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: 1,003,297 

90-day Inhalation Toxicity - Rat (2012) I Page 1 of 11 

Non-guideline 

Signature: .~c./t.t:2-1<. CJdw 

Date ___ o-=.Rr/o;__;.. I.L.:../>o_f:.....::3~c'--

Date

Signature: 

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II DATA EVALUATION RECORD- SUPPLEMENT II 

STUDY TYPE: Subchronic Inhalation Toxicity- Rat; OPPTS 870.3465 [§82-4]; OECD 413. 

PC CODE: 072599 DP BARCODE: D411279 

TEST MATERIAL (PURITY, SHAPE, SIZE): Pure Silver (purity not provided. Some 

oxidized silver nanoparticles may be formed. 14.09 ±1 .71nm) 

SYNONYMS: Not available 

CITATION: Song KS.; Sung, JH; and Ji, JH et al. 2012. Recovery from silver-nanoparticleexposure-induced lung inflammation and lung function changes in Sprague 

Dawley rats. Nanotoxicology doi: 10.3109/17435390.2011.648223 MRID 

49052001 

SPONSOR: Not applicable 

EXECUTIVE SUMMARY: 

Inhalation study (MRID 49052001) was conducted to evaluate the recovery from the nano-sized 

silver particles induced toxicity. In the study, pure silver particle (14.09 ±1.71 nm) were 

administered in Sprague-Dawley (SD) rats (17 male rats and 12 female rats/dose/group) via a 

whole-body inhalation chamber at dose levels of fresh-air control, low-dose (0.66 x 10 

particle/cm3

, 48.76 J.Lg/m\ middle-dose (1.41 x 106 particle/cm3, 117.14 J.Lg/m\ and high-dose 

(3.24 x 106 particle/cm3

, 381.43 J.Lg/m3) for 6 hr/day, 5 days/week, for 12 weeks. After the 12-

week exposure period, an additional12 week- recovery period was followed. 

Four animals/sex/group were used to monitor specific time points: after 12 weeks of exposure, 

after 4 weeks of recovery, and after 12 weeks of recovery. An additional5 male rats were also 

assigned to the 12-week exposure group for a detailed study of the brain translocation. The 

animals were examined daily on weekdays for any evidence of exposure-related effects, 

including respiratory, dermal, behavioral, nasal, or genitourinary changes suggestive of irritancy. 

Body weights were measured at the time of purchase, at the time of grouping, at one day after 

exposure, once a week during the inhalation exposure and recovery, and before necropsy. 
Silver nanoparticles/ (PC Code 072599) 

90-day Inhalation Toxicity - Rat (2012) I Page 2 of 11 

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No test substance-related differences were observed in food consumption rates, or body 

weight/body weight gain changes between the exposed and the control groups. 

For male rats, dose-related decrease in the physiological measurement of tidal volume (TV) that, 

in turn, led to decrease in measures of minute volume (MV) and peak inspiratory flow (PEF) , 

were observed in both exposure and recovery periods in the high-dose and middle-dose groups. 

In contrast, for the female rats, no significant changes were observed in the lung function test 

parameters when comparing the exposed groups with the unexposed control group. 

In the histopathological examination, a significant increase was observed in the incidence of 

mixed cell infiltrate perivascular and chronic alveolar inflammation, including alveolaritis, 

granulomatous lesions, and alveolar wall thickening and alveolar macrophage accumulation in 

both the male and female. During the 12 weeks of recovery, the female rats in all the groups 

showed a gradual clearance of the silver nanoparticles and decreased inflammation. However, 

the male rats in the high-dosed group and middle-dosed group did not achieve a complete 

recovery, exhibiting persistent inflammation throughout the 12-week recovery period (compared 

to the controls). 

Based on the significant increase in the incidence of mixed cell infiltrate perivascular and 

chronic alveolar inflammation in both the male and female, and decreases in physiological 

measures of lung function (respiratory minute volume, tidal volume, and peak inspiratory flow) 

in the males, the NOAEL is determined to be 48.76 Jlg/m3 and LOAEL is 117.14 Jlg/m3

. 

This inhalation study is classified Acceptable/Non-guideline and may be used for regulatory 

purposes; however, they do not satisfy the guideline requirement OPPTS 870.3465 for a 

subchronic inhalation study. 

NOTE: The study presented in this published article is a continuation of previous 28-day and 

90-day inhalation studies conducted by the authors. The 28-day and 90-day studies were also 

reviewed by the Agency (DP BARCODE: D392812) and are identified as following: 

1. Sung, JH, Ji, JH, and Park, JD et al. (2009) Subchronic inhalation toxicity of silver 

nanoparticles. Toxicological Sciences. 108(2);452-461. MRID 47757505. 

