Disilver oxide

Endpoint summary

Description of key information

 

A read-across approach from silver acetate (as source substance) is used for other silver compounds. The Toxicokinetic study confirmed the read-across between silver acetate and other silver compounds. For further details please refer to separate report "CSR Annex 7 Read Across Justification Human Health_Nov 2022" which is attached to the CSR in section 13.

Key information considered:

• Lourens et al. 2022 performed a repeated dose toxicity (90-days) study according to OECD 408 and following GLP principles, with silver acetate administered via the diet to Wistar Han rats at 40, 120 and 320 mg/kg bw/day. Based on the results, the NOAEL was a target dose level of 120 mg/kg bw/day for males and a target dose of at least 320 mg/kg bw/day for female.


• 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.


• Boudreau et al. 2016 conducted a 90-day (OECD 408) oral toxicity study.  Sprague Dawley rats of seven-week-old rats (10 rats per sex per group) were dosed with silver acetate (AgAc) at 100, 200, and 400 mg/kg bw; and controls (water). Rats exposed to AgAc at high dose (400 mg/kg bw/day) presented high morbidity with 70% of female and 100% of male rats being removed prior to the scheduled terminal sacrificed. Clinical findings suggested severe gastrointestinal symptoms, loss of body weight and unthrifty appearance among these animas, likely due to the bactericidal activity of silver ion on the intestinal microbiota. Significant lower mean body weight were observed in female rats administered of 100 and 400 of AgAc; the overall mean body weights were 88.5% and 74.4% of the control groups. Male rats administered with 400 and 200 mg/kg bw/d demonstrated significantly lower mean body weight than the controls, beginning at week 1 and week 3, respectively. body weight of male rats administered 100 mg/kg bw/d were not significantly affected. At the highest dose, the absolute heart and thymus weight were lower when compared with controls.

Lourens et al. 2022 (NOAEL of 120 and 320 mg AgAc/ kg bw/day for males and females respectively) was assessed. However, the study was not selected as starting point to derive the DNEL for silver compounds because the NOAELs decribed in this study were considered not conservative enough. Therefore, it is the LOAEL of 40 mg AgAc/kg bw/day defined by the EOGRTS (F0 generation - exposed during 135 days (in total)) that was used as a starting point.

For further details, please refer to separate report "CSR Annex 2_Derivation of DNEL_2022" which is attached to the CSR in section 13.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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:

20 January 2021-13 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Substance considered to fall within the scope of read-across: justification of a read-across approach for human health endpoints (document attached in IUCLID section 13)

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guideline on Bioanalytical Method Validation, European Medicines Agency (EMA), EMEA/CHMP / EWP/192217/2009

Version / remarks:

21 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance for industry: Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Veterinary Medicine (CVM)

Version / remarks:

May 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 417 Toxicokinetics

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Silver purity: 100%
Silver content: 64.56%
nitrate (NO3): 95 ppm
Copper: 1 ppm
Iron: 1 ppm
Sodium: 3 ppm
Magnesium: 1 ppm

Species:

rat

Strain:

other: Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent
species for nonclinical toxicity test by regulatory agencies.
Species: Rat
Strain: Crl: WI(Han)
Condition: Outbred, SPF-Quality
Source: Charles River Deutschland, Sulzfeld, Germany or Charles River
Laboratories France, L'Arbresle Cedex, France.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks old
- Weight at study initiation: 165-240 g (males), 127-170 g (females
- Fasting period before study: not applicable
- Housing:
Caging: Polycarbonate cages (Makrolon type IV, height 18 cm or Makrolon type 2000P, height 21.5 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
Up to 5 animals of the same sex and same dosing group together. During locomotor activity monitoring, animals are housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
These housing conditions will be maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. The room(s) in which the animals will be kept will be documented in the study records.
Cage Identification:
Color-coded cage card indicating at least Test Facility Study No., group, animal identification number(s).
- Diet (e.g. ad libitum): Animals will have access to the test diets from Day 1 onwards.
During the acclimatization period, animals will have free access to pellets without the test item (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals will not have access to food for a maximum of 2 hours.
The test item diets remain in the food hopper for a maximum of one week. On the day of weighing the remaining food in the food hopper, is replaced with new diet.
Type: Pellets.
Frequency: Ad libitum, except during designated procedures.
- Water (e.g. ad libitum):Municipal tap water, freely available to each animal via water bottles.
- Acclimation period: The animals will be allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.
-animal enrichment:
Animals will be socially housed for psychological/environmental enrichment and may be provided with items such as devices for hiding in, paper and/or objects for chewing, except
when interrupted by study procedures/acivities. Results of analysis for contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants that would interfere with the objectives of the study.

DETAILS OF FOOD AND WATER QUALITY:
Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 °C
- Humidity (%): 40-70%
- Air changes (per hr): ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h light and 12 h dark (except during designated procedures

IN-LIFE DATES:
From: 20 Jan 2021 (Animal arrival, first date data are collected from the study)
To: 7 May 2021 (last date of necropsy

Route of administration:

oral: feed

Details on route of administration:

The oral route of exposure via dietary inclusion was selected because this is a possible route
of human exposure during manufacture, handling or use of the test item.

Vehicle:

unchanged (no vehicle)

Remarks:

oral administration via the diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
Stability in rat diet (powder, SM R/M-Z; supplier SSNIFF®) over the concentration range 50
to 10000 ppm was confirmed for at least 4 weeks at room temperature (Test Facility Study
No. 20274169. The test item diets remain in the food hopper for a maximum of one
week. On the day of weighing the remaining food in the food hopper, is
replaced with new diet. As such, diets may be prepared up to 3 weeks in advance of first use.

- Mixing appropriate amounts with (Type of food):
Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest,
Germany) will be used to prepare pelleted diets. The test item will be grinded using a mortar
and pestle and mixed with some powder feed (premix) in multiple mixing steps without the
use of a vehicle, and subsequently mixed with the bulk of the diet. Water (approximately
15% in total) will be added to aid pelleting. The pellets will be dried for approximately 24
hours at 35°C before storage. The control animals will receive similarly prepared pellets but
without the test item. The diets will be prepared under yellow light.
- Storage temperature of food: room temperature (protected from light)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet preparation samples (week1, week 6 and week 13) will be collected directly after preparation for analysis:
-all groups for concentration
-group 2 and 4 for homogeneity.
All samples are transferred (at room temperature) to the analytical laboratory at the Test Facility for same day analysis, where possible or stored for analysis (at 18-22 °C)within known formulation stability period.
Analyses are performed by using a validated analytical procedure (Test Facility Study No. 20274169).
Acceptance Criteria:
-For concentration: mean sample concentration results within or equal to +/- 20% of theoretical concentration.
-For homogeneity, relative standard deviation (RSD) of concentrations of +/- 10% for each group.
Backup Samples: Duplicate middle samples for Groups 1 and 3 (concentration analysis
only) and duplicate top, middle, and bottom samples for Groups 2 and 4
(concentration and homogeneity analysis) are taken on the same day. Samples are collected for analyses and stored at the Test Facility at ≤-15ºC for possible future analysis.

