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Repeated dose toxicity: other routes

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short-term repeated dose toxicity: other route
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions - non-physiological route of administration (i.v) - reduced number of animals per dose group

Data source

Reference Type:
Systemic and immunotoxicity of silver nanoparticles in an intravenous 28 days repeated dose toxicity study in rats
De Jong, W.H. et al.
Bibliographic source:
Biomaterials (2013),

Materials and methods

Test guideline
according to
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
modified OECD 407
Principles of method if other than guideline:
The animals were exposed to nanosilver particles via intravenous administration. Instead of three dose groups (low, mid and high dose) and one control group, the number of dose groups was increased, at the expense of the group sizes, i.e. while keeping the total number of animals the same. With this design, and by applying the bench mark dose (BMD) approach (EFSA Scientific Opinion)*, an improved characterization of the dose response is obtained without increasing the number of animals. Because of the known accumulation in the spleen (Lankveld et al. 2010)* additional tests with spleen cells were performed for possible immunotoxicological properties of nanosilver. In addition not all organs indicated in OECD 407 were evaluated by histopathology.

* References:
- EFSA Scientific Opinion. Use of the bench mark dose approach in risk assessment. EFSA J 2009;1150:1-72.
- Lankveld DP, Oomen AG, Krystek P, Neigh A, Troost-de Jong A, Noorlander CW, et al. The kinetics of the tissue distribution of silver nanoparticles of different sizes. Biomaterials 2010;31:8350-61.
GLP compliance:
not specified
Limit test:

Test material

Test material form:
other: nanomaterial 20 nm BioPure citrate AgNPs Particle size: 21.0 ± 2.6 nm 100 nm Biopure citrate AgNPs Particle size: 107 ± 7.6 nm Source: NanoComposix, San Diego (USA)
Details on test material:
- Name of test material (as cited in study report): BioPure silver nanoparticles (in 2 mM phosphatte buffer) (from NanoComposix, San Diego, CA, USA)
- Size ± SD:
20 nm diameter: 21.0 ± 2.6 nm
100 nm diameter: 107 ± 7.6 nm
- Coefficient of variation:
20 nm diameter: 12.2%
100 nm diameter: 7.1%
- Size range (min-max diameter):
20 nm diameter: 12.4 - 27.9
100 nm diameter: 92.8 - 128.4
- Number of particles:
20 nm diameter: 3.9 x 10^13 mL^1
100 nm diameter: 3.8 x 10^11 mL^1
- Surface area per particle:
20 nm diameter: 5.49 x 10^16 nm^2
100 nm diameter: 1.37 x 10^16 nm^2
- Silver concentration
20 nm diameter: 2 mg/mL
100 nm diameter: 2.6 mg/mL
- Zeta potential:
20 nm diameter: -40.8 mV
100 nm diameter: -38.7 mV

The nanosilver dispersions provided were characterized with minimal agglomeration or aggregation.

Test animals

other: SPF Wistar
Details on test animals and environmental conditions:
TEST ANIMALS - Wistar derived WU rats
- Source: Harlan Nederland BV, Horst, The Netherlands
- Age at study initiation: 8 weeks of age
- Diet (ad libitum): conventional feed
- Water (ad libitum): drinking water

Husbandry conditions were maintained according to all applicable provisions of the national laws, Experiments on Animals Decree and Experiments on Animals Act.

Administration / exposure

Route of administration:
other: intravenous, via tail vene
other: phosphate buffer
Details on exposure:
Animals were divided in 11 groups and were intravenously injected (tail vein) with either 1 mL nanosilver dispersion or vehicle control. The experiment was performed in two phases, the first being the 20 nm particle treatments followed by the 100 nm particle treatments.
Individual body weights of the rats were used to calculate the individual dose levels.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrations
Doses / Concentrations:
20 nm nanosilver (AgNPs): 0.0082, 0.0025, 0.074, 0.22, 0.67, 2 and 6 mg/kg bw/day
100 nm nanosilver (AgNPs): 6 mg/kg bw/day
No. of animals per sex per dose:
Vehicle control: 2 males/2 females
20 nm nanosilver (0.0082, 0.0025, 0.074 mg/kg bw /day): 2 males/2 females
20 nm nanosilver (0.22, 0.67, 2 and 6 mg/kg bw/day): 3 males/3 females
100 nm nanosilver (6 mg/kg bw/day): 4 males/4 females
Control animals:
yes, concurrent vehicle


Observations and examinations performed and frequency:


- Time schedule for examinations: prior to and weekly during the experiment.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data



- Time schedule for collection of blood: blood samples obtained at autopsy
- Anaesthetic used for blood collection: Yes, isoflurane (Isoflu®, AST Pharma, Oudewater, The Netherlands) in oxygen and subsequently euthanized by drawing blood from the abdominal aorta
- Animals fasted: No data
- How many animals: all
- Parameters examined: white blood cell count, red blood cell count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red blood cell distribution width, haemoglobin distribution width, platelet count, and mean platelet volume
- Blood smears were prepared for visual evaluation.

