Silver nanoparticles/ (PC Code 072599) 

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

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

### RiskAssessment and Science SupportBranch AntimicrobialDivision TXR: 1,003,297 90-day Inhalation Toxicity

- Rat (2012) IPage 1 of 11 Non-guideline 

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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 maybe formed. 14.09 ±1
.71nm) 

### SYNONYMS: Notavailable 

### CITATION: Song KS.; Sung, JH; andJi, 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. Inthe 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 offresh-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 106particle/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 ofexposure, after 4 weeks ofrecovery, and after 12 weeks ofrecovery. An additional5 male rats were also assigned to the 12-week exposure group for a detailed study ofthe braintranslocation. The animals were examined daily onweekdays for any evidence ofexposure-related effects, including respiratory, dermal, behavioral, nasal, or genitourinary changes suggestive ofirritancy. Body weights were measured atthe time ofpurchase, at the time ofgrouping, 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 Non-guideline No test substance-related differences were observed in food consumption rates, or body weight/body weight gain changes betweenthe exposed and the control groups. For male rats, dose-related decrease in the physiological measurement oftidal volume (TV) that, in turn, led to decrease in measures ofminute volume (MV) and peak inspiratory flow (PEF) , were observed inboth 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 withthe unexposed control group. Inthe histopathological examination, a significant increase was observed 

### inthe 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 ofrecovery, the female rats in all the groups showed a gradual clearance ofthe 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 inflammationthroughout the 12-weekrecovery period (compared to the controls). Based onthe significant increase inthe incidence ofmixed cell infiltrate perivascular and chronic alveolar inflammation in boththe male and female, and decreases inphysiological measures oflung function (respiratory minute volume, tidal volume, and peak inspiratory flow) inthe 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 studypresented in this published article is a continuation ofprevious 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 ofsilver nanoparticles. Toxicological Sciences. 108(2);452-461. MRID 47757505. 2. Sung, JH; Ji, JH; and Yoon, 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 Inhalationtoxicity 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. TestMaterial: 90-day Inhalation Toxicity- Rat (2012) IPage 3 of 11 Non-guideline



### Description: Silvernanoparticles 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/dilutionsto be used inthe inhalation studies ofnanosilverparticles. 

### Characterization: The generated study nanosilver particles observedby transmission electron microscopy (TEM) were in spherical shape and nonaggregated/agglomeratedpure silver, not silver oxides. The nanosilver particles distribution withrespect to size in each chamber was measured directly inreal-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 ofthe silver nanoparticles were summarized inTable 1.


### Table 

### 1. Particle Characterization Geometric Geometric Concentration Mean Standard TotalNumber 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) IPage 4 of 11 Non-guideline

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Compound Stability: Notprovided Notprovided Notprovided Notprovided Notrequired CAS # ofTGAI: Structure:Silver nanoparticles/ (PC Code 072599) 90-day Inhalation Toxicity

- Rat (2012) IPage 5 of 11 Non-guideline 

### 2. Vehicle and/orpositive 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. Purchasedfrom 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 adlibitum 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-weekrecovery 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 offresh-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 106particle/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 ofrecovery, and after 12 weeks ofrecovery. An additional5 male rats were also assigned to the 12-week exposure group for a detailed study ofthe braintranslocation. 

### C. METHODS: LungFunction Test: Pulmonary functions offour rats from each dose group were evaluated weekly during the 12-week exposure and 12-weekrecovery periods using a ventilated bias flow whole-body plethysmograph (WBP). The parameters ofpulmonary functions included tidal volume (TV, ml), minute volume (MV, ml/min), respiratory frequency (BPM, breath/min), inspirationtime (Ti, s), expiration time (Te, s), peak inspiration flow (PIF, ml/s), andpeak 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 othertissues The silver concentration was observed inthe lung and other tissues from animal groups exposedto 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 inparaffin, stained with hematoxylin and eosin (H&E), and examined using light microscopy. 

### D. DATAANALYSIS: Statistics- AnANOVAtest and Duncan's multiple range tests were usedto compare the body weights, parameters from the lung function test and silver concentration in tissue obtained for the three dose groups withthose 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 BODYWEIGHT None ofthe rats died. No test substance-related differences were observed infood consumption rates, orbody weight/body weight gain changes between exposed and the control groups. 

### LUNG FUNCTIONTEST (MRID 48552402): For male rats, dose-related decrease in the physiological measurement oftidal volume (TV) that, 

### inturn, led to decrease in measures ofminute 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 ofexposure; a statistically significant decrease was observed after 5, 8, and 9 weeks ofexposure (p 

### < 0.05) (Figure 3A). Meanwhile during the recovery period, a significant decrease in the TV was also observed inthe exposed groups when compared withthe control group after 1-10 weeks ofrecovery, where a statistically significant decrease was observed after 1, 3, 4, 5, 6, 7, 8, and 10 weeks ofrecovery (Figure 3A). This tendency ofa lung function decrease also affected the MV (minute volume) and PEF (peak expiratory flow) during boththe 12-week exposure and 12-weekrecovery 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) afterSilver nanoparticles/ (PC Code 072599) 90-day Inhalation Toxicity

- Rat (2012) IPage 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).