Results:
Accuracy
The concentrations analyzed in the diets of Groups 2-4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).
The analysis performed on Day 2 with the diets prepared for use in Week 1 was not accepted
because the QC samples were not prepared on the correct target levels. Therefore, new QC
samples were prepared and analyzed on Day 8 together with re-analysis of the Week 1 diet
samples, which were acceptable.
For the diets of Group 2 Females (570 ppm) prepared for use in Week 13, the mean accuracy
was above the target concentration (i.e. 148% of target). Therefore, reserve samples of
Group 2 Females prepared for use in Week 13 were analyzed after the end of in-life and these
results were within specification. It was therefore concluded that the diet was accurately
prepared and the initial results to be erroneous.
Samples of Group 1 prepared in Weeks 1, 6 and 13 had a comparable response with the QC
sample blanks response. Hence, it can be concluded that no test item was present in Group 1
diet samples.

Homogeneity
The diets of Groups 2 and 4 prepared for use in Weeks 1 and 6 were homogeneous (i.e.
coefficient of variation ≤ 10%). Furthermore, the diet of Group 4 Males (4960 ppm) prepared
for use in Week 13 was also homogenous. The diets of Group 2 Females (570 ppm) prepared
for use in Week 13 were not homogeneous (i.e. 27% of target). Therefore, reserve samples of
Group 2 Females prepared for use in Week 13 have been analyzed after the end of in-life and
these results were within specification (i.e. coefficient of variation ≤ 10%). It was therefore
concluded that the diet was accurately prepared and the original result was considered to be
erroneous.

Duration of treatment / exposure:

90 days

Frequency of treatment:

ad libitum access to food (dietary administration of the test item), except during designated procedures

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

'group 1'

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

'group2'
Actual Mean over Means Intake [mg test item/kg body weight/day] with (mean range indicated within brackets):
-Males: 41 (33-48)
-females: 42 (37-48)

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

'group 3'
Actual Mean over Means Intake [mg test item/kg body weight/day] with (mean range indicated within brackets):
-Males: 122 (108-141)
-females: 126 (109-142)

Dose / conc.:

320 mg/kg bw/day (nominal)

Remarks:

'group 4'
Actual Mean over Means Intake [mg test item/kg body weight/day] with (mean range indicated within brackets):
-Males: 287 (270-328)
-females: 319 (299-354)

No. of animals per sex per dose:

10 males + 10 females per dosing group, plus 3 male + 3 females for toxicokinetics assessment, plus two spare animals.
Total: 52 males + 52 females

Control animals:

yes

Details on study design:

- Dose selection rationale:
The 90-day study will be performed with a readily soluble silver salt to maximise read-across
possibilities. Silver acetate was selected as the representative silver salt as it was considered
the best option as a soluble silver compound. This selection is based on other sub-chronic
toxicity testing like Price et al (2002), Sprando et al (2017), van den Brule et al (2019). The
alternative candidate, Ag nitrate, has a higher corrosive potential. The EPMF is generating
comparative in vivo TK data for different forms of silver/silver salts and this work is
anticipated to confirm that AgAc can be considered the most bioavailable form of Ag.
The dose levels are based on the results of a 14 day dietary toxicity study in the rat (Test
Facility Study No. 20274197). In the 14 days study groups of 3 males and females were fed
diets containing 1680 ppm (low dose) or 4480 ppm (high dose). The target doses were 120
and 320 mg/kg bw/day respectively. Achieved doses were 150 and 170 mg/kg bw/day for
low dose males and females respectively and 378 and 426 mg/kg bw/day for high dose males
and females respectively. No effects were observed in the low dose group animals and this
has been used to represent the control for assessing effects in the high dose group animals. A
lower body weight gain was observed in high dose males; difference in body weight at
termination was -8.6%. Food consumption was also reduced in males of the high dose (-12%
overall). There were no effects in females. There were also no test item-related clinical
observations, macroscopic post mortem findings or effects on organ weights.
The target high dose of 320 mg/kg bw/day in the 14 day study is considered equally
appropriate for the high dose of the 90 day study. It is anticipated that there will be an effect
on body weight in males and possibly also in females with the extended duration of treatment
for this study. This dose level should ensure that an MTD is not exceeded if the body weights
continue to diverge from control during the course of the 90 day study and is expected to
achieve the requirements of OECD 408 for the highest dose level. The mid dose target level
recommended is 120 mg/kg bw/day, which aims to achieve at least 100 mg/kg bw/day and
this is important for classification and labelling for STOT-RE. It also fulfills the suggested
fold intervals in OECD 408. Based on the recommended fold interval above doses the low
dose suggested is 40 mg/kg/bw/day. Target doses will be achieved by adjusting the dietary
incorporation levels based on body weight and food consumption in each treated group.

- Rationale for animal assignment (if not random):
random assignment

- Fasting period before blood sampling for clinical biochemistry:
Yes (overnight with a maximum of 24 hours)

- Rationale for selecting satellite groups:
not applicable

-Veterinary Care:
Veterinary care will be available throughout the course of the study and animals will be
examined by the veterinary staff as warranted by clinical signs or other changes. In the event
that animals show signs of illness or distress, the responsible veterinarian may make initial
recommendations about treatment of the animal(s) and/or alteration of study procedures,
which must be approved by the Study Director. Treatment of the animal(s) for minor injuries
or ailments may be approved without prior consultation with the Sponsor representative when
such treatment does not impact fulfillment of the study objectives. If the condition of the
animal(s) warrants significant therapeutic intervention or alterations in study procedures, the
Study Monitor will be contacted, when possible, to discuss appropriate action. If the
condition of the animal(s) is such that emergency measures must be taken, the Study Director
and/or attending veterinarian will attempt to consult with the Study Monitor prior to
responding to the medical crisis, but the Study Director and/or veterinarian has authority to
act immediately at his/her discretion to alleviate suffering. The Sponsor representative will be
fully informed of any such events.

Positive control:

no positive controls

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (all main animals)
- Time schedule:
At least once daily from start of administration onwards, up to the day prior to necropsy.
Animals will be observed within their cage unless necessary for identification or confirmation of
possible findings.
For observations that cannot be attributed to an individual animal due to social housing (e.g., watery feces), the observation will be recorded to each animal in the socialized group.
No cage side observations reported since no major observations noted.

DETAILED CLINICAL OBSERVATIONS: Yes (all main animals)
- Time schedule: Weekly; from Week 1 and throughout the study, and on the day of necropsy.

BODY WEIGHT: Yes (all main and TK animals)
- Time schedule for examinations: Weekly; from at least Day 1 and throughout the study. In order to monitor the health status, animals may be weighed more often. (Fasted weight on the day of
necropsy.)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
-All Main and TK animals
-Weekly; from at least Day 1 and throughout the study
- Quantitatively measured per cage
- Compound intake calculated based on nominal concentration of test item in diet against body weight and food consumption.