2. Sung, JH; Ji, JH; and Y oon, JU et al. (2008) Lung Function Changes in Sprague-Dwaley 

Rats after Prolonged Inhalation Exposure to Silver Nanoparticles. Inhalation Toxicology. 

20:567-74. MRID 48552402. 

3. Ji, JH; Jung, JH; and Kim, SS et al. (2007) Twenty-eight-day Inhalation toxicity Study of 

Solver nanoparticles in Sprague-Dawley Rats. Inhalation Toxicology. 19:857-71. MRID 

48552401. 

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

Confidentiality statements were not provided. 
Silver nanoparticles/ (PC Code 072599) 

I. MATERIALS AND METHODS 

A. MATERIALS: 

1. Test Material: 

90-day Inhalation Toxicity- Rat (2012) I Page 3 of 11 

Non-guideline 

Description: Silver nanoparticles were generated by evaporation/condensation 

using a small ceramic heater with a local heating area (Figure 1 ). Figure 2 shows 

the system used to produce different test concentrations/dilutions to be used in the 

inhalation studies of nanosilver particles. 

Characterization: The generated study nanosilver particles observed by 

transmission electron microscopy (TEM) were in spherical shape and 

nonaggregated/agglomerated pure silver, not silver oxides. The nanosilver 

particles distribution with respect to size in each chamber was measured directly 

in real-time using a differential mobility analyzing system (DMAS), the 

geometric mean diameter (GMD), geometric standard deviation (GSD), total 

number concentration, mass concentration and surface area of the silver 

nanoparticles were summarized in Table 1. 

Table 1. Particle Characterization 

Geometric Geometric 

Concentration Mean Standard Total Number Mass Surface 

Diameter Deviation Concentration Concentration Area 

(GMD) (om) (GSD) (particles/cm3

) (JJ.g/mJ) (nm2

/cm3

) 

Low 14.54 1.62 0.66 X 10° 48.76 0.76 X 10~ 

Medium 14.38 1.64 1.41 X 10° 117.14 1.70 X 10~ 

High 15.00 1.75 3.24 X 10° 381.43 6.61 X 10~ 
Silver nanoparticles/ (PC Code 072599) 

90-day Inhalation Toxicity - Rat (2012) I Page 4 of 11 

Non-guideline 

Lot/Batch #: 

Purity: 

- Quartz Tube - • • • ••• • ~tree Matel"ial Clean Air • • • <====:J· 

• • • • ' . . . . Nanoparticle~ 

• 

• • 

-

... 

I Cc1runk heater t 

' ..... __ "" 

' \ 

. 

' . 

\ 

,, 

II ..__ II_ AC Pow~r 

,----------------------------_y _____ -----------------------.., . . l A Ceramic Healer with a local lumting Af(>a : . . . . : 6.2mm. : 

: --;.;, ·~ ~ : i ' :•?~:-.-- i 

i J,:§_ '·, =r- i : Sout'"e ~lat(>n:d • l 

' . '······-·· ······------ ------ --· ------ - - --------------! 

J 

FIG. I. Nnnoparticle generator with small ceramic heater used for inhalation toxicity study. 

Clrnn nir 

IlEPA Fiher 

Nnnopnrtic(e!; ~lonitoring 

E!'\tlosurc chamber -----+High 

-·-·-·-~Medium 

VilC\l~nn Pump 

-··-··•Low 

FIG. 2. System used to distribute particle concentration in inhalation toxicity study. 

Compound Stability: 

Not provided 

Not provided 

Not provided 

Not provided 

Not required 

CAS # of TGAI: 

Structure: 
Silver nanoparticles/ (PC Code 072599) 

90-day Inhalation Toxicity - Rat (2012) I Page 5 of 11 

Non-guideline 

2. Vehicle and/or positive control: Fresh air 

3. Test animals: 

Species: 

Strain: 

Age/weight at study initiation: 

Source: 

Housing: 

Diet: 

Water: 

Environmental conditions: 

Acclimation period: 

B. STUDY DESIGN: 

1. In life dates: 

Rat 

Specific-pathogen-free Sprague-Dawley 

Six-week-old rats; males weighing approximately 171 grams and females 135 grams. 

Purchased from KOATECH Co. (Korea) 

Individually housed in polycarbonate cages (five rats per cage). 

