Registration Dossier

Toxicological information

Neurotoxicity

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Administrative data

Endpoint:
neurotoxicity
Remarks:
subacute
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
other: not rated (non-standard test system)

Data source

Reference
Reference Type:
publication
Title:
Exposure to silver nanoparticles does not affect cognitive outcome or hippocampal neurogenesis in adults mice
Author:
Liu, P. et al.
Year:
2013
Bibliographic source:
Ecotoxicology and Environmental Safety 87, 124 - 130

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
To assess whether silver nanoparticles influence spatial cognition and adult hippocampal neurogenesis, male ICR mice received intraperitoneal administration of silver nanoparticles (10, 25, and 50 mg/kg/bw) or vehicle every day for 7 days. At the end of this time period, Morris water maze test was performed for the spatial learning and memory. Subsequently, mice were injected with bromodeoxyuridine and sacrificed 1 day or 28 days after the last injection in order to evaluate cell proliferation, survival and differentiation in the hippocampus.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: nanomaterial
Details on test material:
- Name of test material (as cited in study report): noncoated silver nanoparticles (Ag-NPs)(diameter: 25 nm)(purchased from Amreco, USA)

Test animals

Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: National Rodent Laboratory Animal resources (Shanghai branch, China)
- Weight at study initiation: 32 - 35 g
- Housing: housed in separate cages
- Diet (ad libitum): standard laboratory food
- Water (ad libitum): water

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
physiological saline
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The suspension of the Ag-NPs was prepared in deionized water, and dispersed by ultrasonic vibration (KQ2200E, Kunshan, China) for 30 minutes in icy water, followed by stirring on a vortex agitator before every use. The morphologies of Ag-NPs were studied by transmission electron microscopy (TEM, JEM-2000EX, JEOL, Japan), and the size statistical distributions were determined by counting more than one hundred Ag-NPs in TEM photographs. Dynamic light scattering (DLS) and zeta potential measurements were carried out at room temperature using a Zetaplus Analyzer (Zetaplus, Brookhaven, USA).
The Ag-NPs were generally spherical in morphology with an average size of 36.3 ± 1.2 nm. The DLS size distribution by number was 51.4 ± 0.5 nm, which was consistent with the TEM results. the value of zeta potential was -19.6 ± 0.7 mV.

Ag-NPs suspensions or the same volume vehicle was administered intraperitonally.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
7 consecutive days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 25, and 50 mg/kg bw silver nanoparticles
Basis:
nominal conc.
No. of animals per sex per dose:
Experimental groups: 15 mice
Control group: 10 mice
Positive control group: 10 mice
Control animals:
yes, concurrent vehicle

Examinations

Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: No data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data
Specific biochemical examinations:
NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No data
CHOLINESTERASE ACTIVITY: No data
Neurobehavioural examinations performed and frequency:
One day after the last administration the Morris water maze test was started (n =10 mice) in order to assess the spatial learning and memory ability (protocol by Vorhees and Williams, 2006)*.

*Reference
- Vorhees, C.V., Williams, M.T., 2006. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat. Protoc. 1, 848 - 858.
Sacrifice and (histo)pathology:
One day after the last administration, five mice from each experimental group were sacrificed, and the hippocampi were taken out for silver content analysis. Hippocampi were rapidly separated from brains, weighed, and digested. Inductively coupled plasma-mass spectrometry (IPS-MS, Elan 9000, PE, USA) was used to analyze the silver concentration in the samples.

24 hours after the Morris water maze, mice (n = 5 in each group/time point) from the control and experimental groups were intraperitoneally injected with bromodeoxyuridine (BrdU; Sigma-Aldrich, St. Louis, MO) at 100 mg/kg/day for 2 consectuve days and sacrificed 1 day or 28 days after the last BrdU injection. Brains were removed and coronal sections were prepared.
Biotinylated-BrdU immunostaining of the sections was carried out (Karishma and Herber, 2002)*. In addition, double immunofluorescence staining for BrdU and neuronal nuclear protein (NeuN), or BrdU and glial fibrillary acidic protein (GFAP) was performed to identify the survival and phenotypes of proliferated cells (Yagity et al., 2001)*.
The total number of BrdU positive cells throughout the entire hippocampus was estimated by using an unbiased stereological procedure in a blinded fashion. BrdU positive cells in the subgranular zone, granular cell layer, and hilus of the bilateral dentate gyrus were exhaustively counted in one out of six serial coronal sections using a conventional light microscope (PD70) with a 100 x objective. When labelled cells were found in clusters, the focal plane was changed to maximize the ability to distinguish individual cells. The number of BrdU positive cells was calculated by multiplying the total number counted by 6 (because every sixth section was used). Fluorescent-stained preparations underwent essentially the same procedure. Every labeled cell within the outlined dentate gyrus was counted. Data are presented as the percentage of BrdU-NeuN or BrdU-GFAP out of the total number of BrdU positive cells. Each group data contained five mice.