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

WATER CONSUMPTION: Yes (all main animals)
- Time schedule for examinations: on regular basis throughout the study. Water consumption is monitored by visual inspection of the water bottles. If inter group differences are noted, consumption is assessed by weight.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretreatment Period (all Main Study animals once (including spare animals) and Administration Period (All Group 1 and 4 Main Study animals during
Week 13. If treatment-related findings are noted, animals in Groups 2 and 3 will also be examined.)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment (all main animals), prior to necropsy
- Anaesthetic used for blood collection: Yes (Sampled prior to necropsy from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands).
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all main animals (4 groups x (10 males + 10 females))
- Parameters checked:
White Blood Cells (WBC)
Neutrophil (absolute)
Lymphocyte (absolute)
Monocyte (absolute)
Eosinophil (absolute)
Basophil (absolute)
Large unstained cells (LUC) (absolute)
Red Blood Cell
Reticulocyte (absolute)
Red Blood Cell Distribution Width (RDW)
Hemoglobin
Hematocrit
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelet
A blood smear will be prepared from each hematology sample. Blood smears are labeled, stained, and stored. These smears will not be examined, but may be evaluated when required to confirm analyzer results.

CLINICAL CHEMISTRY & PLASMA/SERUM HORMONES/LIPIDS: Yes (all main animals)
- Time schedule for collection of blood: end of treatment (Sampled between 7.00 and 10.30 from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The
Netherlands)
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all main animals (4 groups x (10 males + 10 females))
- Parameters checked
Alanine aminotransferase (ALT)
Triglycerides
Aspartate aminotransferase (AST)
HDL and LDL Cholesterol
Alkaline Phosphatase (ALP)
Sodium
Total protein
Potassium
Albumin
Chloride
Total Bilirubin
Calcium
Urea
Inorganic Phosphate (Inorg. Phos)
Creatinine
Triiodothyronine (T3)
Glucose
Thyroxine (T4)
Cholesterol
Thyroid-Stimulating Hormone (TSH)

After receipt of the serum for T3, T4 and TSH analysis it will be divided in two aliquots.
One aliquot will be used for measurement of thyroid hormones TSH using the IMMULITE®
1000 analyser. These aliquots for TSH will be stored in an ultra-low freezer (≤ -75°C) until
analysis. Any sample remaining after TSH analysis will be discarded. The other aliquot will
be used for measurement of T3 and T4 using LC-MS. The aliquots for T3 and T4 will be
collected in uniquely labelled clear 1.4 mL V-bottom Micronic polypropylene tubes and
stored in a freezer (≤ -15°C) until analysis. Measurement of T3 and T4 will be performed
according to the bioanalytical method validated in Test Facility Study No. 20213516. Any
samples remaining after the LC-MS analysis will be returned to storage for the retention
period.
The LC-MS analysis will be based on the following guidelines:
European Medicines Agency (EMA). Guideline on Bioanalytical Method Validation.
EMEA/CHMP/EWP/192217/2009, 01 February 2012.
Guidance for industry: Bioanalytical Method Validation, U.S. Department of Health and
Human Services, Food and Drug Administration, Center for Drug Evaluation and Research
(CDER) and Center for Veterinary Medicine (CVM), May 2018.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the Administration Period. All Main animals during Week 12-13. These tests are performed after clinical observations (including arena observation, if applicable).
- Dose groups that were examined: all main animals
- Battery of functions tested:
• hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength will be recorded as the mean of three measurements.
• locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations will be reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

IMMUNOLOGY: Yes
- Time schedule for examinations: end of treatment (Sampled between 7.00 and 10.30 from the
retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands)
- How many animals: all main animals (4 groups x (10 males + 10 females))
- Dose groups that were examined: all groups
- Parameters checked
On the day of sampling, peripheral blood mononuclear cells (PBMC) will be isolated from
blood samples by gradient density centrifugation using Ficoll Paque™ PREMIUM 1.084.
PBMCs will be counted with a Coulter Counter Z1 (Beckman Coulter Inc, Fullerton, United
States) and diluted to 1 x 106 cells/mL.
The following parameters will be determined in isolated PBMCs using a FACSCanto™ flow
cytometer system (BD Biosciences, Erembodegem, Belgium) according to a validated
method (Project 499468):
Parameter (Abbreviation) // Unit // Markers for identification
T-cells // % Lymphoid cells // CD3+/CD45RA-
T-helper cells // % Lymphoid cells // CD3+/CD4+/CD8-
T-cytotoxic cells // % Lymphoid cells // CD3+/CD4-/CD8+
B-cells // % Lymphoid cells // CD3+/CD45RA+
NK-cells // % Lymphoid cells // CD3-/CD161a+
Ratio T-helper cells/ T-cytotoxic cells (Th/Tc)

The % lymphoid cells of PBMC will be determined using the Forward Scatter and Side
Scatter.

OPHTHALMIC EXAMINATIONS
-Frequency: Pretreatment Period = All Main Study animals once (including spare animals), administration Period = All Group 1 and 4 Main Study animals during Week 13. If treatment-related findings are noted, animals in Groups 2 and 3 will also be examined.
-Procedure: The eyes will be examined using an ophthalmoscope after application of
a mydriatic agent (tropicamide 0.5%).

ESTROUS STAGE DETERMINATION
-Frequency: End of Treatment (on the day of necropsy) a vaginal smear will be taken to determine the stage of estrus from all Main Study animals. This will be done for all females, except for females that have to be euthanized in extremis or die spontaneously.
-Procedure: Estrous stage will be evaluated by examining the vaginal cytology of
the samples obtained by vaginal smears procedures.

COAGULATION
-all main animals
-end of treatment
-overnight fasting (with a maximum of 24 h)
-Sampled prior to necropsy from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands).
-Coagulation Parameters:
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)

BIOANALYSIS AND TOXICOKINETIC EVALUATION
-Bioanalytical Sample Collection from TK animals on day 1 and week 13
-group1 TK animals: between 2-3 am and between 8-9 am, group 2-3-4 TK animals: between 8-9pm, 2-3 am, 8-9 am and 2-3 pm
-0.3 mL sample from jugular vein, K2ADTA as anticoagulant, collection on ice.
-Whole blood samples will be collected and stored within 1 hour in a freezer set to maintain
-75°C. After transfer on dry ice to the bioanalytical laboratory the samples will be stored in a
freezer set to maintain -75°C until analysis.
-Whole blood will be analyzed for concentration of the total Silver (Ag) concentration using a
validated analytical procedure. Analysis will be performed under Analytical Procedure AP.20274188.ICP-R-WB. Statistical analyses including regression analysis and descriptive statistics including arithmetic means and standard deviations, accuracy and precision will be performed.
-Toxicokinetic Evaluation
Toxicokinetic parameters will be estimated using Phoenix pharmacokinetic software. A
non-compartmental approach consistent with the oral route of administration will be used for
parameter estimation. All parameters will be generated from Silver individual concentrations
in whole blood from Days 1 and Week 13 whenever practical.
-Parameters to be Estimated
Parameter // Description of Parameter
AUClast // The area under the concentration versus time curve from the start of dose administration to the last observed quantifiable concentration calculated using the linear trapezoidal method.
AUClast/Dose // The AUClast divided by the dose administered.
AUC(0-24h) // The area under the concentration versus time curve within a 24 hours’ time interval calculated using the linear trapezoidal method.
AUC(0-24h)/Dose // AUC(0-24h) divided by the dose administered.

