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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Publication of a GLP guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
In vivo genotoxicity of silver nanoparticles after 90-day silver nanoparticle inhalation exposure
Author:
Kim, J.S. et al.
Year:
2011
Bibliographic source:
Saf Health Work 2, 34 - 38

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
within the scope of an OECD 413
Deviations:
yes
Remarks:
no information on a positive control was given in the publication
GLP compliance:
yes
Type of assay:
micronucleus assay

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): silver nanoparticles
- silver nanoparticles were generated by a ceramic heater (see also references Ji, 2007 and Sung, 2008)*

* References:
- Ji JH, Jung JH, Kim SS, Yoon JU, Park JD, Choi BS, Chung YH, Kwon IH, Jeong J, Han BS, Shin JH, Sung JH, Song KS, Yu IJ. (2007). Twenty-eight-day inhalation toxicity study of silver nanoparticles in Sprague-Dawley rats. Inhal Toxicol 19, 857-871.
- Sung JH, Ji JH, Yoon JU, Kim DS, Song MY, Jeong J, Han BS, Han JH, Chung YH, Kim J, Kim TS, Chang HK, Lee EJ, Lee JH, Yu IJ. (2008). Lung function changes in Sprague-Dawley rats after prolonged inhalation exposure to silver nanoparticles. Inhal Toxicol 20, 567-574.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS - specific-pathogen-free (SPF) Sprague-Dawley rats
- Source: SLC (Tokyo. Japan)
- Age at study initiation: eight-week-old
- Weight at study initiation: males: 253 g; females: 162 g
- Housing: rats were housed in polycarbonate cages (5 rats per cage)
- Diet (ad libitum): rodent diet (Harlan Teklab, Plaster International Co., Seoul)
- Water (ad libitum): filtered water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 2°C
- Humidity: 55 ± 7%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Vehicle:
- Vehicle(s)/solvent(s) used: clean (dry and filtered) air
Details on exposure:
Silver nanoparticles were generated as described in previous reports (Ji, 2007 and Sung, 2008)*.

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the rats were exposed to the silver nanoparticles in a whole-body-type exposure chamber (1.3 m^3, Dusturbo, Seoul)

- System of generating particulates/aerosols: the generation consisted of a small ceramic heater connected to an AC power (91.6 V) supply and housed within a quartz tube case (Jung et al., 2006)*. The heater dimensions were 50 × 5 × 1.5 mm^3, and a surface temperature of about 1500°C within a local heating area of 5 × 10 mm^2 could be achieved within about 10 seconds. For long-term testing, the source material (about 160 mg, Daedeok Science, Daejeon) was positioned at the highest temperature point. The quartz case was 70 mm in diameter and 140 mm long (Ji et al., 2007). Clean (dry and filtered) air was used as the carrier gas, and the gas flow was maintained at 30.0 L/min (Re = 572, laminar flow regime) using a mass flow controller (AERA, FC-7810CD-4V, Japan). The system produced different concentrations of nanoparticles (high, middle, and low) in three separate chambers. The nanoparticle generator was operated at 30 L/min and this was mixed with the 200 L/min flow rate of the main flow through the high-concentration chamber. Using the mass flow controller for the first particle sampler, a portion of the high nanoparticle concentration was diverted to the middle-concentration chamber and diluted by the mass flow controller flow rate. In the same way, a portion of the middle nanoparticle concentration was also diverted to the low-concentration chamber and diluted by the mass flow controller flow rate. The flow rates for the high, middle, and low doses were 47.02 ± 0.14 lpm, 6.76 ± 0.16 lpm, and 5.42 ± 0.18 lpm (mean ± S.E.), respectively.

- Method of particle size/particle concentration determination: the nanoparticle distribution with respect to size was measured directly using a differential mobility analyzer (nano-DMA, 4220, HCT Co., Ltd. Korea, range 2.5-150 nm) and ultra-condensation particle counter (UCPC, 4312, HCT Co., Ltd. Korea, 3025, 0-108/cm^3 detection range). Nanoparticles from 1.98 to 64.9 nm were measured using sheath air at 5 L/min and polydispersed aerosol air at 1 L/min, with these values being the operational conditions for nano-DMA and UCPC, respectively. The particle numbers per cm^3 in the fresh-air control chamber were measured using a particle sensor (4103, HCT Co., Ltd. Korea) that consisted of channel 1 (below 300 nm) and channel 2 (over 300 nm).

* References:
- Ji JH, Jung JH, Kim SS, Yoon JU, Park JD, Choi BS, Chung YH, Kwon IH, Jeong J, Han BS, Shin JH, Sung JH, Song KS, Yu IJ. (2007). Twenty-eight-day inhalation toxicity study of silver nanoparticles in Sprague-Dawley rats. Inhal Toxicol 19, 857-871.
- Sung JH, Ji JH, Yoon JU, Kim DS, Song MY, Jeong J, Han BS, Han JH, Chung YH, Kim J, Kim TS, Chang HK, Lee EJ, Lee JH, Yu IJ. (2008). Lung function changes in Sprague-Dawley rats after prolonged inhalation exposure to silver nanoparticles. Inhal Toxicol 20, 567-574.
- Jung JH, Oh HC, Noh HS, Ji JH, Kim SS. (2006) Metal nanoparticle generation using a small ceramic heater with a local heating area. J Aerosol Sci 37, 1662- 170.
- Ji JH, Jung JH, Yu IJ, Kim SS. (2007). Long-term stability characteristics of metal nanoparticle generator using small ceramic heater for inhalation toxicity studies. Inhal Toxicol 19, 745-751.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
49, 133 and 515 µg/m^3
Basis:
analytical conc.
No. of animals per sex per dose:
10 male rats/ 10 female rats
Control animals:
yes, sham-exposed
Positive control(s):
no data

