Registration Dossier

Administrative data

Description of key information

Insoluble/poorly soluble silver substances (e.g.  silver metal, incl. nano-forms, disilver oxide, silver halides):
Oral: NOAEL of 30 mg Ag/kgbw/day, as established in an oral 90-day study by Kim et al. (2010).
Inhalation: reference is made to the SCOEL (1993) document on OELs for exposure to metallic silver, in which an OEL of 0.1 mg Ag/m3 (inhalable fraction) is established.
Dermal: route considered to be less relevant
Soluble silver substances (e.g. silver nitrate):
Oral: tentative NOAEL of 95 mg Ag/kg bw/day, based on Tamimi et a. (1998). To be replaced by on-going US NTP 90-day oral toxicity study with silver acetate.
Inhalation: Reference is made to the indicative occupational exposure limit value (IOELV) for soluble silver compounds (0.01 mg Ag/m³), as set by EC Directive 2006/15/EC.
Dermal: route considered to be less relevant
For further details see also separate document “Derivation of DNELs for silver substances”, provided as Annex 2 to the CSR.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented publication.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: specific-pathogen free (SPF) Fisher 344 rats were purchased from Japan SLC Inc. (Japan)
- Age at study initiation: 4 weeks
- Housing: during the acclimation and experimental periods, rats were housed in polycarbonate cages (maximum of 3 rats per cage).
- Diet: ad libitum; rodent diet (Harlan Teklad, USA)
- Water: ad libitum; filtered water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 +/- 1.7
- Humidity (%): 48.4 +/- 6.0
- Photoperiod: 12 hours dark/light cycle
No further details are given.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
aqueous, 0.5 % solution (from Sigma, USA)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: no details

VEHICLE
- Justification for use and choice of vehicle (if other than water): CMC is a standard vehicle in toxicity studies
- Amount of vehicle (if gavage): dosing volumes were 10 mL/kg
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:
daily
Remarks:
Doses / Concentrations:
30 mg/kg/day (low dose)
Basis:
other: actual administered
Remarks:
Doses / Concentrations:
125 mg/kg/day (middle dose)
Basis:
other: actual administered
Remarks:
Doses / Concentrations:
500 mg/kg/day (high dose)
Basis:
other: actual administered
No. of animals per sex per dose:
The rats were divided into 4 groups of 10 rats:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected based on previous observations in a 28-day oral toxicity study by Kim et al. (2008).
No further details are given.
Positive control:
No positive control substance was tested.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: no data

FOOD CONSUMPTION: Yes

FOOD EFFICIENCY: No data

WATER CONSUMPTION: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before necropsy after 13 weeks of treatment
- Anaesthetic used for blood collection: CO2
- Animals fasted: for 24 hours
- How many animals: all animals
- Parameters checked: WBC (white blood cell count), RBC (red blood cell count), Hb (haemoglobin concentration), HTC (hematocrits), MCV (mean corpuscular volume), MCH (mean corpuscular haemoglobin), MCHC (mean corpuscular haemoglobin concentration), RDW (red cell distribution width), PLT (platelet count), MPV (mean platelet volume), NE# (number of neutrophils), NE% (percent of neutrophils), LY# (number of lymphocytes), LY% percent of lymphocytes), MO# (number of monocytes), MO% (percent of monocytes), EO# (number of eosinophils), EO% (percent of eosinophils), BA# (number of basophils), and BA% (percent of basophils) using a blood cell counter.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before necropsy after 13 weeks of treatment
- Animals fasted: for 24 hours
- How many animals: all animals
- Parameters checked: ALB (albumin), ALP (alkaline phosphatase), Ca (calcium), CHO (cholesterol), CRE (creatinine), gamma-GT (gamma-glutamyl transpeptidase), GLU (glucose), GOT (glutamic oxaloacetic transaminase), GPT (glutamic pyruvic transminase), IP (inorganic phosphorus), LDH (lactate dehydrogenase), MG (magnesium), TP (total protein), UA (uric acid), BUN (blood urea nitrogen), TBIL (total bilirubin), CK (creatine phosphokinase), Na (sodium), K (potassium), Cl (chloride), TG (triglyceride), and A/G (ratio of albumin to globulin) using a biochemical blood analyser.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Before necropsy, food was withheld for 24 h and the rats were anesthetised with CO2 gas. The rats were killed by cervical dislocation.

GROSS PATHOLOGY: Yes
- Adrenal glands, bladder, testes, ovaries, uterus, epididymis, seminal vesicle, heart, thymus, thyroid gland, trachea, esophagus, tongue, prostate, lungs, nasal cavity, kidneys, spleen, liver, pancreas, and brain were removed carefully and weighed.

HISTOPATHOLOGY: Yes:
- The organs were fixed in a 10% formalin solution containing neutral phosphate-buffered saline.
- Thereafter, the organs were embedded in paraffin, stained with hematoxylin and eosin, and examined under light microscopy.
Other examinations:
Silver distribution study:
- Tissues were digested with conc. nitric acid by using a microwave digestion system.
- The concentration of silver was analysed with a flameless method using an atomic absorption spectrophotometer equipped with a Zeeman graphite furnace.
- The concentration of silver in tissues was expressed as μg/g wet weight.
Statistics:
Statistical analysis was performed with SPSS (Version 12). Statistical evaluation was performed by analysis of two-tailed Student’s t-test or analysis of variance (ANOVA) following multiple comparison tests with Duncan’s method. The level of statistical significance was set at p < 0.05.
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
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 examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
BODY WEIGHT AND WEIGHT GAIN
- No significant dose-related changes were found in females.
- Body weight of high dose males (500 mg/kg bw/d) was significantly (p<0.05) decreased at weeks 4, 5 and 7 and at study termination.
- Body weights of mid dose males (125 mg/kg bw/d) was significantly (p<0.05) lower at week 10.

