, et al. (2010) Repeated-dose toxicity and inflammatory 

responses in mice by oral administration of silver nanoparticles. Environmental 

Toxicology and Pharmacology. 30:162-168. MRID 48385201. 

SPONSOR: Not applicable 

EXECUTIVE SUMMARY: 

There are two independent studies reported in this paper: 

1. A 28-day mice oral study - to study the potential toxicological effects associated with 

oral exposure to nano-sized silver; and 

2. A 14-day mice size difference toxicity test- To study the effects of size on tissue 

distribution of dosed nano-silver. 

28-day mice oral study: 

In the repeated-dose oral toxicity study, silver nanoparticles of 42 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 on the liver, kidney and intestine at the highest dose (1.0 

mg/kg/day) group. Tissue concentrations of silver (Ag) in the testis, kidney, brain, lungs, 
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OPPTS 870.3050 I OECD 407 

stomach and blood were also determined. In addition, blood chemistry and serum cytokine 

levels were measured. 

At the dose level of 1.0 mg/kg/day, significant increases in serum enzyme level of alkaline 

phosphatase (p <0.01) and aspartate transaminase (p <0.05) were observed in both 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 liver to be one of the target organs 

of concern. In addition, a dose-dependent, statistically-significant increase in the concentration 

of pro-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 in the proportion of B cells was observed in all treatment groups. At the 1.0 

mg/kg/day dose level, NK, NKT, B, and T cell distribution were all increased compared to the 

control group. The ratio of CD4+/CD8+ T cells was decreased indicating an increase of CD8+ T 

cell distribution. 

In the 28-day repeated-dose oral toxicity study in mice with silver nanoparticles of 42 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 in the 

cortex of the kidneys was observed at 1.0 mg/kg/day dose level in both males and females. The 

toxicological significance of these changes is questionable. Therefore, No NOAEL or LOAEL 

values can be determined from this 28-day repeated-dose toxicity study. 

The data of the 28-day repeated dose oral toxicity study in mice are categorized as 

"qualitative" and are not appropriate for quantitative use (i.e., dose selection). The data can 

be discussed in the risk characterization as additional lines of evidence to support risk 

conclusions regarding metabolism, adverse effects of concern, life stage susceptibility, and mode 

of toxic action. It does not satisfy the guideline requirement for a 28-day oral rodent study based 

on Guideline 870.3050. The information on the purity of silver nanoparticles, data on statistical 

significance on cytokine production, B-cell distribution, inflammatory cell infiltrates are not 

provided. Histopathology was conducted only on the liver, kidney and intestine at the highest 

dose (1.0 mg/kg/day) group. The reasoning for observing an increase in the 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-day mice daily single dose study: 

A 14-day oral toxicity study with various sizes (22 nm, 42 nm, 71 nm, and 323 nm) of silver 

nanoparticles in deionized water was conducted at a single dose level of 1.0 mg/kg/day in mice 

(5/group). Statistically significant (p <0.01) amounts of silver nanoparticles were accumulated in 

all the tested organs (brain, lungs, liver, kidneys, and testes) in mice being administered orally 
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OPPTS 870.3050 I OECD 407 

with 22 run, 42 run, and 71 run silver nanoparticles, with the greatest accumulation seen in the 22 

run treatment group. This greatest accumulation at 22 run diameter size of silver nanoparticles 

raises significant concerns about penetration, retention, assimilation, and elimination within the 

organs of silver nanoparticles 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 smallersized nanosilver 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 important in determining the tissue distribution after oral exposure of the 

nano-sized material. 

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

statements are not required. 
Silver nanoparticles/ (PC Code 072599) 

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OPPTS 870.3050 I OECD 407 

I. MATERIALS AND METHODS 

A. MATERIALS: 

1. Test Material: Silver nanoparticles (Cat. No. 484059, Sigma-Aldrich, USA) 

Description: 

Lot/Batch #: 

Purity: 

Compound Stability: 

CAS#: 

2. Vehicle and/or positive 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 of National Veterinary 

Research and Quarantine Service" for caring of the 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 of the 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 of the animals. 

Environmental Temperature: 23 ± l°C 

conditions: Humidity: 55±5% 

Air changes: Not provided. 