- Time schedule for collection of blood: blood samples obtained at autopsy
- Animals fasted: No data
- How many animals: all
- Parameters examined: albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transpeptidase, amylase, creatinin kinase, lactate dehydrogenase, sodium, potassium, glucose, urea, creatinin, cholesterol and total protein.


Sacrifice and pathology:
At 24 hours after the last injection, rats were anaesthetized with isoflurane (Isoflu®, AST Pharma, Oudewater, The Netherlands) in oxygen and subsequently euthanized by drawing blood from the abdominal aorta. After collecting blood, rats were evaluated macroscopically for gross lesions. The following organs were examined and sampled: adrenals, brain, bone marrow, small intestines (duodenum, jejunum, ileum), large intestines (caecum, colon, rectum), heart, kidney, liver, lung, lymph nodes (mesenteric and popliteal), oesophagus, pituitary, spleen, stomach, testis (or ovaria), and thymus. Organ weights were determined except for bone marrow, pituitary and gastrointestinal tract.
Histopathology was performed for the following treatments:
- 20 nm Ag-NP 6 mg/kg bw (3 male and 3 female animals) and 2 mg/kg b.w. (3 male and 3 female animals)
- 100 nm Ag-NP 6 mg/kg b.w. (4 male and 4 female animals)
- phosphate buffer control (6 male and 6 female animals).
Other examinations:
Cells were collected by flushing 4 ml Impuls Cytometer Fluid through the femur. The concentration of nucleated cells was determined in a Coulter Counter.

- Spleen cell distribution: the spleen was weighed and a weighed part of the spleen was collected in tissue culture medium of which spleen cells were isolated. Relative spleen cell distribution was determined by fluorescence-activated cell sorter analysis as described by Tonk et al. (2010)*. Spleen single-cell suspensions were prepared and evaluated for subset distribution by 3-color flow cytometry. The following monoclonal antibodies were used: allophycocyanin (APC)-conjugated mouse anti-rat CD3 (clone 1F4, T lymphocytes), R-phycoerythrin (PE)-conjugated mouse anti-rat CD8a (clone OX-8, T-suppressor/ cytotoxic cells), fluorescein isothiocyanate (FITC)-conjugated mouse anti-rat CD4 (clone OX-35, T helper cells), FITC-conjugated mouse anti-rat CD45RA (clone OX-33, B lymphocytes), and PE-conjugated mouse anti-rat CD161a (clone 10/ 78, NK cells); all from Pharmingen, San Diego, CA, USA. Single-cell suspensions were incubated with the conjugated mAbs for 30 minutes at 4°C in the dark. The cells were washed twice with wash buffer (5% BSA in PBS), resuspended in 0.1% paraformaldehyde in PBS, and analysed on a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA, USA). A total of 10, 000 events were recorded per sample and analyzed.

- Natural killer (NK) cell activity: spontaneous cytotoxicity of spleen cell populations was evaluated in an in vitro 51Cr-release assay for determination of NK cell activity as described previously (de Groot (2010); De Jong et al. (1999))*. Spleen single-cell suspensions were adjusted in a standard medium containing 10% FBS. The YAC target cells (1 x 10^6 cells/100 µL) were labeled with 3.7 MBq Na51/2CrO4 (Amersham, Buckinghamshire, UK) for 45 minutes at 37°C, washed 5x, and resuspended in standard medium containing 10% FBS at 1 x 10^5 viable cells/mL. The NK cell activity assays were performed in 96-well polystyrene U-bottom microplates (Costar, Cambridge, MA, USA) for each sample in triplicate. Aliquots of 1 x 10^4 target cells were added to each well containing the appropriate number of effector cells to give effector cell (E) to target cell (T) ratios (E:T) of 200:1, 100:1, 50:1, and 25:1, to a final volume of 200 µL. Controls included a spontaneous 51Cr-release control (target cells ± medium) and a maximum 51Cr-release control (target cells ± 1% triton). Plates were centrifuged for 5 minutes at 1200 rpm and incubated at 5% CO2, 37°C for 4 hours. Radioactivity in the cell supernatants was measured using a gamma counter (Packard, Tilburg, The Netherlands) as counts per minute (cpm). Percentage of cytotoxicity was calculated as:
%Cytotoxicity = (sample cpm - spontaneous cpm / maximum cpm spontaneous cpm) x 100