FOOD CONSUMPTION
- No significant differences were seen between control and treated groups (data not presented).

WATER CONSUMPTION
- No significant differences were seen between control and treated groups (data not presented).

HAEMATOLOGY
- No treatment-related changes were observed, except a significant increase in monocytes (p<0.05) in high dose females (500 mg/kg bw/d).

CLINICAL CHEMISTRY
- ALP appeared to be incresaed (not significant) in mid and high dose males (125 and 500 mg/kg bw/d).
- ALP was significantly (p<0.01) increased in high dose females (500 mg/kg bw/d).
- Cholesterol was significantly (p<0.01) increased in mid and high dose males (125 and 500 mg/kg bw/d) and high dose females.
- Total bilirubin was significantly (p<0.05) increased in mid dose males (125 mg/kg bw/d), without any dose-related trend.
- In high dose females (500 mg/kg bw/d), magnesium (p<0.01), total protein and inorganic phosporus (p<0.05) were significantly decreased.

ORGAN WEIGHTS
- Left testes weights were significantly (p<0.05) increased in high dose males (500 mg/kg bw/d), possibly reflecting lower terminal body weights.
- Right kidney weights were significantly (p<0.05) decreased in low and mid dose females (125 and 500 mg/kg bw/d), without dose-relation.

HISTOPATHOLOGY: NON-NEOPLASTIC (Incidence in the control, 30, 125, 500 mg/kg bw/d groups)
- Bile duct hyperplasia was observed in 4/10, 7/10, 8/10 and 6/10 males and 3/10, 7/10, 8/10 and 7/10 females, respectively.
- Focal, multifocal or lobular necrosis was noted in 0/10, 4/10, 5/10 and 4/10 males and 0/10, 2/10, 2/10 and 2/19 females, respectively.
- Bile duct hyperplasia with or without necrosis/fibrosis was regarded as minimal and due to silver nanoparticles.
- Minimal or mild renal unilateral or bilateral mineralisation was observed in 5/10, 8/10, 7/10 and 9/10 females, respectively.
- In the intestines, pigmentation of villi was observed in 0/10, 0/10, 8/10 and 8/10 males, respectively.
- Only slight treatment-related pigmentation was seen in high dose females (5/10).

OTHER FINDINGS: SILVER DISTRIBUTION
- There was a statitically significant (p<0.01) dose-related increase of silver deposition in testes, liver, kidneys, brain, lungs and blood of treated rats.
- A two-fold higher accumulation of silver was seen in kidneys of females compared to males.
Dose descriptor:
other:
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: incidence of liver damage by increased levels of alkaline phosphatase and cholesterol
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: incidence of liver damage by increased levels of alkaline phosphatase and cholesterol
Critical effects observed:
not specified

Table: Silver concentration in tissue and blood (μg/g wet weight)

Dose/Sex

Testes

Liver

Kidneys

Brain

Lungs

Blood

0 mg/Kg

 

 

Male

0.04 ± 0.02

0.02 ± 0.01

0.04 ± 0.02

0.02 ± 0.01 a

0.10 ± 0.08

0.001 ± 0.000

Female

 

0.01 ± 0.01

0.03 ± 0.01

0.01 ± 0.01 a

0.05 ± 0.02

0.002 ± 0.002

30 mg/Kg

 

 

Male 

6.56 ± 0.33**

4.20 ± 1.57** a

1.49 ± 0.37** b

0.47 ± 0.18**

1.94 ± 0.64** b

0.111 ± 0.016**

Female 

 

8.56 ± 3.22** a

7.98 ± 0.91** b

0.38 ± 0.05**

4.97 ± 0.90 b

0.087 ± 0.017**

125 mg/Kg

 

 

Male 

11.84 ± 1.62**

10.19 ± 2.09** b

8.82 ± 2.13** b

0.69 ± 0.06**

10.97 ± 3.81**

0.191 ± 0.037** b

Female

 

29.13 ± 9.74** b

37.09 ± 17.44** b

0.77 ± 0.11**

17.64 ± 9.06**

0.122 ± 0.010** b

500 mg/Kg

 

 

Male

23.75 ± 9.13**

68.65 ± 33.59**

99.19 ± 32.82** b

3.54 ± 1.44**

56.04 ± 51.14

0.419 ± 0.083**

Female

 

98.75 ± 31.65**

226.88 ± 55.64** b

3.70 ± 1.17**

45.83 ± 11.43**

0.303 ± 0.099**

 

**Significant difference vs. control, p < 0.01. (ANOVA)

a Significant difference vs. distinction of sex, p < 0.05. (T-test)

b Significant difference vs. distinction of sex, p < 0.01. (T-test)