Based on the data, additional secondary parameters may be determined, if appropriate.
Partial AUCs (between two defined sample times), and corresponding dose-normalized
values, may be derived and reported to aid interpretation. Descriptive statistics (e.g. number,
arithmetic mean, median, standard deviation, standard error, coefficient of variation) will be
reported as deemed appropriate. TK table and graphs will also be generated.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
artery, aorta; bone marrow, sternum; bone, sternum: brain; epididymis; esophagus; eye; adrenal gland; mammary gland; parathyroid gland; pituitary gland; prostate gland; submandibular salivary gland; sublingual salivary gland; seminal vesicle gland (including coagulation gland); thyroid gland; gut associated lymphoid tissue (Examine only if detectable in routine section of intestine); heart; kidney; large intestine, cecum; large intestine, colon; large intestine, rectum; liver; lung; mandibular lymph node; mesenteric lymph node; skeletal muscle; optic nerve (Examine only if detectable in the routine section of the eye); sciatic nerve; ovary; pancreas; skin; small intestine, duodenum; small intestine, ileum; small intestine, jejenum; spinal cord; spleen; stomach; testis; thymus; trachea; urinary bladder; uterus/cervix; vagina

HISTOPATHOLOGY: Yes
artery, aorta; body cavity, nasal; bone marrow, sternum; bone, femur; bone, sternum: brain; epididymis; esophagus; eye; adrenal gland; clitoral gland; harderian gland; lacrimal gland; mammary gland; parathyroid gland; pituitary gland; preputial gland; prostate gland; submandibular salivary gland; sublingual salivary gland; parotid salivary gland; seminal vesicle gland (including coagulation gland); thyroid gland; gut associated lymphoid tissue (Examine only if detectable in routine section of intestine); heart;, kidney; large intestine, cecum; large intestine, colon; large intestine, rectum: larynx; liver; lung; mandibular lymph node; mesenteric lymph node; skeletal muscle; optic nerve (Examine only if detectable in the routine section of the eye); sciatic nerve; tibial nerve; ovary; pancreas; skin; small intestine, duodenum; small intestine, ileum; small intestine, jejenum; spinal cord; spleen; stomach; testis; thymus; tongue; trachea; urinary bladder; uterus/cervix; vagina
Macroscopic abnormalities in the organs listed and in other organs will be sampled at necropsy, processed for histology and examined microscopically.
Tissues are embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Tissues are evaluated histopathologically by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

Optional endpoint(s):

Optional endpoints: No

Statistics:

-Constructed Variables:
Body Weight Gains: Calculated between each scheduled interval.
Food Consumption: Calculated between each scheduled interval.
Test Item Intake: Calculated based on nominal concentration of test item in diet against body weight and food consumption.
Organ Weight Relative to Body Weight: Calculated based on the terminal body weight.

-Descriptive Statistical Analyses
Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences are reported as appropriate by dataset.
-Inferential Statistical Methods
All statistical tests will be conducted at the 5% significance level. All pairwise comparisons are conducted using two sided tests and will be reported at the 1% and 5% levels, unless otherwise noted.
-Parametric/Non-parametric
Levene’s test is used to assess the homogeneity of group variances.
The groups are compared using an overall one-way ANOVA F-test if Levene’s test is not
significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis
test is found to be significant, then pairwise comparison sare conducted using Dunnett’s
or Dunn’s test, respectively.
-Non-Parametric
The groups are compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the above pairwise comparisons are conducted using Dunn’s test.
-ANCOVA
The data corresponding to a response variable of interest and to a related covariate will be
submitted to an analysis of covariance, including only groups with at least three
non-missing paired values and if found to be significant, then pairwise comparisons will be
conducted using Dunnett’s test.
[cfr. section 'any other information on materials and methods incl.tables' for continuation]

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted during daily detailed observations and no findings were noted during the arena observations in this study.
On Days 92 or 93 erected fur was noted in two males at 320 mg/kg body weight/day and in one female at 40 mg/kg body weight/day, which also showed hunched posture. Based on the incidental nature of these clinical signs, they were considered not toxicologically relevant.
The other clinical signs noted during the administration period were scabs, which occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose related trend. At the incidence observed, these were considered to be unrelated to the test item.

Mortality:

no mortality observed

Description (incidence):

No deaths occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
40 and 120 mg/kg body weight/day: slightly lower body weight and body weight gain between Days 1 and 36 (mean body weights 3.9% and 4.3% lower than control on Day 36 at 40 and 120 mg/kg body weight/day, respectively), which did recover thereafter.
320 mg/kg body weight/day: lower body weight and body weight gain throughout the administration period (mean body weight 14.9% lower and mean body weight gain 33% lower than control at the end of the administration period).

Females:
40 mg/kg body weight/day: similar body weight growth curve to those at 320 mg/kg body
weight/day during the initial part of the study. However, a -3.1% difference in mean body
weight was noted between females at 40 mg/kg body weight/day and the control group on
Day 1. When taking this difference on Day 1 into account, the maximum mean difference
between this group and the control group was only 3.3%, which is only minimal and
considered to be not toxicologically relevant.
120 mg/kg body weight/day: no clear effects on body weight and body weight gain
320 mg/kg body weight/day: slightly lower body weight gain seen starting
at Day 50 onwards (mean body weight up to 6.1% lower than control on Day 85
(not statistically significant) and a 14.5% lower mean body weight gain than control at the
end of the administration period).

cfr. Table in section 'Any other information on results incl. tables'

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males:
40 mg/kg/body weight/day: although food consumption was also slightly decreased between Days 8 and 50 (up to 7.0% lower than of control), this is considered not to be related to treatment with the test item in the absence of a similar finding at 120 mg/kg body weight/day.
120 mg/kg body weight/day: no effects on food consumption
320 mg/kg body weight/day: food consumption was decreased throughout the administration period with a maximum of 19.6% lower than of control between Days 57 and 64.

Females:
40 mg/kg body weight/day: food consumption decreased from Day 8 onwards, up to 10% lower than control between Days 22 and 29 and 85 and 91. These differences are considered not to be related to treatment in the absence of a similar finding at 120 mg/kg bw/day.
120 mg/kg body weight/day: no clear effects on food consumption
320 mg/kg body weight/day: food consumption was decreased on several occasions during the administration period (between Days 8 and 15, 36 and 57, 64 and 71 and 78 and 85). The maximum difference from the control group was reached between Days 64 and 71 (11.2% lower than control).

