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:
supporting study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study reported by Tamimi et al. (1998) has its limitations resulting from the route of administration and the uncertainties about the real dose levels given. Animals were exposed by swabbing of the oral cavity with a mouth wash containing silver nitrate at different concentrations. Dose levels are stated in mg/kg without any further explanation.
Qualifier:
according to
Guideline:
other: standard methods that comply with the guidelines of the OECD as summarised in Mosberg and Hayes (1989): Subchronic toxicity testing. In: Principles and methods of toxicology, 2nd. ed., ed. A.W. Hayes, 221-236
Deviations:
not specified
Principles of method if other than guideline:
Male and female rats were used in the study to investigate the subchronic toxicity of a new antismoking (A.S.) mouthwash (0.5 % silver nitrate as the active ingredient). Three different dose levels were used. Animals were treated by swabbing the oral cavity with the mouthwash daily for 30 consecutive days.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: locally inbred; raised at the Animal House of the Faculty of Medicine, University of Jordan
- Age at study initiation: 10 - 12 month old
- Weight at study initiation: between 200 and 250 g
- Fasting period before study: no, only at the end of the experiment overnight.
- Housing: in plastic cages before exposure; individually in stainless steal cage during exposure
- Diet: Purina rat chow, ad libitum
- Water: tap water, ad libitum
- Acclimation period: rats were observed for 1 week before starting the experiment
Route of administration:
other: swabbing of the oral cavity
Vehicle:
other: placebo solution (not specified)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- the moutwash solutions were suplied as 0.5, 8, or 10 % of silver nitrate dissolved in placebo
- the following dose levels were used: 1.5 mg/kg, 15 mg/kg and 150 mg/kg
- the placebo volume (1.88 mL/kg) was equivalent to that of the high dose
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
30 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
1.5 mg/kg (low dose)
Basis:
other: nominal, no details provided
Remarks:
Doses / Concentrations:
15 mg/kg (intermediate dose)
Basis:
other: nominal, no details provided
Remarks:
Doses / Concentrations:
150 mg/kg (high dose)
Basis:
other: nominal, no details provided
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: the low dose was calculated according to the human proposed daily dose (21 ml of 0.5% solution per day); the intermediate dose is 10-fold and the high dose 100-fold higher than the low dose.
Positive control:
no data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: every other day for the first week of the experiment, and weekly thereafter and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Animals fasted: overnight; animals were anesthetised and blood weree obtained directly from the heart
- How many animals: all
- Parameters checked: not stated in method description, only the following are mentioned in the results: platelet counts, haemoglobin, haematocrit, RBC, WBC

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Animals fasted: overnight; animals were anesthetised and blood weree obtained directly from the heart
- How many animals: all
- Parameters checked: not stated in method description, only the following are mentioned in the results: sodium, potassium, calcium, glucose, creatine, albumine, urea nitrogen, total protein, LDH, AIp, AST, ALT

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
At the end of the experiment all animals were fasted overnight. Next morning, the animals were anesthetised and weighed.

GROSS PATHOLOGY: Yes
- The animals were opened by thoracicoabdominal longitudinal incision and the following organs were obtained: gum, tongue, teeth, esophagus, stomach, intestine, spleen, pancreas, liver, kidneys, trachea, lung, heart, brain, testes, and ovaries.
- Wet weight of all organs was recorded.

HISTOPATHOLOGY: Yes
- Tissues were fixed in 10 % formalin for histopathological examination.
Other examinations:
no data
Statistics:
Student's t-test, analysis of variance (ANOVA) and Dunnett's test were used to assess differences between treated and control (placebo) animals. A p-value of < 0.05 was considered statistically relevant.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
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:
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:
CLINICAL SIGNS AND MORTALITY
- Mild diarrhea and teeth staining was observed in treated rats.
- No mortality occured.

BODY WEIGHT AND WEIGHT GAIN
- Body weights at study termination were slightly lower (not significant) in mid and high dose groups (15 and 150 mg/kg bw/d).

HAEMATOLOGY
- Platelet counts showed a statistically significant increase (p<0.05) at 1.5, 15, 150 mg/kg bw/d and was most pronounced in high dose animals.
- No other effects were observed.

CLINICAL CHEMISTRY
- No significant differences were observed.

ORGAN WEIGHTS
- Kidney weights were significantly reduced (p<0.05) in high dose females (150 mg/kg bw/d) compared to controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
- High dose animals (150 mg/kg bw/d) showed mild inflammation in the gum, tongue and esophagus.
- Teeth and tongue showed a dark staining in high dose animals.
- Similar observations were founfd to a lesser extent in intermediate and low dose group animals (15 and 1.5 mg/kg bw/d).
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Beside an effect on platelet counts, which can be regarded as of questionable toxicological relevance, no signs of systemic effects were observed at the high dose level of 150 mg/kg.
Critical effects observed:
not specified
Conclusions:
The main findings observed in the study were local effects (staining of teeth and tongue and mild inflammation of the gum, tongue and oesophagus) observed dose dependently in all dose groups. The pigmentation was most likely due to silver salt precipitation in the tissues.
However, beside an effect on platelet counts, which can be regarded as of questionable toxicological relevance, no signs of systemic effects were observed at the high dose level of 150 mg/kg, which can be regarded as a NOAEL for systemic effects.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed

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).

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:
Tentatively selected in absence of more robust data. To be replaced by on-going US NTP 90-day oral toxicity study with silver acetate.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
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. The OEL is stoichiometrically converted to 0.016 mg AgNO3/m³.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Testing not applicable, since AgNO3 is corrosive.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Testing not applicable, since AgNO3 is corrosive.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Testing not applicable, since AgNO3 is corrosive.

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.