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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study is considered not reliable due to the following serious reporting and experimental shortcomings: - detailed information on application conditions is not reported - only males used - incomplete necropsy/histopathology - number of animals per group too low, since interim sacrifice was conducted - haematology, clinical chemistry, and ophthalmological examination are missing, which should be conducted according to the OECD guideline 411. - reporting of the results is confusing - histopathological figures do not allow a meaningful interpretation due to poor quality - test items insufficiently characterised, no data on purity or impurities

Data source

Reference
Reference Type:
publication
Title:
Sub-chronic dermal toxicity of silver nanoparticles in guinea pigs: Special emphasis to heart, bone and kidneys toxicities
Author:
Korani, M. et al.
Year:
2013
Bibliographic source:
Iranian Journal of Pharmaceutical Research 12 (3): 511 - 519.

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
only males were used; limited necropsy/histopathology
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): silver nanoparticles (purchased from Quantum sphere company, USA)
Three different aqueous solutions were provided by Dr. K Gilani in the Pharmaceutics Lab., Faculty of Pharmacy, Tehran University of Medical Sciences.
To detect the extent and size of silver nanoparticles, transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used by a standard equipment( Siemens with Cu source, 40 K V and 30 mA ). Sample patterns were determined at 5°- 75° (2θ )(Gupta (2007))*.
Particle size (TEM): < 100 nm
XRD: 38, 44, 64.5 degrees (2θ)

Details on AgNPs were published previously: Korani et al. (2011) Int J Nanomedicine 6, 855-862.

*Reference:
- Amit Gupta Generation of C60 nanoparticle aerosol in high mass. J. Aerosol Sci.( 2007) 38: 592-603.

Test animals

Species:
guinea pig
Strain:
other: Hartley-albino
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Pasteur Institute of Iran
- Age: 5 to 6 weeks
- Weight: 350-450 g
- Housing: three guina pigs were housed in stainless steel cages
- Diet (ad libitum): standard laboratory diet, vitamin C
- Water (ad libitum): tap water
- Acclimation period: 14 days before the experiments

ENVIRONMENTAL CONDITIONS
- Temperature: about 22 ± 3 °C
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:
not specified
Vehicle:
not specified
Details on exposure:
TEST SITE
Hair of animals were shaved in the area where the test item and positive control (silver nitrate) were used.
- % coverage: test item was applied to 10 % of the body surface area of experimental animals.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
no data
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 1000, and 10000 ppm silver nanoparticles
Basis:
nominal per unit area
No. of animals per sex per dose:
12 male guinea pigs
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: after performing the acute test and estimating the necessary doses of subchronic toxicity assessments, the test substance were applied.
No mortality was recorded during dermal application of different concentrations of silver nanoparticles in doses up to 10000 μg/mL in preliminary study. Therefore we considered it as a practically non toxic agent in the acute dermal exposure .
Positive control:
100 µg/mL of silver nitrate was used as positive control. The positive control was applied to 10 % of the body surface area of experimental animals.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: cage side observation, neurological and physical examination

DETAILED CLINICAL OBSERVATIONS: No data
DERMAL IRRITATION: No data

BODY WEIGHT: Yes
- Time schedule for examinations: 2 times/week

FOOD CONSUMPTION:
- Food consumption: Yes

FOOD EFFICIENCY:
- 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

WATER CONSUMPTION: Yes

OPHTHALMOSCOPIC EXAMINATION: No data
HAEMATOLOGY: No data
CLINICAL CHEMISTRY: No data
URINALYSIS: No data
NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes

- Determination of tissue levels of silver nanoparticles:
Tissues (heart, skin, kidney, bone, muscle, liver, and spleen) were digested with nitric acid by using flameless method. Then the tissue concentrations of silver were analysed by using an atomic absorption spectrophotometer equipped with a graphite furnace (Perkin Elmer 5100ZL, Zeeman Furnace Module, USA) based on the Abraham TW et al.method (Wan et al., 1991)*.

- Pathological studies:
Heart ,kidney and bone were the three major organs on which this study focused. These organs were removed from 3 animals/group for histopathological studies at three intervals: baseline, midpoint and endpoint . The tissues were fixed in 10% buffered formalin and dehydrated in graded series of alcohol, cleared in xylene and embedded in paraffin wax. Multiple sections from each block were prepared at 5 μm and stained with haematoxylin and eosin for histopathological studies.

*Reference:
- Wan AT Conyers RA, Coombs CJ and Masterton JP. Determination of silver in blood, urine, and tissues of volunteers and burn patients. Clin Chem. (1991) 37: 1683-7.
Statistics:
no data

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
- all animals survived during this study
- no clinical sign of toxicity was recorded in dose groups and control.

BODY WEIGHT AND WEIGHT GAIN
- no significant weight changes were detected during the study.

FOOD CONSUMPTION
- food consumption was not significantly different among control and treatment groups (p = 0.085) .

WATER CONSUMPTION
- water consumption was not significantly different among control and treatment groups (p = 0.087).

HISTOPATHOLOGY: NON-NEOPLASTIC
- Tissue levels of silver nanoparticles:
A close correlation between dermal exposure and tissue levels of silver nanoparticles was found. Silver content in tissues increased dose-dependently and showed the following ranking: kidney>muscle>bone >skin>liver> heart> spleen.
Please also refer to table 1 in the field "Any other information on results incl. tables" below.