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

visual inspection of water consumption throughout the assay did not suggest any effect at any dosing level

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no test item-related ophthalmology findings in Week 13.
The nature and incidence of ophthalmology findings noted during the Pre-treatment Period and in Week 13 was similar among the groups and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
120 and 320 mg/kg body weight/day: an increase in red blood cell distribution width (RDWG) and decrease in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) was observed. The MCV and MCHC changes did not, however, show a clear dose response. At 320 mg/kg body weight/day, hemoglobin concentration (HGB) was also decreased.

Females:
120 and 320 mg/kg body weight/day: an increase in eosinophil (EOS) count and a decrease in MCV (no dose response) and MCH was seen. Furthermore, an increase in RDWG was observed in females at 320 mg/kg body weight/day.

Other values in treated males and females achieving a level of statistical significance, when compared to controls, were considered to have arisen as a result of slightly high or low control values, occurred in the absence of a dose-related distribution and/or were, given the magnitude of change (e.g. MCV in females at 40mg/kg body weight/day), considered to be of no toxicological significance.

cfr Table 'Ratio Hematology Differences from Control Groups' in section 'Any other information on results incl. tables'

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
40 mg/kg/d: Increase in HDL cholesterol
120 mg/kg/d: increases in cholesterol (CHOL) and HDL cholesterol concentration, increase in calcium concentration (not statistically significant)
320 mg/kg/d: increase in alanine aminotransferase (ALT) activity and urea concentration, increases in cholesterol (CHOL) and HDL cholesterol concentration, increase in triglyceride (TRIG) concentration (not statistically significant), increase in calcium concentration (not statistically significant)

Females:
40 mg/kg/d: increase in alkaline phosphatase (ALP) activity
120 mg/kg/d: increase in alkaline phosphatase (ALP) activity, increases in cholesterol (CHOL) and HDL cholesterol concentration, increase in calcium concentration
320 mg/kg/d: increase in alkaline phosphatase (ALP) activity, increases in cholesterol (CHOL) and HDL cholesterol concentration, increase in triglyceride (TRIG) concentration (not statistically significant), increase in calcium concentration

Other remaining differences in clinical chemistry parameters, despite the statistical significance, were considered not to be test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and were of a magnitude of change commonly observed in rats under similar study conditions.

cfr Table 'Ratio Clinical Chemistry Differences from Control Groups' in section 'Any other information on results incl. tables'

Endocrine findings:

no effects observed

Description (incidence and severity):

No test item-related effects were seen on the thyroid hormones (T3, T4 and TSH).

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity was similar between control and the test item groups.
All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

no effects observed

Description (incidence and severity):

There were no clear test item-related immunophenotype alterations.
Differences in immunophenotype results, despite the statistical significance, were considered not test item-related based on the absence of a dose response and the magnitude of change observed.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Differences in mean weights were noted for a few organs starting at 120 mg/kg body weight/day.

Males:
40 mg/kg/d: /
120 mg/kg/d: Slightly higher liver weight (relative to body weight only, no histological correlate)
320 mg/kg/d: Slightly higher liver weight (relative to body weight only, no histological correlate), lower pituitary gland weight (absolute and relative to body weight, no histological correlate), lower absolute epididymis weight (considered secondary to the lower body weight, no histological correlate in the testis or epididymis, the testis weight was not significantly different from the control
animals)

In males at 120 and/or 320 mg/kg body weight/day apparent higher brain, heart and testis weights were noted (statistically significant when expressed relative to body weight only). This was regarded secondary to lower terminal body weight in these groups (-5%, -15%, respectively). Absolute values of these three organs, were not statistically significant from the control animals.

Females:
40 mg/kg/d: /
120 mg/kg/d: /
320 mg/kg/d: Slightly higher liver weight (relative to body weight only, no histological correlate)

Cfr Table 'Mean percent organ weight differences from control groups' in section 'Any other information on results incl. tables'

Gross pathological findings:

no effects observed

Description (incidence and severity):

Dark gray discoloration was noted in all animals in all dose groups in the kidneys, pancreas, and Harderian glands and in most animals in all dose groups in the clitoral/preputial glands (including gray discoloration and gray foci), thymus, urinary bladder, liver, thyroid gland, stomach, small intestine (duodenum, jejunum, and/or ileum), large intestine (cecum, colon, and/or rectum).

More variable response was observed in other tissues including the mandibular salivary gland at all dose levels, spleen (males all dose levels, females starting at 120 mg/kg body weight/day), and the mandibular lymph node starting at 120 mg/kg body weight/day.

In males, a low incidence of dark gray discoloration was noted in the prostate at all dose levels, and the pituitary gland at 320 mg/kg body weight/day only. In females, dark gray discoloration was noted in the lacrimal gland and uterus at all dose levels, and in the skin starting at 120 mg/kg body weight/day. Dark gray discoloration was also noted in a few non-study plan tissues including several lymph nodes in males dosed at 320 mg/kg body weight/day, including axillary, bronchial, hepatic, iliac, inguinal, pancreatic, and popliteal lymph nodes, and the rib from a female dosed at 120 mg/kg body weight/day.

For most tissues, macroscopic gray discoloration correlated well with the presence of blackbrown pigment microscopically. However, for a few tissues, there was no microscopic correlate detected (thyroid gland, mandibular salivary gland, pituitary gland, prostate gland,
and uterus).

The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not specified

Description (incidence and severity):

cfr. gross pathology section

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related black-brown pigment was observed microscopically in all animals at all dose levels in one or more of the following organs: brain (area postrema and subfornical organ), gastrointestinal tract (stomach, small and large intestines), kidney, liver, lymph nodes (mesenteric, mandibular), pancreas, skin, thymus, urinary bladder, harderian gland, preputial/clitoral gland, and lacrimal gland. In the spleen, increased pigment was noted in males at 320 mg/kg body weight/day. In the spleen of females, increased extramedullary hematopoiesis was noted at 320 mg/kg body weight/day. Details of the incidence, severity, and histologic characterization are described by organ below.

Brain: In the brain, pigment (minimal or mild) was noted at all dose levels in the area postrema in all treated animals for which this structure was present in section, and in the subfornical organ of four animals (2 males at 40 mg/kg body weight/day, and one female each at 40 and 320 mg/kg body weight/day) for which this structure was present in section. Both of these structures are circumventricular organs and thus have a specialized capillary structure necessary for their function and are considered to be outside the blood brain barrier, which likely explains the selective presence of the pigment at these locations. Pigment in the area postrema was characterized by the presence of black-brown pigment in/around the blood vessel walls, highlighting these structures. In the subfornical organ, the pigment was less obvious, and was sparce and finely granular black-brown and not clearly associated with the interstitium/blood vessels.
These substructures are not required for evaluation of the brain and there is a normal and acceptable degree of variability in brain sections. These substructures were only available for several animals and noted under a separate organ in the data when present (Brain, area postrema; Brain, subfornical organ).