Photoperiod: 12 hrs dark! light cycle 

Acclimation period: 1-week prior to the initiation of the 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 test to silver nanoparticles. In the 14-day study, various sizes such as 22 

nm, 42 nm, 71 nm, and 323 nm of silver nanoparticles were tested at one single dose of 1.0 

mg/kg/day with 5 mice per group. In 28-day repeated-dose toxicity test, only one size, 42 nm, 

silver nanoparticles at various 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 with de-ionized water. 
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OPPTS 870.3050 I OECD 407 

3. Dose selection rationale: No rationale was provided for studying the various silver 

nanoparticle sizes in the 14-day study or the dose selection rationale with 42 run diameter size of 

silver nanoparticles in the 28-day study. 

4. Treatment preparation, administration, and analysis: Silver nanoparticles were suspended 

with sonication with tetrahydrofurane (99.8%, Fluka)which was evaporated by stirring the 

suspension. The evaporated tetrahydrofurane volume was compensated with the addition of 

deionized water to the suspension for a day or two to finally replace tetrahydrofurane. Thereafter, 

the silver nanoparticles suspension was filtered through varying pore sizes of polycarbonated 

isopore filters to yield different sizes of silver nanoparticles. 

For the 14-day study, the different sizes of silver nanoparticles in diameters were 22 run, 42 run, 

71 run, and 323 run, which were administered orally in a single dose of 1.0 mg/k.g/day. For the 

28-day repeated-dose toxicity study, only the 42 run size silver nanoparticle was administered 

orally at 3 different doses, which were 0.25 mg/k.g/day, 0.5 mg/k.g/day, and 1.0 mg/k.g/day. The 

control group was prepared with the deionized water. 

Results 

Homogeneity(% coefficient of variation) 

Not provided 

Stability (range of% of initial concentration) 

Not provided 

Concentration (range of% of nominal concentration) 

Not provided 

5. Statistics - The results of the treated group were compared using the student's t-test and one 

way ANOV A analysis with the control group for analyzing several parameters like accumulation 

of silver in the organs, serum level of TGF-p, ratio of organ to body weight exposed to varying 

size of silver nanoparticles, and blood biochemical parameters. 

C. METHODS: 

1. Observations: 

la. Cageside Observations- No specific data or information was provided on the frequency of 

mortality and toxicity. 

lb. Clinical Examinations- No specific data or information was provided on the frequency of 

clinical examinations 

lc. Neurological Evaluations- No neurological evaluations were conducted for this study. 
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2. Body weight: Body weight was measured on day 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 in this study. 

5. Hematology & Clinical Chemistry: No specific data or information was provided on 

hematology; however, for biochemical analysis, 1 ml of blood was collected from the retroorbital venous plexus from each mouse using heparinized capillary tubes. From the withdrawn 1 

ml blood, 100 Ill was used for cell phenotype analysis, and the rest was centrifuged at 3000 rpm 

for 10 min for preparing the serum. The serum was stored in the -80 °C freezer prior to blood 

biochemical analyses, which were measured using an auto-analyzer (Hitachi 7180, Hitachi, 

Japan). In the repeated oral toxicity test, blood was obtained from 3 mice of each 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 Serum protein 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 on urinalysis. 

7. Sacrifice and Pathology: Although the author did not specifically state about the histological 

techniques used in this study, the kidneys, livers, and small intestines ofthe control and treated 

groups were fixed with 1 0% neutral buffered formalin and processed using routine histological 

techniques. After the paraffin embedding, which was not explained in the article, 3 llm sections 
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were cut and stained with hematoxylin and eosin for histopathologic evaluation. Injuries, cellular 

changes, and inflammations were examined microscopically. 

All animals after blood drawing were sacrificed and subjected to gross pathological examination 

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

X 

X 

X 

X 

DIGESTIVE SYSTEM CARDIOV ASC.IHEMA T. 

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 evaluated the inflammatory responses by 

measuring the amount of cytokine and lgE production, and the ratio between CD4+ to CD8+ 

cells. 

a. Measurement of cytokines- Enzyme-Linked Immunosorbent Assay (ELISA) kit was used 

for determining the concentrations of each cytokine in the serum. About 1 00 Ill of capture 

antibody was used for coating each well of a 96-well plate and incubated overnight at 4 °C. 

The serum or standard was then added to each individual well after washing and blocking the 

plate with the assay dilution and maintained at room temperature for 2 hours. After another 

wash of the plate, a biotin-conjugate detecting mouse antibody was added to each well and 

incubated at room temperature for 1 hour. 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 linear portion of the generated 

standard curve. 
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b. Measurement of Immunoglobulin E (IgE) - Similar to the measurement of the cytokines, 

the ELISA kit 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 1 hour 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 of the plate, 

detection antibodies were added to each well and incubated for an hour at room temperature. 