- Cytokine production of spleen cells: supernatants of mitogen stimulated cells were harvested for LPS stimulated cells at 24 hours incubation (37°C, 5% CO2), and for Concanavalin A stimulated cells at 48 hours incubation. Interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-10, IL-17, IL-13, interferon (IFN)-γ, TNF-α and granulocyte-macrophage colony-stimulating factor (GM-CSF) were measured in the collected supernatants using a MILLIPLEX Map Kit (Millipore, Billerica, MA, USA).

After collection of blood serum and storage at -20°C immunoglobulin levels were determined in an enzyme linked immunosorbant assay (ELISA) using mouse monoclonal antibodies against rat IgG, IgM and IgE, respectively.

- Tonk EC, de Groot DM, Penninks AH, Waalkens-Berendsen ID, Wolterbeek AP, Slob W, et al. Developmental immunotoxicity of methylmercury: the relative sensitivity of developmental and immune parameters. Toxicol Sci 2010;117: 325-35.
- De Jong WH, Kroese ED, Vos JG, Van Loveren H. Detection of immunotoxicity of benzo[a]pyrene in a subacute toxicity study after oral exposure in rats. Tox
Sc 1999;50:214-20.
Data are presented as means with their standard deviation.
Treatment effects of 20 nm particles were evaluated using the bench mark dose (BMD) approach, by fitting a nonlinear regression model to the data of all individual animals.
Statistical analysis of the effects of the administration of 100 nm Ag-NP was performed with SPSS software (SPSS Inc., Chicago, IL, USA).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
- no clinical signs for toxicity were observed during the 28-days exposure period for treatment with either 20 nm Ag-NP or 100 nmAg-NP.

20 nm Ag-NP:
- male and female rats in the highest dose group (6 mg/kg bw) of the 20 nm Ag-NP showed lower body weights after 4 weeks of intravenous administration of the 20 nm nanosilver.
- males and females did not differ statistically significantly in the dose-response for body weight (apart from background weight).
- CED05 (dose at 5% deviation from control) for a decrease in body weights was 5.26 mg/kg bw and resulted in a confidence interval of (2.9, 24) mg/kg bw, but when evaluating the body weight gain as a percentage of the weight at day 0 it reduced to a CED05 of 3.89 mg/kg bw with a confidence interval of (2.8, 6.1) mg/kg bw.
- maximal response indicates a minor weight difference of 6-8% compared to control animals.

100 nm Ag-NP:
- delay in growth in both male and female animals was observed.

- several parameters associated with red blood cell generation showed a significant decrease in the highest doses administered which could be confirmed by dose response analysis with a CES of 5%. Haematocrit, mean corpuscular cell volume, mean corpuscular cell haemoglobin, and haemoglobin level in red blood cells showed a limited decrease after treatment with the 20 nm Ag-NP. Number of reticulocytes was increased after treatment with the 20 nm Ag-NP.
- after treatment with 100 nm Ag-NP a similar decrease was observed for haematocrit, mean corpuscular cell volume, mean corpuscular cell haemoglobin, and haemoglobin level in red blood cells, which was not significant.
- total number of white blood cell count was not increased, but the absolute number of blood neutrophilic granulocytes showed a significant dose related increase with the 20 nm Ag-NP (p < 0.05 ANOVA). Treatment with 100 nm Ag-NP resulted in similar effects on the blood parameters.

- several clinical chemistry parameters showed a dose related effect including albumin, alkaline phosphatase, alanine transaminase, amylase, aspartate transaminase, creatinin kinase, glucose, lactate dehydrogenase, total protein, and urea.

- heart, kidneys, adrenals, brain, testes and epididymis were weighed and showed no differences in the different dose groups (Ag-NP 20 nm) compared to the control, whereas for liver a weight increase and for the thymus a weight decrease was observed.
- both for the absolute and relative spleen weight an increase was observed with a CED05 of 0.37 mg/kg bw (confidence interval 0.12, 1.04) and 0.25 mg/kg bw (confidence interval 0.14, 0.64), respectively.
- after treatment for 28 days with 100 nm Ag-NP (6 mg/kg bw) in both male and female animals spleen weight was similarly increased compared to control animals.
- bench mark dose analysis showed for the liver an increase in weight for males and females after treatment with 20 nm Ag-NP, CED05 2.20 mg/kg bw (c.i. 1.39, 4.88), and for female animals exposed to 100 nm Ag-NP.