Gastrointestinal tract: Minimal to moderate pigment was present in at least one gastrointestinal segment (most often several) in all males and females at all dose levels.

Stomach: Minimal (and occasionally mild) pigment was present in the superficial lamina propria of the glandular mucosa and correlated with macroscopic dark gray discoloration.

Small intestine: Pigment was present in all segments of the small intestine with a fairly consistent gradation such that the duodenum was the most affected (mild to moderate), then the jejunum (minimal to mild), and lastly the ileum (minimal). A dose related increase in severity was noted for the duodenum.

Large intestine: Pigment was present in all segments of the large intestine with a fairly consistent gradation such that the cecum and colon were approximately equally affected (minimal and mild) and the rectum was the least affected (minimal). This was characterized by the presence of black-brown pigment in the lamina propria.

Kidney: Pigment was noted in the kidney of all animals at all dose levels (mild or moderate) and correlated with macroscopic dark gray discoloration. This was characterized by the presence of black-brown pigment in the interstitial spaces of the outer stripe of the outer medulla, strikingly highlighting this region. To a lesser degree, pigment was also noted in the cortical tubules as rare black brown granules present near the surface of the tubular epithelium, and less commonly, light brown pigment was also present diffusely in the glomerulus.

Liver: Pigment was noted in the liver (minimal to mild) of all animals at all dose levels and correlated with macroscopic dark gray discoloration with a dose-related increase in severity. This was characterized by the presence of finely granular black-brown pigment in the interstitium of the portal areas in and around the blood vessel walls, in the sinusoidal lining cells (mostly of the periportal/midzonal regions), and occasionally within hepatocytes.

Lymph nodes: Pigment was noted in the routinely examined lymph nodes (mesenteric and mandibular) of most animals at all dose levels (minimal to moderate). This was characterized by the presence of finely granular pigment in macrophages within the subcapsular and medullary sinuses. The macrophages containing the pigment were not notably enlarged nor,were the affected lymph nodes enlarged. The mesenteric lymph node (minimal to moderate) was consistently affected more than the mandibular lymph node (minimal to mild) and showed a dose related increase in severity.

Pancreas: Pigment was noted in the pancreas of all animals (minimal) and correlated with macroscopic dark gray discoloration. This was characterized by the presence of black, finely granular pigment present diffusely throughout the interstitium of the exocrine and endocrine pancreas.

Skin: Pigment (minimal) was noted in the skin in of males and females at all dose levels. The pigment was located in the dermis, specifically around the hair bulb, and this correlated with macroscopic dark gray discoloration noted in females. The incidence was somewhat variable in part because the detection depended on the presence of the hair bub in section.

Spleen: In most tissues pigment is not normally present in albino rats making the detection of even small amounts of the test item-related black-brown pigment possible in this study. In the spleen, however, hemosiderin pigment is normally present and the detection of the test item related black-brown pigment on this normal background of brown pigment was not possible. Thus, in the spleen, the diagnosis of pigment was graded on the overall amount, acknowledging it is not possible to clearly differentiate the two types (background pigment vs. test item-related pigment) in routinely stained (hematoxylin and eosin) sections.
In males, there was a slightly higher severity of spleen pigment (up to moderate degree) at 320 mg/kg body weight/day, compared to the control animals (minimal to mild). This could represent either increased amount of the typical pigment present (hemosiderin) or the addition of the test item-related pigment, thus there was no clear microscopic correlate to the gray discoloration noted macroscopically. Minor variation in the amount of extramedullary hematopoiesis was not clearly test item-related and may represent normal biologic variability.
The amount of pigment noted histologically in the spleen of females was comparable between control and treated animals and there was no correlate to the gray discoloration noted macroscopically.

Thymus: Pigment (minimal) was noted in the thymus of most animals at all dose levels. This was characterized by a small amount of finely granular black-brown pigment most often detected in the capsule and correlated with macroscopic dark gray discoloration.

Urinary bladder: Pigment (minimal) was noted in the urinary bladder of all animals at all dose levels. This was characterized by a small amount of finely granular black-brown pigment just below the epithelium in the region of the epithelial basement membrane/superficial lamina propria and correlated with macroscopic dark gray discoloration.

Non-Study plan tissues (examined due to macroscopic observations):
Preputial/Clitoral glands: Pigment (minimal or mild) was present in all dose groups in nearly all animals for which it was examined and correlated with macroscopic dark gray discoloration. This was characterized finely granular black-brown pigment diffusely in the interstitial tissues.

Harderian gland: Pigment was noted in the Harderian gland of all animals histologically (minimal or mild) and correlated with macroscopic dark gray discoloration. This was characterized finely granular black-brown pigment diffusely in the interstitial tissues.

Lacrimal glands: Pigment (minimal) was noted in the lacrimal glands of females at all dose levels for which this tissue was examined and correlated with macroscopic dark gray discoloration. This was characterized finely granular black-brown pigment diffusely in the interstitial tissues.

Lymph nodes: Several non-study plan lymph nodes were examined from the males at 320 mg/kg body weight/day (collected because of macroscopic dark gray discoloration - axillary, bronchial, hepatic, iliac, inguinal, pancreatic, and popliteal) and females at 320 mg/kg body weight/day (popliteal) which also correlated with microscopic pigment, similar to the mandibular and mesenteric lymph nodes described above.

Remaining test item-related findings:
Spleen: In the spleen of females at 320 mg/kg body weight/day a slightly higher incidence and severity of extramedullary hematopoiesis was observed compared to the control females, up to mild degree. In males, the extramedullary hematopoiesis was comparable in control and test item-treated animals.

There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings or were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no neoplastic, test item-related histologic changes. Changes were considered to be incidental findings or were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of these.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Coagulation:
There were no test item-related findings in males at 40 mg/kg body weight/day and females at
40, 120 and 320 mg/kg body weight/day.
Prothrombin time (PT) was slightly shortened in males at 120 and 320 mg/kg body weight/day (0.95 and 0.94x of control respectively).