Finally with another wash the plate was further incubated with color development solution, 

and the reaction was terminated by the addition of 2M H2S04. The absorbance was measured 

with an ELISA reader at 450 nm and IgE was calculated from the linear portion of the 

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 

of the antibodies, blood lymphocytes were blocked with Fc-block. In addition, cells were 

incubated in the dark with 10 J..Ll of the appropriate flurochrome-conjugated antibody for 20 

min at 4 °C, and were washed with a 500 J..Ll Fluroscence Activated Cell Sorter (F ACS). The 

blood was lysed and rewashed with F ACS at room temperature. Thereafter, each of the 

samples was fixed with 1% paraformaldehyde until further analysis. The flow cytometry 

analysis was performed on the F ACSCalibur 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 of toxicity- No cageside observations and clinical examinations were provided 

for this study. 

2. Mortality- No mortalities were reported in this study. 

3. Neurological Evaluations- No neurological evaluations were conducted in this study. 

B. BODY WEIGHT AND WEIGHT GAIN: Body weight changes were not provided for the 

28-day study period with varying doses of 42 nm size of silver nanoparticles. However, the body 

weight change provided for the 14-day study with varying sizes of silver 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 on food consumption. 
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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 1 below. In the 28-day repeated dose toxicity test, 

the levels of aspartate 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 order to 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 significant but an increase in distribution ofNK cells and B cells were 

reported in both 14-day study with 22 nm silver nanoparticles and 28-day study at 1.0 

mg/kg/day dose level. When compared to controls with the CD4+/CD8+ ratio of3.99 in the 

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, in the 28-day 

study, at 1.0 mg/kg/day dose, the CD4+/CD8+ ratio was 2.76 versus 3.80 of controls. 
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Table 1. Comparison of Clinical Chemistry and Inflammatory Responses at Dose Level of 

1.0 mglkglday Treatment Group with 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) ** 13.75 ± 0.57 1.44 

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 

aN ot 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 stated that the organ weights of liver, 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 on the ratio of organ/body weight. 
Silver nanoparticles/ (PC Code 072599) 

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0.05 ..... .. .... .. . 

.lS 

.gl 0.04 

Q) 

~ 0.03 

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i ... 

~ 0.02 

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Liver Kidney Testis 

28-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 of organ/body weight after administration of different sized silver nanoparticles in a 14-day oral 

toxicity study. 

2. Tissue deposition of the Silver- The brain seemed to be the major site of silver 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 of silver was present in all the tested organs: brain, 

lungs, liver, kidneys, and testes when exposed to 22 nm. At 42 nm size silver nanoparticles, 

lungs, kidneys, and testes had significant (p < 0.01) silver accumulation, and the liver and 

kidneys at 71 nm silver nanoparticles size had significant (p <0.01) amount of silver, when 

compared to controls. However, at 323 nm size of silver nanoparticles, no deposition was 

observed in any of the organs tested (see Table 2 below). 

The organs in the 28-day repeated oral toxicity study were not tested for silver accumulation. 
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Table 2. Accumulation of Silver in the Organs in a 14-day Oral Study ofVarious Sized 

Nanosilver Particles 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 

a Not detected. 

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

3. Microscopic pathology- In the 28-day repeated-dose study, oral administration of silver 

nanoparticles at 1.0 mg/kg/day, a slight cell infiltration was observed in the kidney's cortex 

region in males and females. No other significant histopathological changes were reported in the 

treated groups of the 28-day study. 

III. INVESTIGATORS' DISCUSSION AND CONCLUSIONS: In the 14-day study, the 

author described that the oral administration of 22 nm size silver nanoparticles were easily 

translocated into the blood stream and accumulated significantly (p < 0.01) within the organs. 

The brain and kidneys had significant amount of silver nanoparticles deposition when exposed to 

22 nm, 42, and 71 nm silver nanoparticles. The authors indicated the size dependent penetration 

along with the brain, in the lungs and testes. 

In the 28-day repeated-dose toxicity test, 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 indicated that this increase was solely due to the silver 

nanoparticles administered without any vehicle. In addition, the author suggested that since the 

study did not find any histopathological changes in the liver despite the increase in the enzymes 

secretion, further studies are required on the same. Also, the concentration of pro-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 in the 28-day 

repeated-dose study at 1.0 mg/kg/day dose compared to controls. The author did not determine 

any NOAEL or LOAEL values for either the 14-day or 28-day repeated-dose toxicity study. 