Examinations

Tissues and cell types examined:
The number of micronucleated polychromatic erythrocytes (MNPCEs) among every 2000 polychromatic erythrocytes (PCEs) per animal was examined within a day using a fluorescent microscope (Leica, Germany). Since normochromatic erythrocytes (NCE) become opaque when using a fluorescence stain, one additional slide per animal was stained with May-Grünwald and Giemsa solutions. To evaluate the bone marrow toxicity, the ratio PCE / PCE + NCE was calculated based on a total of 200 erythrocytes using these slides (Schmid, 1976)*.

*Reference
- Schmid W. The micronucleus test for cytogenetic analysis. In: Hollaender A, editor. Chemical mutagens: principles and methods for their detection. Vol 4. New York: Plenum Press; 1976. p. 31-53.
Details of tissue and slide preparation:
TREATMENT / SAMPLING TIMES / DETAILS OF SLIDE PREPARATION:
The rats were killed 24 hours after the last administration, then the femurs were removed and the bone marrow collected in 1.5 mL tubes containing 1 mL of foetal bovine serum, and these tubes were then centrifuged for 5 minutes at 1,000 rpm. Two smears were prepared, which were allowed to dry in air prior to fixation with methanol and staining with an acridine orange solution. One drop of a 0.04 mM acridine orange solution in a phosphate buffer was placed on the fixed cells and covered with a coverslip. Since normochromatic erythrocytes (NCE) become opaque when using a fluorescence stain, one additional slide per animal was stained with May-Grünwald and Giemsa solutions.

METHOD OF ANALYSIS:
A fluorescent microscope (Leica, Germany) was used.
Statistics:
The statistical analyses were performed using SPSS 12.1, and the data was expressed as the mean ± S.D. An X^2 test and a one-way analysis of variance (ANOVA) were applied to test all the data. A value of p < 0.05 indicated statistical significance.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- the frequency of micronucleated polychromatic erythrocytes (MN PCEs) in every 2000 PCEs for the male rats was 0.13, 0.21, and 0.18 percent for the groups exposed to low, middle, and high concentrations of silver nanoparticles, respectively, while that for the control was 0.14 percent.
- the frequency of MN PCEs in every 2000 PCEs for the female rats was 0.09, 0.08, and 0.13 for the groups exposed to low, middle, and high concentrations of silver nanoparticles, respectively, while that for the control was 0.14 percent.
- a dose-related increase was found in the number of MN PCEs in the male rats
- no significant treatment-related increase of MN PCEs was detected in the male and female rats when compared to the corresponding negative controls
- no statistically significant difference in the PCE / (PCE + NCE) ratio, representing the absence of bone marrow cytotoxicity, was observed in the male and female rats after silver nanoparticle exposure when compared with the control.

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

Any other information on results incl. tables

OBSERVATIONS

- all the animals appeared normal and remained healthy until the bone marrow was harvested. There were no significant changes in the body weights of the male rats.

- a dose-dependent deposition of silver nanoparticles was found in the blood, stomach, brain, liver.kidneys, lungs, and testes, indicating that the silver nanoparticles were systemically distirbuted in the mammalian tissues.

Table 1: Frequency of MN PCEs and PCE / (PCE + NCE) ratio in bone marrow of male rats

Dose

No. of rats

(Male)

Frequency of MN PCEs in every 2000 PCEs (Mean ± SE, %)

PCE / (PCE + NCE) (Mean ± SE, %)

0

10

0.14 ± 0.10

0.36 ± 0.10

30

10

0.13 ± 0.09

0.39 ± 0.07

300

10

0.21 ± 0.09

0.31 ± 0.05

1000

10

0.18 ± 0.13

0.30 ± 0.08

MN PCE: micronucleated polychromatic erythrocytes

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

Table 2: Frequency of MN PCEs and PCE / (PCE + NCE) ratio in bone marrow of female rats

Dose

No. of rats

(Male)

Frequency of MN PCEs in every 2000 PCEs (Mean ± SE, %)

PCE / (PCE + NCE) (Mean ± SE, %)

0

10

0.14 ± 0.08

0.29 ± 0.08

30

10

0.09 ± 0.06

0.30 ± 0.09

300

10

0.08 ± 0.06

0.35 ± 0.08

1000

10

0.13 ± 0.10

0.31 ± 0.08

MN PCE: micronucleated polychromatic erythrocytes

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
According to the authors, there were no statistically significant differences in the micronucleated polychromatic erythrocytes or in the ratio of polychromatic erythrocytes among the total erythrocytes after silver nanoparticle exposure when compared with the control. They also stated that the present results suggest that exposure to silver nanoparticles by inhalation for 90 days does not induce genetic toxicity in male and female rat bone marrow in vivo.