Conclusions:
The target organ for the silver nanoparticles was found to be the liver in both the male and female rats after 90-day of exposure to silver nanoparticles. Significant dose-related changes were found in alkaline phosphatase and cholesterol levels of male and female rats at and above 125 mg/kg bw/d, indicating slight liver damage. Histopathology revealed slightly higher incidences of bile-duct hyperplasia with or without necrosis, fibrosis and/or pigmentation in treated animals together with a dose-dependent accumulation of silver in all tissues examined.
A NOAEL of 30 mg/kg and LOAEL of 125 mg/kg was established in the present study.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Publication of a GLP guideline study reliable with restrictions Deviations from the guideline OECD 413 (1981): - no explicit statement is made in the publication if the actual concentration of the test substance was held as constant as practicable. No raw data of the actual concentration was presented, no indication was made if exposure started after a test atmosphere equilibration period. However, supporting publications by Jung, 2006 and Ji, 2007 specifically address and confirm the generation of a stable test atmosphere for this study in the same laboratory. - the temperature and humidity values are reported for the acclimatisation period only, but not explicitly for the test period - there was no indication that the animals were observed during the 6-hour exposure period. - animals were not observed every day, but on weekdays only - apparently, the animals were ophthalmologically examined, but it was not described when the examination took place and if all animals were examined. It seemed that at least the high dose group was examined, since it was stated that one animal from the high dose died during this examination, but no further information could be found regarding this examination. - it was not explicitly mentioned, if a full gross necropsy was performed. - baseline data was not given for haematological and biochemistry tests
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
, adopted 1981-05-12
Deviations:
yes
Remarks:
, please refer to "Rationale for reliability incl. deficiencies" above
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS - specific-pathogen-free
- Source: SLC (Tokyo, Japan)(originally derived from the Charles River SD in 1968)
- Age at study initiation: 8 week-old rats
- Weight at study initiation: males: approximately 253 g; females: approximately 162 g
- Fasting period before study: not stated
- Animals were not provided with food during the 6-hour exposure period.
- Housing: during the acclimation and experimental periods, the rats were housed in polycarbonate cages (five rats per cage).
- The animals were housed in individual wire cages during the exposure period.
- Diet (ad libitum, except during exposure): a rodent diet (Harlan Teklab, Plaster International Co., Seoul, South Korea)
- 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
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: no data, please refer to "Any other information on materials and methods incl. tables" below.
Details on inhalation exposure:
Silver nanoparticles were generated as described in previous reports (Ji et al., 2007a,b; Jung et al., 2006)*.

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, South Korea).

- System of generating particulates/aerosols: the generation consisted of a small ceramic heater connected to an alternating current power supply and housed within a quartz tube case. The heater dimensions were 50 X 5 X 1.5 mm^3, and a surface temperature of about 1500°C within local heating area of 5 X 10 mm^2 could be achieved within about 10 s. For long-term testing, the source material (about 160 mg) was positioned at the highest temperature point. The quartz tube case was 70 mm in diameter and 140 mm long. Clean (dry and filtered) air was used as the carrier gas, and the gas flow maintained at 30.0 L/min (Re = 572, laminar flow regime) using a mass flow controller (MFC, AERA, FC-7810CD-4V, Japan). This generator has been shown to generate nanoparticles from 2 to 65 nm in diameter which do not agglomerate in air. X-ray diffraction analysis using an X-ray diffractometer utilizing CuK2 radiation showed that particles generated are metallic silver, not silver oxide.
The system produced different concentrations of nanoparticles (high, middle, and low) in three separate chambers. For the high-concentration chamber, the nanoparticle generator was operated at 47 L/min and mixed with 200 L/min of clean ambient air. A portion of the high nanoparticle concentration was then diverted to the middle-concentration chamber using the MFC for the first dilution (6.76 ± 0.16 liter per minute (Lpm), mean ± SE), and a portion of the middle nanoparticle concentration then diverted to the low-concentration chamber using the second MFC (5.42 ± 0.18 Lpm).

- Method of particle size/particle concentration determination: the nanoparticle distribution with respect to size was measured directly in real-time using a differential mobility analysing system (DMAS); combining a differential mobility analyser (Short type-DMA, 4220, HCT Co., Ltd, Korea; range 5 - 150 nm) and condensation particle counter (CPC, 4312, HCT Co., Ltd, 0 - 10^8/cm^3 detection range). Nanoparticles were measured using sheath air at 5 L/min and polydispersed aerosol air at 1 L/min for DMA and CPC, respectively. The particle concentration in the fresh-air control chamber was measured using a particle sensor (4123, HCT Co., Ltd) that consisted of two channel; 300 - 1000 nm and over 1000 nm.
The filters used to sample the fume particles were coated with carbon, mounted on an electron microscope grid (200 mesh, Veco, Eerbeek, the Netherlands), and visualized under a transmission electron microscope (TEM, Hitachi 7100, Japan). The diameters of 800 randomly selected particles were measured at a magnification of 100,000, and the silver particles analysed using an energy-dispersive x-ray analyzer (EDX-200, Horiba, Japan) at an accelerating voltage of 75 kV.