Rodent diet (Harlan Teklab, Plaster International Co., Seoul, South Korea) except 

during exposure 

Filtered water ad libitum 

Temperature: 21.6 ± 1.2 oc 

Humidity: 43.7 ± 6.8% 

Air changes: Not provided 

Photoperiod: 12 hrs dark/12 hrs light 

2 weeks 

Six -week old rats exposed to silver nanoparticles for 6 h/day, 5 days/week for 12 weeks 

and followed with 12-week recovery period 

2. Animal assignment and treatement: 

Six-week old Sprague-Dawley (SD) rats were divided into four (17 male rats and 12 

female rats/dose/group) via a whole-body inhalation chamber at dose levels of fresh-air 

control, low-dose (0.66 x 106 particle/cm3

, 48.76 J..lg/m\ middle-dose (1.41 x 106 

particle/cm3

, 117.14 J..lg/m\ and high-dose (3.24 x 106 particle/cm3

, 381.43 J..lg/m3

) for 6 

hr/day, 5 days/week, for 12 weeks. After the 12-week exposure period, an additional12 

week- recovery period was followed. 

Four animals/sex/group were used to monitor specific time points: after 12 weeks of 

exposure, after 4 weeks of recovery, and after 12 weeks of recovery. An additional5 

male rats were also assigned to the 12-week exposure group for a detailed study of the 

brain translocation. 

C. METHODS: 

Lung Function Test: Pulmonary functions of four rats from each dose group were 

evaluated weekly during the 12-week exposure and 12-week recovery periods using a 

ventilated bias flow whole-body plethysmograph (WBP). The parameters of pulmonary 

functions included tidal volume (TV, ml), minute volume (MV, ml/min), respiratory 

frequency (BPM, breath/min), inspiration time (Ti, s), expiration time (Te, s), peak 

inspiration flow (PIF, ml/s), and peak expiration flow (PEF, ml/s). 
Silver nanoparticles/ (PC Code 072599) 

90-day Inhalation Toxicity- Rat (2012) I Page 6 of 11 

Non-guideline 

Silver concentration in lung and other tissues 

The silver concentration was observed in the lung and other tissues from animal groups 

exposed to the silver nanoparticles for 12 weeks. The lungs were analyzed for silver 

concentration with a Zeeman graphite furnace atomic absorption spectrophotometer 

(Perkin Elmer 5100 ZL,Zeeman Furnace Module, USA) using the National Institute 

for Occupational Safety and Health (NIOSH) 7300 method (NIOSH 2003). 

Histopatological evaluation 

After the relevant exposure and recovery periods, the rats were anaesthetised with 

pentobarbitol and blood was collected from the abdominal aorta. The lungs, trachea, and 

other organs, including the liver and brain, were removed and fixed in a 10% formalin 

solution containing neutral phosphate buffered saline. The specimens were embedded 

in paraffin, stained with hematoxylin and eosin (H&E), and examined using light 

microscopy. 

D. DATA ANALYSIS: 

Statistics- An ANOVA test and Duncan's multiple range tests were used to compare the 

body weights, parameters from the lung function test and silver concentration in tissue 

obtained for the three dose groups with those obtained for the control group. The 

significance level was set at p < 0.05 and p < 0.01. 

II. RESULTS AND DISCUSSION 

ANIMAL OBSERVATION, FOOD CONSUMPTION, AND BODY WEIGHT 

None of the rats died. No test substance-related differences were observed in food 

consumption rates, or body weight/body weight gain changes between exposed and the 

control groups. 

LUNG FUNCTION TEST (MRID 48552402): 

For male rats, dose-related decrease in the physiological measurement of tidal volume 

(TV) that, in turn, led to decrease in measures of minute volume (MV) and peak 

inspiratory flow (PEF) , was observed in both exposure and recovery periods in the highdose and middle-dose groups (See Figure 3). For the male rats, the TV showed a 

decreasing tendency in the exposed groups when compared with the control group after 

5-12 weeks of exposure; a statistically significant decrease was observed after 5, 8, and 9 

weeks of exposure (p < 0.05) (Figure 3A). Meanwhile during the recovery period, a 

significant decrease in the TV was also observed in the exposed groups when compared 

with the control group after 1-10 weeks of recovery, where a statistically significant 

decrease was observed after 1, 3, 4, 5, 6, 7, 8, and 10 weeks of recovery (Figure 3A). This 

tendency of a lung function decrease also affected the MV (minute volume) and PEF 

(peak expiratory flow) during both the 12-week exposure and 12-week recovery periods 

(Figures 3B and C), where the MV decreased significantly (p < 0.01--0.05) after 2, 5, 8, 9, 

10, and 11 weeks during the exposure period and after 1, 2, 6, and 10 weeks during the 

recovery period (Figure 3B), while the PEF decreased significantly (p < 0.01--0.05) after 
Silver nanoparticles/ (PC Code 072599) 

90-day Inhalation Toxicity - Rat (2012) I Page 7 of 11 

Non-guideline 

1, 5, 8, 9, 10, 11, and 12 weeks during the exposure period and throughout the recovery 

period (Figure 3C). 

