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Genetic toxicity: in vivo

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in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid - liquid: suspension
Details on test material:
AgNPs dispersed in distilled water were purchased from HiQ-Nano (Arnesano, Lecce, Italy) and the characterisation of the dispersion was provided by the manufacturer
Specific details on test material used for the study:
TEM images of AgNPs were recorded by a JEOL Jem 1011 microscope. TEM samples were prepared by dropping a dilute solution of NPs in water on carbon-coated copper grids (Formvar/Carbon 300 Mesh Cu). Dynamic light Scattering (DLS) measurements were performed on a Zerasizer Nano ZS90 (Malvern, USA). The optical absorbance spectra of AgNPs were measured with a Spectrophotometer Nanodrop 2000c at a resolution of 1 nm. Measurement were made at 25°C in aqueous solutions at pH7 and in these conditions the formation of silver ions (Ag+) was minimal (De Matteis et al., 2015)

Test animals

Details on test animals or test system and environmental conditions:
46 adult male and female outbred CD1 mice (31.4+/- 3.4 g male mice and 27.4 +/- 1.7g female mice, 5/6 week old) were purchased from Charles River (Calco, Lecco, Italy). They were housed two/cage, under standard laboratory conditions (22 +/- 0.5°C, 50-60% relative humidity, 12h of dark-light alternation with 12-14 air changes per hours) with water and food (2018 Global Diet purchased from Mucedola, Milan, Italy) available ad libitum. After 1week of acclimatization, mice were divided into 4 treatment groups. (5 mice/sex/group).

Administration / exposure

Route of administration:
oral: gavage
distilled water
Details on exposure:
AgNPs of 20nm of diameter were orally administered for 3 days to male and female adult CD-1 mice
The concentration of supply volume was calculated in order to supply volumes of 2mL/100g of bw.
Mice were checked daily for their health status, individual body weight and food consumption were recorded, dose levels of AgNPs were adjusted according to the weight gain for each animals during the treatment.
Duration of treatment / exposure:
3 consecutive days
Frequency of treatment:
once a day
Post exposure period:
Sacrifice 2 hours after treatment
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 mice/sex/group
Control animals:
yes, concurrent vehicle
Positive control(s):
Group of 3 mice/sex was treated with 100 mg/kg bw/day of methyl methanesulphonate (MMS) as positive control for genotoxicity assays.


Tissues and cell types examined:
Target tissues and analysis:
Blood: biodistribution, genotoxicity, serum biomarkers
Liver: biodistribution (SP-ICP-MS, TEM), genotoxicity, histopathology
Kidney: biodistribution, genotoxicity, histopathology
Duodenum: biodistribution (SP-ICP-MS, TEM), genotoxicity, histopathology
Spleen: biodistribution, genotoxicity, histopathology
Details of tissue and slide preparation:
A portion of liver, spleen, duodenum and one kidney were put in a petri dish, washed with cold mincing buffer (phosphate buffered saline, PBS, containing 25 mM Na2EDTA and 10% Dimethyl Sulfoxide, DMSO) and kept on ice.
Cells of liver and kidney were mechanically isolated (Medimachine, Becton Dickinson).
Duodenum was rinsed with mincing buffer, cut longitudinally and scraped with a spatula.
Spleen was minced in 3mL di RPMI medium 1640 (GIBCO). Cellular suspension (except spleen) was divided in 2 aliquots: one for biodistribution analysis and the second, used for Comet and MN, was flowed through 50 µm filter (Filcons, BD Bioscences) and centrifuged 8 min at 500 round per minutes (rpm) at 4°C; pellets were resuspended in cold PBS. Cells derived from spleen were suspended in 3mL of RPMI 1640, stratified on 2mL di Hystopaque1077 and centrifuged in PBS 1X and centrifuged at 1000 rpm for 10 min at 4°C; cell pellet was counted and divided in two aliquots: 1x10^6 cells were used for Comet assay and remaining cells were cultured in RMPI 1640 plus 15% of in-activated Foetal Calf Serum (Hyclone) for Micronucleus assay. Two or more cultures depending on the number of splenocytes were set up with at least 2x 10^6 cells for each animals.
Evaluation criteria:
Increase DNA damage evaluation in blood, liver, kidney, spleen and duodenum of male and female mice exposued to AgNPs in comparison with control.
For all cells derived from different tissues a total 150 cells from two different slides were scored fro each tissue/animal. For cells derived from spleen: 150 comets were randomly scored using a dedicated image analysus system connected to a fluorescence microscope equipped with appropriate filters and connected to a computer though a charge-coupled device camera.