* References:
Ji, J.H., Jung, J. H., Yu, I. J., and Kim, S.S. (2007a). Long-term stability characteristics of metal nanoparticle generator using small ceramic heater for inhalation toxicity studies. Inhal. Toxicol. 19, 745 - 51.
Ji, J. H., Jung, J. H., Kim, S. S., Yoon, J. U., Park, J. D., Choi, B. S., Chung, Y. H., Kwon, I. H., Jeong, J., Han, B.S., et al. (2007b). Twenty-eight days inhalation toxicity study of silver nanoparticles in Sprague-Dawley Rats. Inhal. Toxicol. 19, 857 - 871.
Jung, H. H., Oh, H. C., Noh, H.S., Ji, J.H., and Kim, S.S. (2006). Metal nanoparticle generation using a small-sized ceramic heater with a local heating area. J. Aerosol Sci. 37, 1662 - 1670.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Please refer to "Details on inhalation exposure" above.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
0 µg/m^3
Basis:
other: control
Remarks:
Doses / Concentrations:
49 µg/m^3
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
133 µg/m^3
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
515 µg/m^3
Basis:
analytical conc.
No. of animals per sex per dose:
10 males/10 females
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: as there is no current data on workplace air concentrations of silver nanoparticles or silver nanoparticle concentrations released from consumer products, the concentrations used in this study are difficult to relate human exposures. Concentrations used in this study were based on the current ACGIH silver dust threshold limit value (TLV) of 0.1 mg/m3 (ACGIH, 2001)*. As such, the low, middle, and high doses were 1/2, 1, and 5 times the ACGIH silver dust TLV, respectively, in terms of mass dose. In addition, the high dose used in this study is nearly 500-fold higher than the ACGIH silver dust TLV in terms of surface area.

Reference:
American Conference of Governmental Industrial Hygienists (ACGIH). (2001). Documentation of the TLVs and BEIs. ACGIH, Cincinnati, OH.
Positive control:
not used
Observations and examinations performed and frequency:
ANIMAL OBSERVATIONS: Yes
- Time schedule: daily on weekdays
- Observations checked: any evidence of exposure-related effects, including respiratory, dermal, behavioural, nasal, or genitourinary changes suggestive of irritation.

BODY WEIGHT: Yes
- Time schedule for examinations: at purchase, at the time of grouping, once a week during the inhalation exposure and before necropsy.

FOOD CONSUMPTION: Yes
- Time schedule: weekly

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: no data
- Dose groups that were examined: not explicitly stated, but it is assumed that all groups were checked

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 13-week experiment (before necropsy); blood was drawn from the abdominal aorta and collected in heparinized vacutainers.
- Anaesthetic used for blood collection: Yes; anesthetized with pentobarbital
- Animals fasted: Yes, food was withheld for 24 hours
- How many animals: all
- Parameters checked: white blood cell count, red blood cell count, haemoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red-cell distribution width, platelet count, mean platelet volume, number of neutrophils, percent of neutrophils, number of lymphocytes, percent of lymphocytes, number of monocytes, percent of monocytes, number of eosinophils, percent of eosinophils, number of basophils, and percent of basophils

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the 13-week experiment (before necropsy); blood was drawn from the abdominal aorta and collected in heparinized vacutainers.
- Anaesthetic used for blood collection: Yes; anesthetized with pentobarbital
- Animals fasted: Yes, food was withheld for 24 hours
- How many animals: all
- Parameters checked: albumin, alkaline phosphatase, calcium, cholesterol, creatinine, gamma-glutamyl transpeptidase, glucose, glutamic oxalacetic transaminase, glutamic pyruvic transaminase, inorganic phosphorus, lactate dehydrogenase, magnesium, total protein, uric acid, blood urea nitrogen, total bilirubin, creatine phosphokinase, sodium, potassium, chloride, triglyceride, and ratio albumin to globulin

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
ORGAN WEIGHTS AND HISTOPATHOLOGY:
After collecting the blood, the rats were sacrificed by cervical dislocation, and the adrenal glands, bladder, testes, ovaries, uterus, epididymis, seminal vesicle, heart, thymus, thyroid gland, trachea, oesophagus, tongue, prostate, lungs, nasal cavity, kidneys, spleen, liver, pancreas, and brain all removed carefully. Organs were weighed and fixed in a 10% formalin solution containing neutral phosphate-buffered saline. The organs were embedded in paraffin, stained with hematoxylin and eosin. All organs from all animals were examined under light microscopy.

Other examinations:
DETERMINATION OF TISSUE SILVER:
After wet digestion using a flameless method, the tissue concentrations of silver were analysed using an atomic absorption spectrophotometer equipped with a Zeeman graphite furnace (Perkin Elmer 5100ZL, Zeeman Furnace Module, Waltham, MA) based on the NIOSH 7300 method (NIOSH (1999), NIOSH Manual of Analytical Methods. Method No. 7300, 7604. National Institute for Occupational Safety and Health, Cincinnati, OH).
Statistics:
All the results were expressed as the means ± SE. An ANOVA test and Duncan's multiple range tests were used to compare the body weights, bronchoalveolar lavage cell distributions, and parameters from the lung function test obtained for the three dose groups with those obtained for the control rats. Histopathological results were analysed by Chi-squared analysis. Level of significance was set at p < 0.05 and p < 0.01.
Clinical signs:
no effects observed
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 examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
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
No gross effects were observed during the 90-day exposure period. One animal from the high-dose group died during the opthalmological examination.

BODY WEIGHT AND WEIGHT GAIN
There were no significant changes in body weights of male rats. Although female rats showed a significant body weight difference between high and middle dose groups, there were no significant dose-related changes.

FOOD CONSUMPTION
No significant differences were observed in food consumption between the exposed rats and the control groups.