A 14 

12 

c--

e 

- 10 ~ .a 

~ 8 -

-e 

I= 

6 

4 

0 

c 

4 8 12 16 

Time(week) 

e g 

.- Unexposed 

~Low 

......- Mtldle _,_ High 

20 24 

FIG. 3A. Title volume (ml) change in male rats exposed to silver nanoparticles and during a 12-week 

recovery period. a: p < 0.05, high vs. low and middle groups; b: p < 0.01, high vs. unexposed and 

middle groups; c: p < 0.05, unexposed vs. middle group; d: p < 0.05, unexposed vs. other groups; e: p 

< 0.05, unexposed vs. middle and high groups; f: p < 0.05, unexposed and high vs. low groups g: p < 

0.01, unexposed vs. other groups (Adapted from Figure 5 of the report). 

B 1100 

1000 

900 

800 

e 

d 

b 

b 

b 

..._Unexposed 

-o- Low 

--.-Middle 

-6- High 

0 4 8 12 1 6 20 24 

Time(week) 

FIG. 3B. Lung Function- minute volume (mVmin) change in male rats exposed to silver 

nanoparticles and during a 12-week recovery period. a: p < 0.01, unexposed vs. other groups; b: 

p < 0.05, unexposed vs. low group; c: p < 0.05, low vs. high group; d: p < 0.01, unexposed and low vs. 

middle and high groups; e: p < 0.05, low vs. unexposed groups; f: p < 0.05, low vs.middle and high 

groups(Adapted from Figure 5 of the report). 
Silver nanoparticles/ (PC Code 072599) 

90-day Inhalation Toxicity - Rat (2012) I Page 8 of 11 

Non-guideline 

c 

90 

t 

::a 75 E 

-

6 

;: 

~ 60 

0 

1! 

30 - &:posure per oo-_.,___ FleOO'IIel)' per oo~ 

0 4 8 12 16 20 24 

Time(week) 

FIG. 3C. Lung Function- Peak Expiratory Flow (ml/sec) change in male rats exposed to silver 

nanoparticles and during a 12-week recovery period. a: p < 0.01, unexposed vs. other groups; b: p < 

0.01, low vs. other groups; c: p < 0.01, low vs. unexposed and high groups; d: p < 0.01, unexposed and 

high vs. low and middle groups e: p < 0.01, low and middle vs. unexposed and high groups f: p < 0.05, 

low vs. unexposed and middle groups; g: p < 0.05, low vs. middle and high groups; h: p < 0.05, 

exposed vs. low and middle groups; i: p < 0.05, unexposed vs. low group (Adapted from Figure 5 

of the report). 

In contrast, for the female rats, no significant changes were observed in the lung function 

test parameters when comparing the exposed groups with the unexposed control group . 

IDSTOPATHOLOGICAL EVALUATION 

In the histopathological examination of lungs, as summarized in Table 2. a significant 

increase in the incidence of mixed cell infiltrate perivascular and chronic alveolar 

inflammation, including alveolaritis, granulomatous lesions, and alveolar wall thickening 

and alveolar macrophage accumulation in both the male and female. During the 12 weeks 

of recovery, the female rats in all the groups showed a gradual clearance of the silver 

nanoparticles and decreased inflammation. However, the male ·rats in the high-dosed 

group and middle-dosed group did not achieve a complete recovery, exhibiting persistent 

inflammation throughout the 12-week recovery period (compared to the controls). 
Silver nanoparticles/ (PC Code 072599) 

90-day Inhalation Toxicity - Rat (2012) I Page 9 of 11 

Non-guideline 

Table 2: Summary of Results from MRID 49052001 (Song, KS, et al 2012) Subchronic Inhalation Study w/ Lung Function Recovery 

0 48.76 J.lg/m3 117.14 J.Lg/mj 381 J.lg/m3 

c3 ~ c3 ~ c3 ~ c3 ~ 

Lung Function Measurement: 

TV/MV/PEF (treatment & recovery) ! (p<0.05) ! (p<0.05) 

Tissue nAg Accumulation: 

Liver, kidneys, spleen, testes, ovaries, blood, eyes j (p<O.Ol) j (p<O.Ol) j (p<O.Ol) j (p<O.Ol) 

(treatment) 

j (p<O.Ol) j (p<O.Ol) j (p<O.Ol) j (p<O.Ol) 