According to the OECD guideline 489, the parameters considered were: the percentage of total DNA in the comet tail and the hedgehogs or ghosts (%G), identified by visual inspections or when their all intensity value was higher than 80%. Media of medians of each group were considered to analyze DNA damage and error standard of the mean.
Statistical analysis was performed with JMP 10 (SAS Institute, Inc.) Cary, NC, USA). The GraphPad Prism 6.0 software was used to perform all graphics. Non parametric Kruskal-Wallis test was applied to all the continuous variables to test the statistical significances of the differences among groups, followed by the post-hoc Dunn's test comparison, where appropriate. For genotoxicity data, one-way ANOVA was performed followed by the post-hox Dunnett's test comparison among treated groups and controls, where appropriate.
For categorical variables (histological endpoints), data were expressed as proportions of quantal data; pairwaise comparisons of treated groups with control group were analyzed by means of two-tailed Fischer exact test. To identify dose-response trends, the Mantel-Haenszelx2 trend test was used.
Significance level was set at P<0.05 for all the analyses.

Results and discussion

Test results
Key result
no statistically significant increased DNA damage was observed in blood, liver, kidney, spleen and duodenum of male and female mice exposed to AgNPs
no effects
No adverse signs of toxicity or mortality were observed in LD, MD and HD treated mice, Body weight gain, feed consumption, absolute and relative organ weights were unaffected by the treatment both in female and male mice
Vehicle controls validity:
Positive controls validity:
mice treated with MMS the % mean of T.I. and % G. were significantly higher than control

Any other information on results incl. tables

AgNP biodistribution in organs and blood:

The SP-ICP-MS method used to analyze dimensional distributions (nm) and concentration (particle/mL) of AgNPs in organs and blood was firstly validated by the measurements of stability, recovery and repeatability, inorder to guarantee that no aggregation/agglomeration or dissolution of particles was present in the different matrices after TMAH extraction. 

Dose-response trend and comparable curves between sexes were present in AgNP biodistribution in blood, liver, kidney and spleen with higher AgNP concentration in female mice in blood and kidney, and in male mice in liver and spleen. In duodenum, no dose-response trend was present in both sexes.

TEM analysis:

Hepatic and duodenum cells of high dose treated male and female mouse showed no pathological ultrastructural changes in nuclei, cytosol and organelles, and cells from control. tissues were comparable with treated tissues. In liver, AgNPs were located either inside membrane  bound structures or dispersed in the cytosol. In the intestinal cells, AgNPs were located insinde membrane bound structures or dispersed in cytosol along microvilli and the presence was comparable in treated and control mice. Both in liver and in duodenum, no AgNPs were detected in the nucleus. Both in liver and duodenum of female mice, AgNPs appeared to be more abundant and better distributed with well-defined membrane in comparison to the same tissues of male mice.


Liver and kidney biomarkers:

Liver serum biomarkers showed, in female mice, significantly reduced levels of GOT and GPT in low dose and mid-dose level, respectively. No statistical sisgnificance alterations were present in male mice. Kidney serum biomarkers showed, in male mice, significantly increased levels in high dose no effects were recorded in female mice.


Histopathological and histomorphometrical analysis:

No significant differences were present in liver, kidney, duodenum and spleen of AgNPs treated male and female mice compared to control.

Applicant's summary and conclusion

In conclusion, AgNP-20nm (up to 300 mg/kg bw/d) did not induce genotoxic damage (DNA damage) after 3 days of oral acute exposure of adult in male and female mice.
Executive summary:

This comet assay has been conducted according to the OECD 489 guideline and under GLP principle.

After 1week of acclimatization, mice were divided into 4 treatment groups. (5 mice/sex/group). CD1 mice (male and female) have been treated with 3 different doses (50, 150, 300 mg/kg bw/d) of AgNP-20nm during 3 consecutive days. The vehcile control group has been treated with distilled water and the positive control group (3 mice/sex) has been treated with MMS.

2 hours after the last treatment mice were sacrified and target tissues were analysed for DNA damage. General toxicity was assessed by kidney and liver biomarkers (GOT, GPT, CREA).

In conclusion, AgNP-20nm (up to 300 mg/kg bw/d) do not induce genotoxic damage (DNA damage) after 3 days of oral acute exposure of adult in male and female mice.