Details on results:

Toxicokinetic Evaluations:
The exposure to Silver increased in a less than dose-proportional manner for males and females on Day 1 and in Week 13. On Day 1, an increase in target dose from 40 to 320 mg/kg body weight resulted in a 0.2/0.2-fold increase in AUClast/Dose (expected ratio of 1.0), for males/females respectively. In Week 13, an increase in target dose from 40 to 320 mg/kg body weight/day resulted in a 0.4/0.3-fold increase in AUC(0-24h)/Dose (expected ratio of 1.0), for males/females respectively.
At all dose levels, (slightly) higher AUClast/Dose values were observed in Week 13 compared to Day 1. Therefore, it can be concluded that slight accumulation of Silver seemed to occur after administration with Silver acetate. This effect was most pronounced at a target dose level of 320 mg/kg body weight/day.
On Day 1, a slight trend towards higher exposure levels in females compared to males was observed, whereas in Week 13, no clear sex differences were noted for AUClast/Dose at all dose levels.
The whole blood concentrations of Silver in the control animals were all below the LLOQ, confirming the absence of exposure to silver acetate in control animals.
cfr. detailed values in table 'Summary of Silver Toxicokinetic Parameters in Male and Female Wistar Han Rat Whole Blood Following Dietary Administration of Silver Acetate on Day 1 and in Week 13' in section 'Any other information on results incl. tables'

Dose descriptor:

NOAEL

Effect level:

>= 320 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Critical effects observed:

no

Table: summary of body weight evolution of male and female animals (in g)

Male	Mean body weight (day1)	Mean body weight (day91)	% difference compared to control
0 mg/kg bw/day	203.1 (SD 13.6, N=10)	413.9 (SD 37.4, N=10)	-
40 mg/kg bw/day	207.9 (SD 19.6, N=10)	400.3 (SD 19.6, N=10)	-3.3%
120 mg/kg bw/day	212.6 (SD 7.6, N=10)	396.7 (SD 27.2, N=10)	-4.2%
320 mg/kg bw/day	211.0 (SD 7.8, N=10)	352.3 (SD 27.3, N=10)**	-14.9%
Female
0 mg/kg bw/day	151.2 (SD 9.1, N=10)	241.1 (SD 19.1, N=10)	-
40 mg/kg bw/day	146.5 (SD 10.4, N=10)	229.1 (SD 17.1, N=10)	-5.0%
120 mg/kg bw/day	153.9 (SD 9.0, N=10)	240.9 (SD 20.5, N=10)	-0.1%
320 mg/kg bw/day	149.9 (SD 10.6, N=10)	226.8 (SD 16.9, N=10)	-5.9%
Anova & Dunnett: ** = p ≤ 0.01

 

Table: Ratio Hematology Differences from Control Groups (Numerical values indicate fold change of the treated group mean value relative to the control group mean)
Dose	40 mg/kg bw/day		120 mg/kg bw/day		320 mg/kg bw/day
Sex (M(ale), F(emale))	M	F	M	F	M	F
eosinophil (EOS)	-	-	-	1.6*	-	1.8**
red blood cell distribution width (RDWG)	-	-	1.08*	-	1.11**	1.14**
mean corpuscular volume (MCV)	-	0.97**	0.93**	0.95**	0.93**	0.95**
mean corpuscular hemoglobin (MCH)	-	-	0.91**	0.95**	0.91**	0.94**
mean corpuscular hemoglobin concentration (MCHC)	-	-	0.98*	-	0.97*	-
hemoglobin concentration (HGB)	-	-	-	-	0.95**	-
Legend: - = no test item effect, *p<=0.05, **p<=0.01

 

Table: Ratio Clinical Chemistry Differences from Control Groups (Numerical values indicate fold change of the treated group mean value relative to the control group mean)
Dose	40 mg/kg bw/day		120 mg/kg bw/day		320 mg/kg bw/day
Sex (M(ale), F(emale))	M	F	M	F	M	F
alanine aminotransferase (ALT) activity	-	-	-	-	1.49**	-
alkaline phosphatase (ALP) activity	-	1.45*	-	1.75**	-	1.84**
urea concentration	-	-	-	-	1.34**	-
cholesterol (CHOL) concentration	-	-	1.46**	1.39*	1.68**	2.03**
HDL cholesterol concentration	1.22*	-	1.49**	1.42**	1.82**	2.15**
triglyceride (TRIG) concentration	-	-	-	-	1.32	1.36
calcium concentration	-	-	1.05	1.07*	1.07	1.05*
Legend: - = no test item effect, *p<=0.05, **p<=0.01

 

Table: Mean Percent Organ Weight Differences from Control Groups
Dose	40 mg/kg bw/day		120 mg/kg bw/day		320 mg/kg bw/day
Sex (M(ale), F(emale))	M	F	M	F	M	F
Terminal Body Weight	-3	-5	-4	0	-15	-6
PITUITARY GLAND
absolute	-9	-9	-7	-6	-24**	-11
relative to body weight	-5	-4	-2	-6	-10*	-5
LIVER
absolute	-4	-5	0	4	-1	8
relative to body weight	-1	0	6*	4	16**	14**
EPIDIDYMIDES
absolute	-5	/	-5	/	-14**	/
relative to body weight	-2	/	-1	/	1	/
Legend: *p<=0.05, **p<=0.01

 

Summary of Coagulation values
Male	PT (sec)	APTT (sec)
0 mg/kg bw/day	17.43 (SD 0.31, N=10)	21.63 (SD 0.77, N=10)
40 mg/kg bw/day	16.93 (SD 0.47, N=10)	21.81 (SD 1.31, N=10)
120 mg/kg bw/day	16.50 (SD 0.56, N=10)**	21.86 (SD 0.69, N=10)
320 mg/kg bw/day	16.41 (SD 0.83, N=10)**	20.46 (SD 1.80, N=10)
Female
0 mg/kg bw/day	16.76 (SD 0.94, N=10)	18.56 (SD 1.29, N=10)
40 mg/kg bw/day	15.99 (SD 0.63, N=10)	17.82 (SD 1.97, N=10)
120 mg/kg bw/day	15.97 (SD 0.29, N=10)	18.73 (SD 1.57, N=10)
320 mg/kg bw/day	16.04 (SD 0.40, N=10)	19.06 (SD 1.53, N=10)
Kruskal-Wallis & Dunn: ** = p ≤ 0.01

 

Summary of Silver Toxicokinetic Parameters in Male and Female Wistar Han Rat Whole Blood Following Dietary Administration of Silver Acetate on Day 1 and in Week 13

Parameters	Target dose level (mg/kg bw/day)
	40		120		320
Sex	M	F	M	F	M	F
Day 1	N=3	N=3	N=3	N=3	N=3	N=3
Average actual test item intake (mg/kg bw/day)	48	48	149	144	337	373
t(last) in h	18	18	18	18	18	18
AUC(last) in h*ng/mL	7610	11100	11000	13300	9830	17300
AUC(last)/Dose in h*kg*ng/mL/mg	158	231	74.1	92.2	29.2	46.4
Week 13	N=3	N=3	N=3	N=3	N=3	N=3
Average actual test item intake (mg/kg bw/day)	38	38	113	112	279	308
t(last) in h	18	18	18	18	18	18
AUC(last) in h*ng/mL	10400	11000	11800	14200	34100	28200
AUC(last)/Dose in h*kg*ng/mL/mg	274	291	105	127	122	91.5
AUC(0-24h) in h*ng/mL	13800	14700	15700	18900	45100	38200
AUC(0-24h)/Dose in h*kg*ng/mL/mg	362	387	139	169	162	124

Conclusions:

The administration of Silver acetate by dietary inclusion was well tolerated in
Wistar Han rats at target dose levels up to 320 mg/kg body weight/day in a 90-day study according to OECD 408. Non-adverse test item-related macroscopic gray discoloration and microscopic pigment were observed in several tissues and organs of all animals starting at 40 mg/kg body weight/day, which was regarded to be due to accumulation of silver. The only other histopathology finding was a non-adverse minor increase in incidence and severity of extramedullary hematopoiesis noted in the spleen of females at 320 mg/kg body weight/day. Finally, minimal and non-adverse changes in clinical pathology parameters were noted mainly at 120 and 320 mg/kg body weight/day.
At 320 mg/kg body weight/day, lower body weight gain and lower food consumption were observed in males and females, which were considered to be adverse in males only.
Based on these results, the No-Observed-Adverse-Effect level (NOAEL) was considered to be at a target dose level of 120 mg/kg body weight/day for males and a target dose level of at least 320 mg/kg body weight/day for females.

