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Epidemiological data

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Administrative data

Endpoint:
epidemiological data
Type of information:
other: human data
Adequacy of study:
supporting study
Reliability:
other: not rated acc. to Klimisch
Rationale for reliability incl. deficiencies:
other: Well-documented longitudinal study

Data source

Reference
Reference Type:
publication
Title:
In vivo human time-exposure study of orally dosed commercial silver nanoparticles
Author:
Munger, M.A. et al.
Year:
2013
Bibliographic source:
Nanomedicine: Nanotechnology, Biology, and Medicine (in press), 1 - 9

Materials and methods

Study type:
cohort study (prospective)
Endpoint addressed:
repeated dose toxicity: oral
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Commercial 10- and 32-ppm nanoscale silver particle solutions were studied in a single-blind, controlled, cross-over, intent-to-treat, design. Healthy subjects (n = 60) underwent metabolic, blood counts, urinalysis, sputum induction, and chest and abdomen magnetic resonance imaging. Silver serum and urine content were determined.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: nanoform
Details on test material:
- Name of test material (as cited in study report): silver nanoparticles (manufactured by American Silver, LLC. (Alpine, Utah, USA))
- Average silver nanoparticle hydrodynamic diameter: 59.8 nm ± 20 nm.


Method

Type of population:
other: volunteers
Ethical approval:
confirmed and informed consent free of coercion received
Remarks:
all patients provided written informed consent
Details on study design:
The study was conducted in accordance with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Guidelines for Good Clinical Practice and the Declaration of Helsinki, and received approval from the University of Utah Institutional Review Board.

SETTING: two intent-to-treat studies were conducted at the University of Utah Lung Health Study Clinic and Center for Clinical and Translational Sciences at the University of Utah Hospital.

STUDY POPULATION
- Selection criteria: each subject underwent a screening evaluation to assess enrollment eligibility. Females of child-bearing potential, defined as women physically capable of becoming pregnant, whose career, lifestyle, or sexual orientation precluded intercourse with a male partner and women whose partners were using 2 barrier birth control methods or hormonal contraceptive method were allowed to participate. Subjects with a history of any heavy metal allergy; asthma, chronic bronchitis or emphysema; or renal impairment defined by a creatinine clearance ≤30 ml/minute; or significant acute or chronic disease as determined by the investigators were excluded. Subjects unable to complete the study were excluded from analysis and replaced.
- Total number of subjects participating in study: 62 subjects
- Total number of subjects at end of study: 60 subjects; two of the 62 subjects were discontinued, one due to inability to draw blood (subject never received study product) and one due to hospitalization for pulmonary embolism (subject received 12 days of placebo diluent only).
- Sex/age:
10 ppm: 17 males and 19 females (total: 36); mean age ± SD: 52 ± 11 years
32 ppm: 18 males and 6 females (total: 24); mean age ± SD: 41 ± 15 years
Exposure assessment:
not specified
Details on exposure:
TYPE OF EXPOSURE: the silver nanoparticle study product was manufactured by a published AC high voltage (10(3)-10(4)) aqueous electrolysis of de-ionized water using silver metallic electrodes. Silver is in the form of zero-valent elemental silver particles coated with silver oxide, with manufacturer's claims to particle size ranging between 5-10 nm (10 ppm; lot #122810) or claims to a mean of 32.8 nm, range of 25-40 nm (32 ppm: lot #071511), respectively.
Subjects received silver nanoparticle colloidal solution diluent (e.g. sterile water [no silver nanoparticles]) followed by the active silver solution. A 72-hour washout period preceded the dosing cross-over. Each subject received 15 mL of study material daily from a pre-mixed oral syringe. Each dose
administration was observed by study personnel to ensure compliance. Subjects were blinded to the study product received.

SILVER ION CONTENT
The study silver solution (lot #09280, 32 ppm) was analysed for silver ion content using a liquid chromatography ICP-MS (LC-ICPMS) Agilent 7500ce (Agilent Technologies, Inc., Santa Clara, California USA).

TYPE OF EXPOSURE MEASUREMENT:
- At baseline and the end of each time-period, subjects underwent a medical and drug history, complete physical examination, comprehensive metabolic panel, blood count with differential and urinalysis. Blood and urine were collected for serum and urine silver concentrations at trough concentrations (≥24 hours post-dose) for the 3- and 7-day time periods at 10 ppm and at peak concentration (≤2 hours post-dose) for the 14-day 10 ppm dose and for the 32 ppm study population. Silver concentrations in serum and urine were determined by using ICP-MS (NMS Laboratories, Willow Grove, USA). Calibration silver samples in dilute nitric acid and controls in human serum matrix were used in each ICP-MS assay cohort. The ICP-MS assay dynamic range of silver samples for this study was 0-40 μg/L. Sputum was collected by induction protocol within 24 hours of the last dose for each time-period, as previously described in Gibson et al. (1998).
- Sputum analysis: hydrogen peroxide concentrations were determined using a modification of a method (Cordray et al. (2007)*. Peroxiredoxin protein expression was measured as described in Cameron and Aust (1999)*. Determination of RNA expression using quantitative real-time polymerase chain reaction (qPCR) was determined by methods of Deering-Rice et al. (2012)*.
- A cardiac and abdominal MRI was obtained at the end of each phase of each time period. Patients were examined on a 1.5-T 32-channel superconducting magnetic resonance system (Magnetom Avanto, Siemens Medical Solutions).

EXPOSURE LEVELS: the average daily ingestion of the elemental silver colloid formulation is estimated to be 100 μg/day for 10 ppm, and 480 μg/day for 32 ppm silver.

EXPOSURE PERIOD: a dose-time escalation dosing scheme was employed. Study one used 10 ppm oral silver particle dosing with 3-, 7-, and 14-day time periods; study two used 32 ppm for 14 days.

