Silver

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study with acceptable restrictions - colchicine was intraperitoneally injected 16.5 hours after test item exposure and the animals were sacrificed 18 hours after exposure. Therefore, the colchicine exposure was too short (OED 475 foresees 3 -5 hours for mice). - mitotic index was not determined - no individual data was reported

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

In vitro and in vivo genotoxicity of silver nanoparticles

Author:

Ghosh, M. et al.

Year:

2012

Bibliographic source:

Mutation Research 749, 60 - 69

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

please refer to "Rationale for reliability incl. deficiencies" above

GLP compliance:

not specified

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

Silver

EC Number:

231-131-3

EC Name:

Silver

Cas Number:

7440-22-4

Molecular formula:

Ag

IUPAC Name:

Silver

Test material form:

solid: nanoform

Details on test material:

- Name of test material (as cited in study report): silver nanoparticles (from Sigma-Aldrich, St. Louis, MO, USA)
- Particle size: ≤100 nm
- Surface area: 5.0 m^2/g
- Density: 10.49 g/cc
- Purity: 99.5%

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS - Swiss albino mice
- Source: obtained from departmental animal house
- Age at study initiation: 8 - 12 weeks old
- Weight at study initiation: 25 - 30 g
- Housing: housed in polyvarbonate cages, bedded with rice usk
- Diet: standard rodent pellet (M/S Hindustan Lever foods, India)
- Water (ad libitum)
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 22°C
- Humidity: 50 - 60%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: water

Details on exposure:

CHARACTERIZATION OF SILVER NANOPARTICLES
Silver nanoparticle powder was characterized using transmission electron microscopy (Jeol JEM-2100 LaB6, 200 kV), scanning electron microscopy, energy-dispersive X-ray spectroscopy (EDX) (Hitachi S-415A Electron microscope, Tokyo, Japan at 25 kV) and by X-ray diffraction (XRD) analysis. The spectra were recorded in a PW.3040/60 PANanlytical X-ray diffractometer Almelo, Netherlands (Cu kα radiation, λ 1.54443) running at 45 kV and 30 mA. The diffracted intensities were recorded from 2 to 99 2θ angles. Zeta potential of silver nanoparticles in solution was measured by laser diffractometry using a Nano Size Particle Analyzer (Zen 1600 Malvern, USA). Absorption maxima of silver nanoparticles in solution were scanned by UV-Vis spectrophotometer (Beckman Coulter DU 700, California, USA) for agglomeration at the wavelength of 20 - 800 nm.

TEM image of silver nanoparticles revealed the particles to be in the size range of 75 - 130 nm, with an average size of ~125 nm. SEM image of the silver nanoparticles revealed an average size of 120 nm; most of the nanoparticles being in the size range of 90 - 180 nm. The purity of the substance was confirmed by EDX analysis. The EDX of the nanoparticle dispersion confirmed the presence of elemental silver. Absence of major peaks of any other elements confirmed the purity of the sample.
Inspection of the XRD patterns of silver nanoparticles reveals the existence of sharp diffraction lines at low angles (2 - 99°). The silver nanoparticles exhibited peaks of siilver at 2θ = 38°, 44°, 64°, 78° and 81° that can be indexed to the (111), (200), (220), (311) and (222) facets of silver, respectively.
Dynamic light scattering measurements performed on the stock suspension of silver nanoparticles showed a tendency of forming agglomerates. Zeta potential of -4.86 mV revealed the unstable nature of the aqueous suspension.
UV visible spectra of silver nanoparticles revealed of absorption maxima between 420 and 440 nm. With an increase in the silver nanopartilces concentration, the intensity and the maximum wavelength of the peak increased. The large change in the optical spectra implied agglomeration of silver nanoparticles with increase in concentration.

TREATMENT SCHEDULE
Single intraperitoneal injection of silver nanoparticles were given to the mice 18 hours before sacrifice.

Duration of treatment / exposure:

single

Frequency of treatment:

once

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 20, 40 and 80 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5 male mice

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: single intraperitoneal injection 18 hours before sacrifice
- Doses / concentrations: 20 mg/kg bw

Mitomycin C
- Route of administration: single intraperitoneal injection 18 hours before sacrifice
- Doses / concentrations: 2.50 mg/kg bw

Examinations

Tissues and cell types examined:

One hundred well spread metaphase plates were scored from each animal per treatment set, at random. The types of aberrations were scored and recorded strictly in accordance with the method of Tice and Ivett (Tice and Ivett, 1985)*. The metaphase cells were scored at 1000x magnification, with selection being based on uniform staining quality, lack of overlapping chromosomes and chromosome number (40 ± 2 chromosomes). Each chromosome aberration recorded was of following types: G'. G''. as chromatid and isochromatid gaps: B'. B'', as chromatid and chromosome breaks, RR as a chromatid rearrangement. Responses were evaluated as the percentage of aberrant damaged metaphase cell (%DC) and as the number of aberrations per cell /CA/cell). Chromatid and chromosome gaps were recorded but were not included in calculations. For a count of the number of CA/cell, chromatid and chromosome breaks, and chromatid rearrangements (dicentric, ring exchanges) were taken as one, regardless of the number of breakage events involved.