*References
- Karishma, K.K., herbert, J., 2002. Dehydroepiandrosterone (DHEA) stimulates neurogenesis in the hippocampus of the rat, promotes survival of newly formed neurons and prevents corticosterone-induced suppression. Eur. J. Neurosci. 16. 445 - 453.
- Yagita, Y., Kitagawa, K., Ohtsuki, T., Takasawa, K., Miyata, T., Okano, H., Hori, M., Matsumoto, M., 2001. Neurogenesis by progenitor cells in the ischemic adult rat hippocampus. Stroke 32, 1890 - 1896.
Positive control:
One group received scopolamine (3 mg/kg bw) as a positive control for the behavioural studies.
Statistics:
All data were expressed as means ± SEM. Data obtained over training days from the hidden platform trial and repeated acquisition test were analysed by repeated measures ANOVA. Data from probe trial and neurogenesis were analysed by one-way ANOVA followed by a Tukey-Kramer post hoc test. Values of p < 0.05 were considered statistically significant.

Results and discussion

Results of examinations

Clinical signs:
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Clinical biochemistry findings:
not examined
Behaviour (functional findings):
no effects observed
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
no effects observed
Other effects:
not examined
Description (incidence and severity):
Migrated information from 'Further observations for developmental neurotoxicity study'



Details on results (for developmental neurotoxicity):not applicable (migrated information)
Details on results:
NEUROBEHAVIOUR
The results showed significant main effects of group (F(4,45) = 166.144; p < 0.001) and day (F(4,180) = 48.027; p < 0.001) in the place navigation test, but mice exposed to Ag-NPs at doses 10, 25, and 50 mg7kg bw did not show a significant longer escape latency to locate the platform compared to the control mice (p > 0.05). Swimming velocity was also not affected by Ag-NPs throughout the testing. Probe trials revealed no difference in the number of platform crossing and percentage of time spent searching in the target quadrant between control and experimental groups (p > 0.05). Spatial working memory was assessed as the mean performance in the second trial over days. There were significant main effects of group (F(4,45) = 67.534; p < 0.001) and trial (F(4,180) = 78.048; p < 0.001), however, no significant difference between control and Ag-NPs exposed groups were found (p > 0.05). These data suggested that Ag-NPs exposure did not induce dysfunction in spatial or memory performance.

GROSS PATHOLOGY
Silver accumulated in the hippocampus in a dose-dependent manner (F82,12) = 5.848; p < 0.05), indicating that the peritoneally absorbed silver from nanoparticles is able to enter the blood circulation and be distributed to hippocampus.

NEUROPATHOLOGY
To determine whether exposure to Ag-NPs impairs hippocampal progenitor proliferation, the expression level of BrdU positive cells were investigated by immunohistochemical staining 1 day after the last BrdU injection. Results showed that a number of brownly stained BrdU positive cells were evident in the dentate gyrus in all four groups of animals. The number of BrdU positive cells in Ag-NPs treated mice was not altered significantly compared with that in control mice (F(3,16) = 0.593; p > 0.05).
To further assess the effect of Ag-NPs on survival and differentiation of the newly generated cells, the total number of BrdU, BrdU-NeuN, and BrdU-GFAP positive cells was estimated using immunofluorescence staining 28 days after the last adminstration of BrdU. The results revealed that most of the BrdU positive cells had been incorporated into the granule cell layer. No overall difference on survival and differentiation were noted between groups (F(3,16) = 1.902, F(3,16) = 0.326, and F(3,16) = 0.566; p > 0.05), despite variability in the data likely precludes detecting a difference in the number of BrdU-GFAP positive cells. These data indicate that exposure to Ag-NPs did not modify hippocampal neurogenesis.

Effect levels

Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified

Applicant's summary and conclusion

Conclusions:
The authors stated that both reference memory and working memory were not impaired in Ag-NPs exposed groups. In addition, no differences were observed in hippocampal progenitor proliferation, new born cell survival or differentiation. According to the authors, the data reveal that exposure to Ag-NPS dose not affect spatial cognition or hippocampal neurogenesis in mice.