HAEMATOLOGY
There were no significant dose-related differences in the haematology values among groups.
To evaluate aggregation of red blood cells or blood coagulation attributable to silver nanoparticles, erythrocyte aggregation, activated partial thromboplastin time, and prothrombin time were tested. Only the percent of aggregation in the high-dose females showed a statistically significant difference compared with the controls.

CLINICAL CHEMISTRY
There were no significant dose-related differences in the blood biochemical parameters.

ORGAN WEIGHTS
No significant organ weight changes were observed in either the male or female rats after the 90 days of silver nanoparticle exposure.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver:
- minimal bile-duct hyperplasia was identified in 0/10, 0/10, 1/10, and 4/9 of the control, low, middle, and high dose males, respectively.
- one high-dose male had minimal bile-duct hyperplasia with minimal portal mineralization.
- the higher incidence of bile-duct hyperplasia in the high dose males, with or without mineralization suggested a minimal test article-related effect.
- minimal bile-duct hyperplasia was present in 3/10, 2/10, 4/10, and 8/10 of the control, low, middle, and high dose females, respectively.
- single-cell hepatocellular necrosis, characterised by increased cellular eosinophilia and shrunken condensed nucleo, was noted in 3/10 high dose females.
- one high dose female exhibited moderate bile-duct hyperplasia with concurrent moderate centrilobular fibrosis, minimal single-cell hepatocyte necrosis, mild pigment accumulation, and moderate multifocal necrosis.
- the higher incidence of bile-duct hyperplasia, with or without necrosis, fibrosis, and/or pigmentation, in high dose females suggested a test-article-related effecte, which was slightly mor obvious than in the males.
Please also refer to "Any other information on results incl. tables" below (Table 1 and 2).

Lung:
- examination revealed a high incidence of minimal alterations, including some chronic alveolar inflammation, a mixed cell perivascular infiltrate, and alveolar macrophage accumulation in high dose male and female animals when compared with the controls.
Please also refer to "Any other information on results incl. tables" below (Table 1 and 2).

Nasal pathways:
- no histopathologic findings in the nasal pathways were considered test article related.

Kidneys:
- incidence of minimal tubular basophilia was noted in all the groups, including the controls, and thus not considered to be test article exposure related.
- tubular basophilia was more prevalent in males compared with the females.
- although tubular dilatation, cast formation, mineralization, and inflammation were noted occasionally in the control and/or treated animals, they were considered to be within the range of expected background spontaneous change.

Heart:
- minimal degeneration/necrosis was observed in all the groups, including the controls, indicating that this alteration was not test article related.
- the change was more obvious in the males.
- the finding is a common spontaneous background change.

Harderian gland/prostate
- inflammation was noted occasionally, which is a common background spontaneous finding and thus not considered to be test article related.

OTHER FINDINGS
Silver distribution in tissue:
- silver concentration in lung tissue from groups exposed were statistically significant (p < 0.01) and increased with dose.
- a clear dose -dependent increase in silver concentration in the blood for both genders.
- dose-dependent increase in the liver silver concentration for both genders.
- silver concentration in the olfactory bulb was higher than in brain, and increased in a dose dependent manner in both the male and female rats (p<0.01).
- silver concentration in the kidneys showed a gender difference with the female kidneys containing two to three times more silver acculmulation than in male kidneys.
- because gender difference in silver accumulation was noted in kidneys, the kidney function was measured based on the N-acetylglutamate and protein in urine. No significant difference was noted among the dose groups and between genders, except for an increase of protein in the urine from the high-dose male rats (high-dose group: 2.57± 0.13 g/g creatinine; control group: 1.89 ± 0.11 g/g creatinine; p < 0.05).
Please also refer to "Any other information on results incl. tables" below (Table 3 and 4).
Dose descriptor:
NOAEC
Effect level:
133 other: µg/m^3
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEC
Effect level:
515 other: µg/m^3
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Histopathology

Table 1: Histopathological findings for male rats

Group

Control

Low

Middle

High

Number of animals

10

10

10

9

 

N

%

N

%

N

%

N

%

Liver

No microscopic findings

 

 

10/10

100

10/10

100

9/10

90

5/9

55.6

 

Abnormality*

 

 

0/10

0

0/10

0

1/10

10

4/9

44.4

 

Necrosis

Multifocal

Minimum

0/10

0

0/10

0

0/10

0

1/9

11.1

 

Hyperplasia*

Bile duct

Minimum

0/10

0

0/10

0

1/10

10

4/9

44.4

 

Vacuolation

Hepatocellular

Minimum

0/10

0

0/10

0

0/10

0

1/9

11.1

 

Mineralization

Portal

Minimum

0/10

0

0/10

0

0/10

0

1/9

11.1

Lungs

No microscopic findings

 

 

5/10

50

3/10

30

3/10

30

0/9

0

 

Abnormality

 

 

5/10

50

7/10

70

7/10

70

9/9

100

 

Accumulation

Macrophage, alveolar

Minimum

3/10

30

5/10

50

5/10

50

8/9

88.9

 

Inflammation**

Chronic, alveolar

Minimum

2/10

20

3/10

30

2/10

20

8/9

88.9

 

Infiltrate

Mixed cell perivascular

Minimum

3/10

30

4/10

40

6/10

60

7/9

77.8

 

Hemorrhage

Alveolar

Minimum

1/10

10

0/10

0

0/10

0

0/9

0

 

Osseous foreign body

 

 

0/10

0

0/10

0

0/10

0

1/9

11.1

 