*Reference:
- R.R. Tice, J.L. Ivett. Cytogenetic analysis of bone marrow damage, in: R.D. Irons (Ed.) Toxicology of the Blood and Bone Marrow. Raven Press, New York, 1985, pp. 119 - 140.

Details of tissue and slide preparation:

TREATMENT / SAMPLING TIMES / DETAILS OF SLIDE PREPARATION:
For bone marrow chromosome analysis, 16.5 hours after the administration of the test compounds the animals were injected intraperitoneal with 0.04% of colchicine, on an individual weight basis (1 mL/100 g body weight), and 90 minutes later they were sacrificed. Bone marrow samples were collected 18 hours after exposure to silver nanoparticles, when the cells were in their first metaphase. Bone marrow cells were routinely processed by the standard procedure (Preston et al., 1987; Das et al, 2004)*. The animals were sacrificed by cervical dislocation. Bone marrow cells from the femur bones of the same animal were flushed into a centrifuge tube with phosphate-buffered saline (PBS, pH 7.4). The cells were pelleted by centrifugation, and incubated in 0.075 M KCl for 30 minutes at 37°C. This was followed by fixation in ice-cold 3:1 methanol:glacial acetic acid for 10 minutes. Each bone marrow sample was washed twice in fixative, slides prepared and air dried. The slides were coded and stained in 8% Giemsa in Sorenson's phosphate buffer (pH 6.8).

*References:
- R.J. Preston, B.J. Dean, S. Galloway, H. Holden, A.F. McFee, M. Shelby: Mammalian in vivo cytogenetic assays. Analysis of chromosome aberrations in bone marrow cells, Mutat. res. 189 (1987) 157 - 165.
- A. Das, A. Mukherjee, J. Chakrabarti, Sanguinarine: an evaluation of in vivo cytogenetic activity, Mutat. res. 563 (2004) 81 - 87.

Statistics:

Data are presented as means with standard deviation (mean ± S.D.) and one way analysis of variance (ANOVA) test was done by using Sigma Stats 3 software (SPSS Inc., Chicago, IL, USA). For all statistical analysis the level of significance was established at P ≤ 0.05.

Results and discussion

Test results

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The aberrations scored were mainly found to be of chromatid breaks, while in animals treated with the positive compound (mitomycin C) both chromatid and chromosome type of aberrations were recorded. ANOVA test revealed the frequency of aberrant cells and the number of breaks per cell to be signifcantly higher (P ≤ 0.05) than the control.

Please also refer to the table in the field "Any other information on results incl. tables" below.

Any other information on results incl. tables

Table 1: Chromosomal aberrations of mice bone marrow cells following treatment with different doses of silver nanoparticles.

Test chemical	Dose (mg/kg)	Total chromosome aberrations^a					% aberrant cells^b	Number of aberration^c/cell
		G’	G’’	B’’	B’’	RR
Negative control (distilled water)	-	6	-	5	-	-	2.00 ± 0.81	0.02 ± 0.01
Silver nanoparticles	10	7	1	41	4	-	17.07 ± 4.6*	0.18 ± 0.01
	20	10	3	48	5	-	19.10 ± 1.01*	0.21 ± 0.04
	40	12	1	65	2	-	23.33 ± 4.38*	0.27 ± 0.03
	80	8	3	36	1	-	15.66 ± 6.48*	0.15 ± 0.02
Mitomycin C	2.5	26	12	130	41	2	17.00 ± 1.08*	0.59 ± 0.04

G'. G'': chromatid and isochromatid gaps; B'. B'': chromatid and isochoromosome breaks; RR: chromatid rearrangements.

a = 50 metaphase cells/animal (5 animals/dose).

b = Percentage of cells with damaged metaphase (excluding gaps).

c = Number of chromosome aberration/cell (excluding gaps)

* P ≤ 0.05

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): positive
According to the authors, silver nanoparticles can be classified as clastogen. The aberrations scored were mainly found to be of chromatid breaks.