4-wk recovery (spleen) (spleen; ovaries) (not testes & eyes) (not eyes) 

j (p<O.Ol) j (p<O.Ol) 

12-wk recovery (liver, spleen, eyes) (spleen, liver) 

nAg Lung Clearance: 

T112 (days) 28.5 38.7 84.9 112.9 42.3 40.4 

MRT.n (days) 13.8 11.5 33.9 35.4 28.5 28.4 

Histopath: n=5 n=4 n=5 n=4 n=5 n=4 

Minimal alveolar macrophage accumulation 0 0 1 0 3 0 5 4 

(treatment) 

0 0 0 0 3 0 5 4 

Minimal alveolar Chronic inflammation (treatment) 

1 2 1 0 3 0 5 4 

Minimal perivascular mix cell infiltration (treatment) 

2 0 0 1 1 1 2 2 

4-wk recovery (n=4) 

1 0 0 0 3 2 2 0 

12-wk recovery (n=4) 

- -- - -
Silver nanoparticles/ (PC Code 072599) 

90-day Inhalation Toxicity - Rat (2012) I Page 10 of 11 

Non-guideline 

SILVER CONCENTRATION IN TISSUES (LUNG AND OTHERS) 

A statistically significant (p < 0.01) dose-dependent increase in the silver concentration 

was observed in the lung tissue from the male and female groups exposed to the silver 

nanoparticles for 12 weeks. Although these silver concentrations did gradually clear from 

the lung tissue during the 12-week recovery period, the silver concentrations in the highdose group were not completely cleared (Table 2). A statistically significant (p < 0.01) 

dose-dependent increase in the silver concentration was observed in all tissues from the 

male and female groups exposed to the silver nanoparticles for 12 weeks, except for the 

female brain. While the tissue silver concentrations gradually reduced to basal levels in 

most tissues after 12 weeks of recovery, a significant accumulation compared with the 

control group was still noted (p < 0.01) in the liver, spleen, and eyes from the male rats 

(Table 2). The silver concentrations in the kidneys also showed a gender difference, with 

the female kidneys containing five times more accumulated silver than the male kidneys 

(Table 3). 

III. DISCUSSION AND CONCLUSIONS: 

A. INVESTIGATORS CONCLUSIONS: 

Although signs of inflammation were present in the middle-dose group after 12 weeks of 

exposure and recovery and the middle dose could be suggested for LOAEL in this study, 

it is supposed that the minimal accumulation of macrophages and inflammation in the 

alveolar were negligible and regarded as a minor response as often observed in the 

control group as shown in the previous study (Sung et al. 2009). The previously 

suggested 90-day NOAEL of 100 mg/m3 (Sung et al. 2009) was reconfirmed by the 

results of the present 12-week exposure study, where a NOAEL of 117 mg/m3 showed 

no significant lung damage after a 12-week recovery period. 

B. REVIEWER'S COMMENTS: 

For male rats, dose-related decrease in the physiological measurement of tidal volume 

(TV) that, in turn, led to decrease in measures of minute volume (MV) and peak 

inspiratory flow (PEF) , was observed in both exposure and recovery periods in the highdose and middle-dose groups. In contrast, for the female rats, no significant changes were 

observed in the lung function test parameters when comparing the exposed groups with 

the unexposed control group. 

In the histopathological examination, a significant increase in the incidence of mixed cell 

infiltrate perivascular and chronic alveolar inflammation, including alveolaritis, 

granulomatous lesions, and alveolar wall thickening and alveolar macrophage 

accumulation in both the male and female. During the 12 weeks of recovery, the female 

rats in all the groups showed a gradual clearance of the silver nanoparticles and decreased 

inflammation. However, the male rats in the high-dosed group and middle-dosed group 

did not achieve a complete recovery, exhibiting persistent inflammation throughout the 

12-week recovery period (compared to the controls). 
Silver nanoparticles/ (PC Code 072599) 

90-day Inhalation Toxicity - Rat (2012) I Page 11 of 11 

Non-guideline 

Therefore, based on the significant increase in the incidence of mixed cell infiltrate 

perivascular and chronic alveolar inflammation in both the male and female, and 

decreases in physiological measures of lung function (respiratory minute volume, tidal 

volume, and peak inspiratory flow) in the males, the NOAEL is determined to be 48.76 

J.1g/m3 and LOAEL is 117.14 J.1g/m3

• 

STUDY CLASSIFICATION: This inhalation study is classified Acceptable/Nonguideline and may be used for regulatory purposes; however, they do not satisfy the 

guideline requirement OPPTS 870.3465 for a subchronic inhalation study.