Hyperplasia

Respiratory epithelium

level I

 

0/10

0

0/10

0

0/10

0

1/9

11.1

* p < 0.05, compared with control, ** p < 0.01, compared with control

Table 2: Histopathological findings for female rats

Group

Control

Low

Middle

High

Number of animals

10

10

10

10

 

N

%

N

%

N

%

N

%

Liver

No microscopic findings

 

 

7/10

70

5/10

50

5/10

50

1/10

10

 

Abnormality*

 

 

3/10

30

5/10

50

5/10

50

9/10

90

 

Necrosis

Multifocal

Minimum

2/10

20

0/10

0

0/10

0

0/10

0

 

 

 

Moderate

0/10

0

0/10

0

0/10

0

1/10

10

 

 

Focal

Minimum

0/10

0

0/10

0

1/10

10

0/10

0

 

 

Single-cell hepatocellular *

Minimum

0/10

0

0/10

0

0/10

0

3/10

30

 

Hyperplasia*

Bile duct

Minimum

3/10

30

2/10

20

4/10

40

8/10

80

 

 

 

Moderate

0/10

0

0/10

0

0/10

0

1/10

10

 

Granuloma

Multifocal

Minimum

0/10

0

2/10

20

0/10

0

0/10

0

 

Vacuolation

Hepatocellular

Minimum

0/10

0

1/10

10

0/10

0

0/10

0

 

Fibrosis

Centrilobular

Mild

0/10

0

0/10

0

0/10

0

1/10

10

 

Pigment

Centrilobular

Mild

0/10

0

0/10

0

0/10

0

1/10

10

Lungs

No microscopic findings

 

 

3/10

30

5/10

50

6/10

60

2/10

20

 

Abnormality

 

 

7/10

70

5/10

50

4/10

40

8/10

80

 

Accumulation

Macrophage, alveolar

Minimum

7/10

70

4/10

40

4/10

40

6/10

60

 

Inflammation**

Chronic, alveolar

Minimum

3/10

30

2/10

20

0/10

0

8/10

80

 

Infiltrate**

Mixed cell perivascular

Minimum

0/10

0

0/10

0

1/10

10

7/10

70

* p < 0.05, compared with control, ** p < 0.01, compared with control

Silver distribution in tissue

Table 3: Tissue content of silver in male rats (mean± SE) (Unit: ng/g tissue wet weight)

 

Control

Low

Middle

High

Liver

0.70 ± 0.20 (3)

3.52 ± 0.98 (5)

13.75 ± 2.28 (5)

132.97 ± 22.87* (4)

Kidneys

0.85 ± 0.20 (5)

1.63 ± 0.33 (5)

3.58 ± 0.41** (5)

9.49 ± 0.86* (4)

Olfactory bulb

0.51 ± 0.38 (3)

6.44 ± 0.77 (5)

17.10 ± 1.61 (5)

30.48 ± 2.15*** (4)

Brain

1.12 ± 0.34 (2)

3.45 ± 0.73 (4)

7.89 ± 0.95** (5)

18.63 ± 1.24* (4)

Lungs

0.77 ± 0.25 (5)

613.57 ± 66.03† (5)

5450.29 ± 904.17** (5)

14645.42 ± 2630.24* (4)

Whole blood

0.09 ± 0.02 (7)

0.68 ± 0.08 (10)

1.82 ± 0.20** (10)

4.31 ± 0.37* (9)

Note. (): number of samples.

*p < 0.01, high-dose versus other groups.

** p < 0.01, middle-dose versus unexposed and low-dose groups.

*** p < 0.01, high-dose versus other groups (dose dependent).

†p < 0.05, male versus female in low-dose group.

Table 4: Tissue content of silver in female rats (mean± SE) (Unit: ng/g tissue wet weight)

 

Control

Low

Middle

High

Liver

0.90±0.31 (5)

4.55±1.40 (5)

12.07±2.50 (5)

71.08±24.50* (5)

Kidneys

0.94±0.18 (4)

2.61±0.57 (5)

11.81±4.27 (4)

37.66±7.04*(5)

Olfactory bulb

2.26±0.74 (4)

7.43±0.75 (5)

13.75±1.32 (5)

32.84±2.74*** (5)

Brain

0.66±0.26 (4)

4.09±0.46 (5)

10.22±1.19** (5)

19.97±2.41* (5)

Lungs

1.01±0.10 (3)

295.92±78.50 (5)

4241.17±641.10** (5)

20585.63±1880.31* (5)

Whole blood

0.05±0.01 (5)

0.85±0.14 (10)

2.10±0.22** (10)

6.86±0.60*,(10)

Note. (): number of samples.

*p < 0.01, high-dose versus other groups.

** p < 0.01, middle-dose versus unexposed and low-dose groups.

*** p < 0.01, high-dose versus other groups (dose dependent).

†p < 0.05, female versus male in low-dose group.

p < 0.01, female versus male in high-dose group.