Executive summary:

A GLP-compliant study was performed to determine the potential toxicity of Silver acetate, when given via diet for 90 days to the Wistar Han rat (according to OECD 408).

Target dose levels for groups 1 to 4 were 0, 40, 120 and 320 mg silver acetate/kg bodyweight/day, respectively. The target dose level was reached for Group 2 and 3 animals and Group 4 females. Group 4 males had an actual mean dose level of 287 mg/kg body weight/day as a consequence of a dose related reduction in food consumption/palatability for Group 4 males. For the identification of Group 4 males in this report the target dose level (320 mg/kg body weight/day) is used.

Chemical analyses of dietary preparations were conducted in Weeks 1, 6 and 13 to assess accuracy and homogeneity. Dietary analyses confirmed that formulations of test item in diets were prepared accurately and homogeneously.

 

The following parameters and endpoints were evaluated in this study: mortality, clinical signs, functional observation tests, body weights, food consumption, ophthalmology, estrus stage determination, clinical pathology parameters (hematology, coagulation, clinical chemistry, thyroid hormones and immunophenotyping), toxicokinetic parameters, gross necropsy findings, organ weights, and histopathologic examinations.

 

At 40 mg/kg body weight/day, a non-adverse test item-related increase in alkaline phosphatase activity (ALP) in females and HDL cholesterol concentration in males was noted.

 

At 120 mg/kg body weight/day, non-adverse test item-related changes in clinical pathology included increased eosinophil count in females, increased red blood cell distribution width in males, along with a decreased mean corpuscular volume and mean corpuscular hemoglobin in both sexes. In addition, mean corpuscular hemoglobin concentration was decreased in males only. Moreover, slightly shortened prothrombin time (PT) was observed in males. Furthermore, increased ALP activity in females and increased (HDL) cholesterol and calcium concentrations in males and females were noted. At necropsy, a test item-related higher relative liver weight was observed in males, which was without microscopic correlate and therefore considered to be non-adverse.

 

At 320 mg/kg body weight/day, a test item-related lower body weight and body weight gain was seen in males throughout the administration period. In females, a slightly lower body weight and/or body weight gain was seen starting at Day 50 onwards. The food consumption in males was decreased throughout the administration period, while in females it was decreased during six out of thirteen weeks. The effects on food consumption at 320 mg/kg body weight/day and consequently on body weight are at the severity observed considered to be adverse for males and not adverse for females. At clinical pathology, non-adverse changes included increased eosinophils (females), increased red blood cell distribution width (both sexes) and decreased mean corpuscular volume (both sexes), mean corpuscular hemoglobin (both sexes), mean corpuscular hemoglobin concentration (males) and hemoglobulin concentration (males). In addition, a slightly shortened PT was observed in males. Clinical chemistry analysis revealed increased alanine aminotransferase activity and urea concentration in males, ALP activity in females and (HDL) cholesterol, triglyceride and calcium concentrations in males and females. Test item-related non-adverse organ weight differences consisted of slightly higher liver weight in males and females and slightly lower pituitary gland and epididymis weight in males, which were without a microscopic correlate. Microscopic evaluation showed a slightly higher incidence and severity of extramedullary hematopoiesis in the spleen in females compared to the control females. At the severity observed, this finding was considered to be not adverse.

 

At necropsy, test item-related non-adverse macroscopic gray discoloration was present in all animals at all dose levels. Affected organs included: kidneys, harderian gland, clitoral or preputial gland, thymus, urinary bladder, liver, thyroid gland, stomach, small and large intestines, mesenteric and mandibular lymph nodes, mandibular salivary gland, skin, lacrimal gland, pituitary gland, uterus, and prostate gland. Test item-related microscopic black-brown pigment (ranging from minimal to moderate degree) was present in tissues from all animals at all dose levels. Affected organs included the brain (area postrema and subfornical organ), kidney, liver, lymph nodes (mesenteric, mandibular), pancreas, skin, stomach, small and large intestines, thymus, urinary bladder, harderian gland, preputial/clitoral gland, and lacrimal gland. In the spleen at 320 mg/kg body weight/day increased pigment was noted in males. The presence of the pigment microscopically was caused by the silver accumulation and was not associated with any other tissue alterations and was therefore considered to be a non-adverse change.

 

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical signs, functional observation, ophthalmoscopy, thyroid hormones and immunophenotype parameters).

 

There was no evidence of exposure to Silver in control animals. All Silver acetate-treated animals were exposed to Silver confirming administration and absorption. Exposure to Silver, expressed as AUC/Dose, increased less than dose proportionally over the used target dose accumulation of Silver seemed to occur after administration with Silver Acetate. This effect was most pronounced at a target dose level of 320 mg/kg body weight/day. On Day 1, a slight trend towards higher exposure levels in females compared to males was observed, whereas in Week 13, no clear sex differences in exposure were observed during the study.

 

In conclusion, administration of Silver acetate by dietary inclusion was well tolerated in Wistar Han rats at target dose levels up to 320 mg/kg body weight/day. Non-adverse test item-related macroscopic gray discoloration and microscopic pigment were observed in several tissues and organs of all animals starting at 40 mg/kg body weight/day, which was regarded to be due to accumulation of silver. The only other histopathology finding was a non-adverse minor increase in incidence and severity of extramedullary hematopoiesis noted in the spleen of females at 320 mg/kg body weight/day. Finally, minimal and non-adverse changes in clinical pathology parameters were noted mainly at 120 and 320 mg/kg body weight/day.

At 320 mg/kg body weight/day, lower body weight gain and lower food consumption were observed in males and females, which were considered to be adverse in males only.

Based on these results, the No-Observed-Adverse-Effect level (NOAEL) was considered to be at a target dose level of 120 mg/kg body weight/day for males and a target dose level of at least 320 mg/kg body weight/day for females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

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:

no study available

Additional information

 

 

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 (Mota et al. 2021)*. The available animal studies with silver compounds do not indicate any specific target organ toxicity. For this reason, silver compounds do not meet any of the classification criteria for effects associated with repeated dosing.

*Mota, L.; Dinis-Oliveira, R.J. Clinical and Forensic Aspects of the Different Subtypes of Argyria. J. Clin. Med. 2021, 10, 2086.