- macroscopic examination of the rats revealed enlarged brownish colored spleens and livers, and enlarged and dark coloured lymph nodes in the highest dose groups.

- pigment was observed in various organs including spleen, liver, and lymph nodes.
- after administration of 20 nm Ag-NP the pigment in the spleen was mainly observed in the red pulp, while after 100 nm Ag-NP the pigmentwas observed in both the red and white pulp of the spleen.
- pigment was observed in the sinusoids of the lymph nodes (mesenteric and popliteal lymph node).
- in the liver the pigment (nanoparticles) was present in the Kupffer cells lining the walls of the venous sinusoids.
- pigment was observed in venous endothelial cells and in small foci of inflammatory cells. These foci are also present (without pigment) in control animals and are generally considered background histopathology in the rat strain used.
- pigment was observed in the kidney, adrenal glands and in most of the intestinal tract (jejunum, ileum, caecum, colon and rectum).
- in the lung small granulomas were observed partly caused by the injection technique as indicated by the presence of hair fragments. Some granulomas contained black pigment after injection of 100 nm Ag-NP.

An increase in IgM and IgE serum levels was seen after treatment with both 20 nm and 100 nm Ag-NP. In the spleen T cell, B cell and NK cell populations showed an increase after treatment with 20 nm Ag-NP. Similarly, for 100 nm Ag-NP an increase in T, B and NK cell populations was observed. This increase in cell numbers can be considered responsible for the weight increase of the spleen. Mitogen responses to B (LPS) and T (Concanavalin A) cell mitogens showed increased responses per spleen due to the increased cell number Interferon-γ, TNF-α, IL-6 and IL-10 cytokine production were decreased after treatment with 20 nm Ag-NP, whereas IL-1β production was increased. For the 100 nm Ag-NP treatment only the IL-10 production was decreased.
Both nanoparticles (20 nm and 100 nm Ag-NP) induced an almost complete suppression of the NK cell activity at all effector-to-target ratios (200:1, 100:1, 50:1, 25:1) investigated. The suppression of NK cell activity by the 20 nm Ag-NP started at a dose of 0.22 mg/kg bw, but was most pronounced at doses of 2 mg/kg and 6 mg/kg bw. The CED05 for the suppression of NK cell activity was 0.06 mg/kg bw with a confidence interval (0.04, 0.11) when evaluated per NK cell culture, and 0.12 mg/kg bw with a confidence interval of (0.04, 0.29) when evaluated per spleen.

- Bone marrow: no differences in cell count were observed after treatment with 20 nm or 100 nm Ag-NP.

Effect levels

Dose descriptor:
other: Discussion of effects by the authors
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Effect level not specified (migrated information)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

According to the authors, treatment with a maximum dose of 6 mg/kg body weight was well tolerated by the animals, but for 20 nm and 100 nm Ag-NP growth retardation was observed during the treatment. A severe increase in spleen size and weight was present which was due to an increased cell number. Both T and B cell populations showed an increase in absolute cell number, whereas the relative cell numbers remained constant. At histopathological evaluation brown and black pigment indicating Ag-NP accumulation was noted in spleen, liver, and lymph nodes. Ag-NP was also detected incidentally in other organs. Clinical chemistry indicated liver damage (increased alkaline phosphatase, alanine transaminase, and aspartate transaminase) that could not be confirmed by histopathology. Hematology showed a decrease in several red blood cell parameters.
There was almost complete suppression of the natural killer (NK) cell activity in the spleen at high doses. Other immune parameters affected were: decreased interferon-γ and interleukin (IL)-10 production by concanavalin-A stimulated spleen cells, increased IL-1β and decreased IL-6, IL-10 and TNF-α production by lipopolysaccharide stimulated spleen cells, increase in serum IgM and IgE, and increase in blood neutrophilic granulocytes. For the spleen weight a critical effect dose of 0.37 mg/kg bw could be established. The lowest critical effect dose (CED) for a 5% change compared to control animals was observed for thymus weight (CED05 0.01 mg/kg b.w.) and for functional immune parameters, i.e. decrease in NK cell activity (CED05 0.06 mg/kg b.w.) and LPS stimulation of spleen cells (CED05 0.04 mg/kg b.w.).