Conclusions:
The results of this subchronic 90-day silver nanoparticle inhalation toxicity indicated that lungs and liver were the major target tissues for prolonged silver nanoparticle accumulation.
The NOAEC of 133 µg/m^3 is based on test article-related effects seen at higher (515 µg/m^3) exposure concentrations (minimal bile-duct hyperplasia in males and females, chronic alveolar inflammation and macrophage accumulation in the lungs of males and females, and erythrocyte aggregation in females) of nano-silver (test atmosphere characterised by a count median diameter of 18 nm).
This rat NOAEC was corrected to human equivalent concentrations (HEC) for typical, commercial non-nano silver metal powders (D50 in µm range) based on particle size considerations and modelled comparative fractional deposition in the respiratory tract (rats/humans). The resulting HECs under various considerations are all >> 3 mg/m³ (EBRC, 2012). In contrast, the current OEL set by SCOEL based on human data and taken forward as the DNEL is 0.1 mg/m^3. Since this is more than one order of magnitude lower than the HEC based on animal data with nano-silver, the more conservative approach based on human data as derived by SCOEL was adopted as the DNEL.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
133 µg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral exposure:

Studies on soluble silver substances:

 

Reliable data on oral RDT studies with soluble silver substances are currently scarce. One study reported by Tamimi et al. (1998) has severe limitations resulting from the route of administration (oral swabbing), and the corresponding uncertainties concerning the actual dose levels. Further, this study was performed with silver nitrate, which is a very highly soluble substance and also of corrosive nature. Despite these limitations, a tentative NOAEL of 150 mg AgNO3/kg bw/day, corresponding to 95 mg Ag/kg bw/day can be derived from this study.

 

Hadrup et al. (2012) conducted a subacute (28-day) oral toxicity study comparing the effects of silver acetate to those of nanoscale silver (see also below). Silver acetate was tested at only one dose (14 mg/kg bw/d, corresponding to 9 mg silver/kg bw/d) and in female rats only (for data on silver nanomaterials, see below). Oral exposure to silver in ionic form (as acetate) was reported by the authors to be associated with lower body weight gain, an increase in ALP and a decrease in urea concentrations in plasma and lower absolute and relative thymus weights. In lack of other more conclusive data, this study despite its limitations indicates a tentative LOEL for ionic silver of 9 mg/kg bw/d.

 

However, the applicant is aware of an experimentally completed subchronic (90-day) study with silver acetate currently in its reporting phase (Boudreau, 2012), involving dose levels of 100, 200 and 400 mg/kg bw/d. Based on preliminary reporting, the oral administration of silver acetate at doses of 200 mg/kg bw/day (ca. 133 mg Ag/kg bw/day) and higher induced significant decreases in body weight and increased morbidity in rats, with severe gastroenteritis listed as cause of death. It is anticipated that the extensive design of this study will provide a more robust basis for the establishment of an NOAEL/LOAEL than the studies listed above.

 

Studies on metallic silver, incl. nano-forms:

Studies on metallic (micron-sized) silver are not available. Instead, several studies have recently been conducted with silver nanomaterials:

Two studies were conducted by Kim et al (2008 and 2010) using silver nanomaterial as test item; in the absence of other relevant and reliable data on non-nano silver, these studies were used as a worst case, although it must be noted that the silver nanomaterial employed in some of these studies is inadquately characterised (e.g. no information on coating):

In a 28d oral toxicity study (Kim et al., 2008), treatment of male and female rats at doses of 30, 300 and 1,000 mg/kg bw/d revealed effects on red blood cell parameters at and above 300 mg/kg bw/d, as well as increases of alkaline phosphatase, cholesterol and total protein levels at dose levels of 300 and 1000 mg/kg bw/d. Histopathological examinations (no raw data reported, no dose-relation) are said by the authors to indicate bile duct hyperplasia. A tentative NOAEL of 30 mg/kg bw/d may be derived from this study.

In another sub-acute study by Hadrup et al. (2012), rats were dosed orally for 28 days with PVP-stabilised silver nanomaterial at levels of 2.25, 4.5 and 9.0 mg/kg bw/d. Based on the absence of any effects up to the highest dose tested, an NOAEL of 9 mg/kg bw/d can be established.

In a 90d oral study involving doses of 30, 125 and 500 mg/kg bw/d (Kim et al., 2010), significant dose-related changes were found in alkaline phosphatase and cholesterol levels of male and female rats at and above 125 mg/kg bw/d. Histopathology revealed mild bile-duct hyperplasia with or without necrosis, fibrosis and/or pigmentation in treated animals. The authors derived a NOAEL of 30 mg/kg bw/d.

 

In addition, the applicant is aware of an experimentally completed subchronic (90-day) study with three differently sized silver nanomaterials (10, 75 and 110 nm) currently in its reporting phase, involving dose levels of 9, 18 and 36 mg/kg bw/d (Boudreau, 2012). Based on the available very brief preliminary reporting, the oral administration of silver nanomaterials indicates a tentative NOAEL of 36 mg/kg bw/d (highest dose tested).

In a 90d oral study, adult male rats received doses of 50, 100 and 200 mg/kg bw/d as PVP coated AgNP (Lafuente et al 2016). Clear treatment-related effects, achieving statistical significance, were not evident for testis and epididymal organ weights; sperm count; sperm motility; and sperm viability. Sperm morphological anomalies tended to be more frequent in treated groups (some results achieving statistical significance), but a dose-response was absent and background biological variation is expected for this parameter. Testicular and epididymal histopathology was mainly normal with only minimal tubular desquamation evident at the highest dose. The NOAEL is set at 200 mg/kg bw/d.

The NOAEL at 30 mg/kgbw/day reported in the fully published 90-day study by Kim et al (2010) provides the most reliable starting point for the derivation of a DNEL for metallic silver and is taken forward to the quantitative hazard assessment (see separate “DNEL report”, attached as Annex 2 to this CSR).