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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 ofthe report). B 1100 1000

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### Time(week) FIG. 3B. Lung Function- minutevolume (mVmin) change in male rats exposed to silver nanoparticles and during a 12-weekrecovery 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 ofthe report).Silver nanoparticles/ (PC Code 072599) 90-day Inhalation Toxicity

- Rat (2012) I Page 8 of 11 Non-guideline 

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### Time(week) FIG. 3C. LungFunction- PeakExpiratory 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. othergroups; 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 ofthe report). In contrast, for the female rats, no significant changes were observed in the lung function test parameters when comparingthe exposed groups withthe unexposed control group . 

### IDSTOPATHOLOGICALEVALUATION Inthe histopathological examination oflungs, as summarized in Table 2. a significant increase in the incidence ofmixedcell infiltrateperivascular and chronic alveolar inflammation, including alveolaritis, granulomatous lesions, and alveolar wall thickening and alveolar macrophage accumulation inboththe male and female. During the 12 weeks ofrecovery, the female rats in all the groups showed a gradual clearance ofthe silver nanoparticles and decreased inflammation. However, the male· rats inthe high-dosed group and middle-dosed group did not achieve a complete recovery, exhibiting persistent inflammationthroughout the 12-weekrecovery 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 ofResults from MRID 49052001 (Song, KS, et al 2012) Subchronic Inhalation Study w/LungFunction Recovery 0 48.76 J.lg/m3 117.14 J.Lg/mj 381 J.lg/m3 

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### ! (p<0.05) 

### ! (p<0.05) Tissue nAgAccumulation: 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-wkrecovery (spleen) (spleen; ovaries) (nottestes & eyes) (not eyes) j (p<O.Ol) j (p<O.Ol) 12-wkrecovery (liver, spleen, eyes) (spleen, liver) nAgLung Clearance: T 112 (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-wkrecovery (n=4)

- -- - -Silver nanoparticles/ (PC Code 072599) 90-day Inhalation Toxicity

- Rat (2012) I Page 10 of 11 Non-guideline 

### SILVERCONCENTRATION IN TISSUES (LUNG AND OTHERS) A statistically significant (p 

### < 0.01) dose-dependent increase inthe silver concentration was observed inthe 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-weekrecovery period, the silver concentrations inthe highdose group were not completely cleared (Table 2). A statistically significant (p 

### < 0.01) dose-dependent increase inthe silver concentration was observed in all tissues from the male and female groups exposedto the silver nanoparticles for 12 weeks, exceptfor the female brain. While the tissue silver concentrations gradually reduced to basal levels in mosttissues after 12 weeks ofrecovery, 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 inthe kidneys also showed a gender difference, with the female kidneys containing five times more accumulated silver thanthe male kidneys (Table 3). 

### III. DISCUSSION AND CONCLUSIONS: A. INVESTIGATORS CONCLUSIONS: Although signs ofinflammation were present in the middle-dose group after 12 weeks of exposure and recovery and the middle dose could be suggested for LOAEL inthis study, it is supposed that the minimal accumulation ofmacrophages and inflammation in the alveolar were negligible and regarded as a minor response as often observed in the control group as shown inthe previous study (Sung et al. 2009). The previously suggested 90-dayNOAEL of 100 mg/m3 (Sung et al. 2009) was reconfirmed by the results ofthe present 12-weekexposure study, where a NOAEL of 117 mg/m3 showed no significant lung damage after a 12-weekrecovery period. 

### B. REVIEWER'S COMMENTS: Formale rats, dose-related decrease inthe physiological measurement oftidal volume (TV) that, inturn, led to decrease in measures ofminute volume (MV) and peak inspiratory flow (PEF) , was observed in both exposure and recovery periods inthe highdose and middle-dose groups. Incontrast, for the female rats, no significant changes were observed in the lung function test parameters when comparingthe exposed groups with the unexposed control group. Inthe histopathological examination, a significant increase inthe incidence ofmixed cell infiltrate perivascular and chronic alveolar inflammation, including alveolaritis, granulomatous lesions, and alveolar wall thickening and alveolar macrophage accumulation inboththe male and female. During the 12 weeks ofrecovery, the female rats in all the groups showed a gradual clearance ofthe silver nanoparticles and decreased inflammation. However, the male rats inthe high-dosed group and middle-dosed group did not achieve a complete recovery, exhibiting persistent inflammationthroughout the 12-weekrecovery period (compared to the controls).Silver nanoparticles/ (PC Code 072599) 90-day Inhalation Toxicity

- Rat (2012) IPage 11 of 11 Non-guideline Therefore, based onthe significant increase inthe incidence ofmixed cell infiltrate perivascular and chronic alveolar inflammation inboththe male and female, and decreases inphysiological measures oflung function (respiratory minute volume, tidal volume, andpeak inspiratory flow) inthe males, 

### theNOAEL 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 satisfythe guideline requirement OPPTS 870.3465 for a subchronic inhalation study.