*Reference:
- Gibson PG, Wlodarczyk JW, Hensley MJ, Gleeson M, Henley RL, Cripps AW, Clancy RL. Epidemiological association of airway inflammation with asthma symptoms and airway hyperresponiveness in childhood. Am J Respir Crit Care Med 1998; 158: 36-41.
- Cordray P, Doyle K, Edes K, Moos PJ, Fitzpatrick FA. Oxidation of 2-cys-perosiredoxins by arachidonic acid peroxide metabolites of lipoxygenases
and cycloxygenase-2*. J Biol Chem 2007;282(45):32623-9.
- Cameron MD, Aust SD. Degradation of chemicals by reactive radicals produced by cellobiose dehydrogenase from Phanerochaete chyrsoporium.
Arch Biochem Biophys 1999;367(1):115-21.
- Deering-Rice CE, Johansen ME, Roberts JK, Thomas KC, Romero EG, Lee J, et al. Transient receptor potential vanilloid-1 (TRPV1) is a mediator of lung toxicity for coal fly ask particulate material. Mol Pharmacol 2012;81(3):411-9.
Statistical methods:
Average effect analysis was employed to assess toxicity. A ± 4 SD range was applied as the statistical rule based on the concept that a normal distribution contains nearly all individual observations within a ±3 SD interval. A mixed effects linear regression model with repeated measurements from the crossover periods nested within subjects was used to determine effect differences. In this model, the baseline value was included as a covariate. The crossover period was the primary predictor variable. To test for a linear trend across exposure time, the 3-, 7-, and 14-days a mixed effects model using exposure time as the predictor was fitted. The P value for the time variable in such a model represents a linear dose–response significance test. All reported P values were from a two-sided comparison.
The power of the study to detect toxicity was sufficient for individual observation analysis and the usual mean difference analysis. Combining the 10 ppm and 32 ppm silver colloidal solutions, for a total sample size of n = 60, based on a binomial probability, using 1-Prob (observed incidence = 0|true incidence = 0.027), the total sample size of n = 60 provided 80% probability of observing toxicity in at least one study subject if the true toxicity incidence is 2.7%. For the analysis examining paired sample mean differences between placebo (diluent only) and active silver solution, the n = 24 subjects with maximum exposure of 32 ppm silver colloidal solution for 14 days provided 80% power to detect a mean difference of 0.48 standard deviations (SD), using a two-sided alpha 0.05 comparison and assuming a correlation of r = 0.70 between the placebo and active phases. Given the reference range is ±2 SDs, the effect sizes of 0.48 SD and 0.30 SD represented detectable effects well within normal laboratory reference ranges.

Results and discussion

Results:
CLINICAL FINDINGS
- No clinically important changes in weight, BMI, systolic or diastolic blood pressure or heart rate were noted. However, heart rate significantly declined by 2.3 beats per minute for the total group.
- Results of the complete metabolic panel showed no significant or clinically important changes in laboratory finding across the total population.
- Blood urea nitrogen and alanine aminotransferase tests from the 10 ppm dose were analyzed to be statistically significant, but nothing in the 32 ppm dose cohort was noted. When the 95% CI limits of these significant tests are added to the mean value of the active period, representing a statistical comparison to the normal reference range limits, all values remain within the normal reference range. There was no significance in any metabolic test, suggesting that increasing dosing from 10 to 32 ppm does not elicit silver toxicity.
- Comparison of the red blood cell count (RBC) between active vs. placebo solutions was significant at the 10 ppm dose, but not at the 32 ppm dose. There were no clinically important changes in any blood count value including erythrocytes, granulocytes, or agranulocyte counts. Exposure time was not associated with changes in blood counts.
- No significant or clinically important changes were evident in the complete urinalysis. Although there were individual subject positive tests for urine ions, proteins, blood cells, and some other solutes, these changes remained unchanged in comparison between the active and placebo solutions.

SERUM SILVER AND URINE FINDINGS
- Serum and urine silver concentrations were determined at different time variables. There was no detection of serum silver from subjects at trough concentrations throughout the 3- and 7-day time periods at 10 ppm. Peak serum silver concentration was detected in 42% of subjects in the 14-day 10 ppm dosing showing a mean of 1.6 ± 0.4 mcg/L. The 32 ppm dose mean concentration was detected in 92% of subjects at 6.8 ± 4.5 mcg/L. No silver was detected in the urine, independent of dose or time period.

SPUTUM REACTIVE OXYGEN SPECIES (HYDROGEN PEROXIDE) AND PRO-INFLAMMATORY CYTOKINE RNA FINDINGS
Quality paired samples allowing determination of ROS concentrations and pro-inflammatory cytokine RNA expression were analyzed in 72% and 83% of 10 ppm and 32 ppm study samples, respectively. No statistically significant change in markers of hydrogen peroxide production or peroxiredoxin protein expression was detected. Analysis of IL-8, IL-1α, IL-1β, MCP1 and NQO1 also showed no statistical difference between the active silver and placebo solutions.

MRI FINDINGS
Eighteen 10 ppm and eleven 32 ppm subjects underwent a post 3-14 day, respective active and placebo solution cardiac and abdominal MRIs. No morphological or structural changes were noted between active and placebo solutions.

Applicant's summary and conclusion

Conclusions:
The authors stated that no clinically important changes in metabolic, hematologic, or urinalysis measures were identified. No morphological changes were detected in the lungs, heart or abdominal organs. No significant changes were noted in pulmonary reactive oxygen species or pro-inflammatory cytokine generation. According to the authors, in vivo oral exposure to these commercial nanoscale silver particle solutions does not prompt clinically important changes in human metabolic, hematologic, urine, physical findings or imaging morphology.