 

Inhalation Exposure:

Studies on soluble silver substances:

No relevant studies involving repeated inhalation exposure to soluble silver substances are available.

Studies on non-nano metallic silver:

Studies on metallic (micron-sized) silver are not available. For this reason, for purposes of inhalation exposure risk assessment, reference is made to the SCOEL (1993) document on OELs for metallic silver, in which the OELs are derived based on occupational exposure data. Endpoint records for these reports are included in the technical dossier. In brief, the conclusions are derived on the grounds that local argyria may result from dust of metallic silver or aerosols of silver salts, whereas systemic argyria is observed after ingestion of silver compounds only. Local argyrosis, manifesting itself for example as deposition in the conjunctivae of the eyes, seems to be the most sensitive effect (Buckley et al, 1965; Rungby J, 1986; Green and Su, 1987). These effects are irreversible, but they are cosmetic rather than damaging to health. In a study conducted by Wölbling et al. (1988), no cases of argyria were observed in workers exposed to metallic silver (0.003-0.54 mg/m3), but argyrosis was observed in workers exposed to silver compounds in the range of 0.005-0.38 mg/m3(Roseman et al, 1979 and 1987; Pifer et al, 1989). Perrone et al (1977) observed 4 argyria cases of the eye in workers exposed to 0.022 mg/m3(respirable dust) / 0.31 mg/m3(total dust) of metallic silver.

In the absence of other reliable data on exposure to metallic silver, it appears appropriate in the context of REACH to refer to the currently applicable OELs when deriving DNELs for the inhalation route:

For metallic/poorly soluble silver substances: DNEL = OEL = 0.1 mg Ag/m³, as per SCOEL, 1993: The Scientific Committee on Occupational Exposure Limits (SCOEL). Recommendations from Scientific Expert Group on Occupational Exposure Limits for Metallic Silver. Document No. SEG/SUM/25B 1993).

For soluble silver substances: DNEL = OEL = 0.01 mg Ag/m³, as perthe indicative occupational exposure limit value (IOELV) for soluble silver compounds (EC Directive 2006/15/EC).

 

Studies on silver nanomaterials:

In a reliable 28 -day inhalation toxicity study (Ji et al., 2007) with nanosilver (average particle size ca. 12-15 nm) in Sprague-Dawley rats, no significant health effects were observed up to the highest test concentrations of 61 µg/m³.

In a follow-up reliable 13-week inhalation study with silver nano-particles (median diameter ca. 18 nm) rats were exposed for 6 hours per day for 5 days per week for 13 weeks to concentrations of 49, 133 and 515 µg/m³ (Sung et al., 2009). A NOAEC of 133 µg/m³ was established, based on test article-related effects seen at the highest exposure concentrations of 515 µg/m³: minimal bile-duct hyperplasia in males and females, chronic alveolar inflammation, macrophage accumulation in the lungs of males and females, and erythrocyte aggregation in females. A supplementary publication to the same study (Sung et al, 2008), further detailed investigations of lung function and inflammation parameters supports this NOAEC.

Another study by the same group (Song et al. 2013) used basically the same study design albeit with slightly modified exposure concentrations (49, 117 and 381 µg/m³), but including 4- and 12-week recovery periods. The NOAEC of 117 µg/m³ obtained in this study is considered to support the NOAEC of 133 µg/m³ from the study described above.

The above 90d (rat, inhalation) NOAEC of 133 µg/m³ can be recalculated to a human equivalent concentration (HEC) of 190 µg/m³ based on particle size considerations and modelled comparative fractional deposition in the respiratory tract (rats/humans) taking into consideration morphological and physiological differences between rats and humans. By applying an overall assessment factor AF=15, a rounded DNELinhalation-nano= 10 µg/m³ can be obtained (for details, refer to separate DNEL report attached as Annex 2 to this CSR), which is applicable to exposures to silver nanomaterials in the size range of 20 nm. However, when taking the above into account and, based on laboratory simulation test with airborne silver nanomaterial, assuming that the majority of workplace particulates during handling of silver nanomaterials may be expected in the inhalable size range (for details, refer to HEC calculation as reported within Annex 2 to this CSR), then the maintenance of the current DNEL/OEL of 0.1 mg/m3for metallic silver should ensure that exposure to silver metal nanomaterials is covered also, assumed to be protecting against systemic (argyria) and local (lung inflammation potentially caused by very fine aerosols) effects.

 

Dermal Exposure:

No studies involving repeated dermal exposure to soluble or insoluble silver substances are available.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
For purposes of inhalation exposure risk assessment (systemic effects), reference is made to the SCOEL (1993) document on OELs for exposure to metallic silver, in which an OEL of 0.1 mg/m3 is derived based on occupational exposure reports attributable to the inhalable fraction.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Key study

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Waiver

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Waiver

Justification for classification or non-classification

The only relevant effect observed in humans with chronic exposure to silver substances is argyria, which is visibly detectable as a discolouration of skin and eyes, and also with tissue deposition of silver, but which to date is also not known to be associated with any adverse health effects. The available animal studies with soluble silver substances as well as silver nanomaterials likewise do not indicate any specific target organ toxicity. For this reason, silver substances - soluble substances and metal (nano or non-nano forms) - do not meet any of the classification criteria for effects associated with repeated dosing.