- Toxic responses of bone (bone samples of a total of 27 animals):
Abnormal inflammatory responses were found in all treated groups and osteoclasts were formed in these animals in a dose dependent manner. Separated lines and marrow space narrow were observed in three different dose levels of silver nanoparticles when the alterations were compared with negative group.
Please also refer to table 2 in the field "Any other information on results incl. tables" below.

- Toxic responses of heart (heart samples of a total of 26 animals):
Abnormal changes were detected in dose groups as well as silver nitrate group. However 4 major signs of toxicity (inflammation, presence of clear zone around nucleus, cardiocyte deformities, congestion and hemorrhage) were magnified in the high dose group. Increased dermal dose of silver nanoparticles caused cardiocyte deformity.
Please also refer to table 3 in the field "Any other information on results incl. tables" below.

- Toxic responses of heart (kidney samples of a total of 28 animals):
Six major toxic responses were observed and scoring was performed according to the following classification: Inflammation, gluomeral adhesion to Bowman’s capsule, proximal convoluted tubule degeneration, capsular thickening, membranous thickening and increased mesangial cells. Inflammatory reactions and glomeral adhesion to Bowman's capsule were identified in all dose groups. These reactions were magnified in a dose-dependent manner. Besides these toxic reactions, increased mesangial cells, increased membranous thickening and increased capsular thickening were detected too. The highest levels of degeneration proximal convoluted tubule and distal convoluted tubule were seen in the middle and high-dose groups.
Please also refer to table 4 in the field "Any other information on results incl. tables" below.

Effect levels

Dose descriptor:
LOAEL
Effect level:
100 other: ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Tissue levels of silver nanoparticles in comparison to AgNo3and negative control (ng/g).

Dose Groups

Heart

(n=26)

Skin

(n=22)

Kidney

(n=28)

Bone

(n=27)

Muscle

(n=28)

Liver

(n=27)

Spleen

(n=26)

Control negative

8.46+ 6.85

9.36 ± 2.28

8.78 ±2.64

7.7736+1.96

6.109+1.06

11.5 ± 1.29

5.36+1.37

Control Positive

(AgNO3)

14.34 + 6.85

18.84+ 7.72

16.6 ± 9.92

9.3377+2.75

7.124+1.39

24.06 ± 2.05

7.50+2.46

100 ppm silver nanoparticles

9.09 +6.85

18.84+2.002

19.03 ± 9.92

12.025+1.97

14.316+5.68

24.06 ± 3.80

12.07+2.46

1000 ppm silver nanoparticles

15.65+5.96

29.96+9.69

24.06 ± 15.36

12.557+2.75

14.026+7.84

26.148 ± 7.21

11.88+3.23

10000 ppm silver nanoparticles

22.66+8.73

31.02+14.3

35.95 ± 12.94

32.325+9.1

33.63+3.31

30.324 ± 2.84

20.87+8.85

 

Table 2: Silver nanoparticles induced bone toxicity after dermal application in guinea pig.

Dose Groups

Inflammation

Osteoclasts

Separated Lines

Marrow space narrow

Control negative

-

-

-

-

Control Positive

(AgNO3)

+

+

+

+

100 ppm silver nanoparticles

+

+

+

+

1000 ppm silver nanoparticles

+

++

+

+

10000 ppm silver nanoparticles

+

+++

++

++

Severe (+++), moderate (++), mild (+), none(-)

Table 3: Silver nanoparticles induced heart toxicity after dermal application in guinea pig

Dose Groups

Inflammation

Cardiocyte deformity

Clear zones around nucleus

Congestion & hemorrhage

Control negative

-

-

-

-

Control Positive

(AgNO3)

+

+

-

+

100 ppm silver nanoparticles

+

+

+

+

1000 ppm silver nanoparticles

+

++

+

+

10000 ppm silver nanoparticles

++

++

++

++

 

Table 4: Silver nanoparticles induced nephrotoxicity after dermal application in guinea pig.

Dose Groups

Inflammation

PCT Degeneration

Adhesion of glomerular epithelial cells to BC

Capsular thickening

Membranous

thickening

increased Mesangial cells

Control negative

-

-

-

-

-

-

Control Positive

(AgNO3)

+

++

-

+

-

+

100 ppm silver nanoparticles

+

++

+

+

-

+

1000 ppm silver nanoparticles

+

++

+

+

+

+

10000 ppm silver nanoparticles

+

+++

++

+

++

++

BC: Bowman's capsule, DCT : Distal Convoluted Tubule, PCT: Proximal convoluted tubule, GL: Glomerulus

Applicant's summary and conclusion

Conclusions:
LOAEL (guinea pigs; males): 100 ppm (nominal)
Beside inflammatory reactions kidneys of all silver-treated groups showed as main finding degeneration of the proximal convoluted tubule with increasing severity. Abnormal inflammatory responses in bones were found in all silver-treated groups and formation of osteoclasts occurred in a dose-dependent manner. Heart responses to increasing dermal doses of silver nanoparticles consisted of inflammation, cardiocyte deformity, congestion and haemorrhage.
Taken together, dermal application of very high doses of silver nanoparticles caused histopathological alterations in the kidney, bone and heart of guinea pigs indicating systemic bioavailability of silver. No NOAEL could be identified.