A. REVIEWER COMMENTS: The agency agrees that the size of the silver nanoparticles 

along with the route of exposure renders its translocation into the blood stream and deposition 

within the organs. In this case, the smallest, 22 nm diameter size silver nanoparticles 

demonstrated the greatest accumulation in all the tested organs: the brain, lungs, kidneys, testes, 

and liver compared to the other sizes tested, and the accumulation was significantly (p < 0.01) 
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higher than controls. The largest silver nanoparticle tested was 323 run in diameter, which did 

not accumulate in any of the tested organs. All the relatively smaller sizes (22, 42, 71 run) to 323 

run had significantly (p < 0.01) accumulated in the brain and kidneys, but the accumulation in the 

brain reduced with increase in silver nanoparticles size. This penetration as indicated by the 

author raises concerns about the retention and elimination of the nanoparticles after it has 

accumulated within the body. Additional investigations must adhere to understanding the 

biotransformation of silver nanoparticles at small sizes and low-doses. Also, clarification on 

observing a sudden decrease in silver nanoparticles accumulation in the liver and kidneys at 42 

run must be provided. 

In the 28-day repeated-dose toxicity study, as suggested by the author, further analysis of the 

liver is required to comprehend the statistically significant increase in the liver enzyme despite 

not observing any histopathological changes/damages to the liver. Due to the lack of data 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 on the same. 

The reduced ratio of CD4+/CD8+ in both the 14-day and 28-day repeated dose toxicity study as 

mentioned by the author is an "increase in the CD8+ cell distribution". This increase in the 

CD8+ T cells means that the suppressor-cytotoxic T cells are more in numbers, which indirectly 

signifies that the silver nanoparticles affected the CD4+ T cells by reducing their numbers. Thus, 

the increase in CD8+ T cell distribution as specified by the author is due to what? Moreover, for 

the production of CD8+ T cells, which are predominantly activated by a set of reactions from the 

CD4+ T cells, specifically T-helper cells are necessary. Thus, the author must provide substantial 

reasoning to explicitly describe this increase in CD8+ cell distribution in the study by either 

providing an initial count of both CD4+ and CD8+ cells before commencing the experiment on 

their ratio to lucidly understand the actual effect of silver nanoparticles on the same. 

In the 28-day repeated-dose oral toxicity study in mice with silver nanoparticles of 42 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 on the liver. A slight cell infiltration in 

the cortex of the kidneys was observed at 1.0 mg/kg/day dose level in both males and females. 

The toxicological significance of these changes is questionable. Therefore, No NOAEL or 

LOAEL values can be determined from this 28-day repeated-dose toxicity study. 

B. STUDY DEFICIENCIES: 

Major deficiencies for the 28-day repeated-dose toxicity test: 

1. The purity of silver nanoparticles was not provided 

2. Additional data on statistical significance level of cytokine production, B-cell 

distribution, and inflammatory cell infiltrates in kidney at 1.0 mg/kg/day dose level. 

3. No information on neurological evaluations and hematology were provided 
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4. Increase in cell distribution of CD8+ as stated by the author from analyzing the 

CD4+/CD8+ ratio in 28-day repeated-dose study is required. 

5. Several other organs that are mandatory for sacrifice and pathology must be studied for 

this 28-day repeated-dose study. Histopathology was conducted only on the liver, kidney 

and intestine at the highest dose (1.0 mg/kg/day) group. 

6. Data must be provided on urinalysis, hematology, and detailed gross necropsy. 

7. Need for studying the inflammatory responses in mice in this study was not elucidated in 

the introduction section of the article. The author must provide supporting evidence on 

the same. 

Minor deficiencies: 

8. Information or data is required on test substance homogeneity, food consumption, food 

efficiency, ophthalmological examination, cage-side observation, and mortalities. 

C. STUDY CLASSIFICATION: 

The data of the 28-day repeated dose oral toxicity study in mice are categorized as 

"qualitative" are not appropriate for quantitative use (i.e., dose selection), and the data can 

be discussed in the risk characterization as an additional lines of evidence to support risk 

conclusions regarding metabolism, adverse effects of concern, life stage susceptibility, and mode 

of toxic action. It does not satisfy the guideline requirement for a 28-day oral rodent study based 

on Guideline 870.3050. The information on the purity of silver nanoparticles, data on statistical 

significance on cytokine production, B-cell distribution, inflammatory cell infiltrates are not 

provided. Histopathology was conducted only on the liver, kidney and intestine at the highest 

dose (1.0 mg/kg/day) group. The reasoning for observing an increase in the 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-day mice study identifies the effects of size on tissue distribution of dosed nanosilver.