Silver

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The evaluation of reproductive toxicity of silver metal (massive and powder) was performed via the weight of evidence approach. The approach was built on:

• confident and reliable reproductive toxicity studies using nanosilver and soluble silver salts (like AgAc
• the comparative in vivo TK study justifying a correction factor to adjust the difference in oral absorption/ bioavailability between silver metal (massive and powder)
• the observed Cu depletion as presumed Mode of Action behind the observed reproductive effects, which has been demonstrated in the comparative in vivo TK study to not appear up to limit dose levels of Ag metal powder.

A strong weight of evidence using the available data demonstrates that the adverse effects on fertility and reproduction after repeated exposure to nanosilver and soluble silver salts are not expected for silver metal (massive and powder) based on the bioavailability considerations and its direct interaction with the Cu-depletion mode of action.

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

To be completed when FINAL report available

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study has been conducted according to OECD guideline and under GLP conditions

Justification for type of information:

Weight of evidence approach described in 'Silver metal (massive and powder): Weight of Evidence' (document attached in IUCLID section 13 - "CSR Annex 11 - Weight of Evidence Justification for Silver metal - human health endpoints).

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals:
According to updated ECHA Guidance, it is recommended that the default position for the duration of the premating exposure period should be 10 weeks to cover the full cycles of spermatogenesis/sperm maturation and folliculogenesis before the mating—thus permitting an optimal assessment of any effects on fertility.

- Basis for dose level selection
The dose level has been determine by a dose range finding study. The study was not designed to meet any particular regulatory requirements. The study design was recommended by LabCorp (previously Covance) as a dose-range finding study for the OECD 443 Extended One Generation Reproductive Toxicity Study. The work performed generally followed Good Laboratory Practice principles.

- Inclusion/exclusion of extension of Cohort 1B Uses leading to exposure of consumers or professionals, including potential consumer exposure from articles, are foreseeable. However, the matching conditions specified within Annex IX/X; 8.7.3 (Column 2) are not met. The substance is not considered to be genotoxic and is not classified as a mutagen. From appropriate toxicokinetic/biomonitoring data in animals and humans, there are no indications that the absorbed internal dose will only achieve a steady-state condition after extended exposure. A categorisation as PBT or vPvB is not applicable; and taking account of physico-chemical parameters and other considerations, no accumulation in the body is expected. For water-soluble silver forms, toxicokinetic data show that they are absorbed and cleared relatively rapidly (with the exception of a minor fraction represented by tissue-associated complexes of silver selenide and silver sulphide – which represent very insoluble and biologically inert depots). Please also refer to further detailed justification under ‘Background information used to conclude on the study design’. Based on the available data which can be categorised as robust, adverse effects on the endocrine system are not apparent (including considerations of the oestrogen-axis, androgen-axis and thyroid hormone activity). Therefore, the conditions for an extension of cohort 1B are not fulfilled. In terms of the expected sensitivity and predictivity performance of the proposed basic test design, the conclusions of the OECD report covering the retrospective analysis of 2-generation reproductive toxicity data (OECD 2012, Series on Testing and Assessment No. 176) are also taken into account, viz. it was stated that most of the national coordinators and subject matter experts agreed that extensive retrospective analyses demonstrated that the production of a second generation would rarely affect hazard characterisation outcomes either for risk assessment or for hazard classification of chemicals. However, it should be noted that during conduct of the proposed EOGRTS, in-study triggers for production of a F2 generation remain a possibility if warranted by initial outcomes.

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B:
Cohorts 2A and 2B are to be proposed in the case of a particular concern relating to a potential for (developmental) neurotoxicity; as described in Annex IX/X; 8.7.3 (Column 2). No definitive triggers for the inclusion of Cohorts 2A and 2B (developmental neurotoxicity) were identified. This assessment is based on evaluation of indicators such as the absence of signs of morphological or functional neurotoxicity from robust/TG-conform repeat dose toxicity studies in adult animals, the available pre-natal/post-natal information, and also takes account new transplacental toxicokinetic data in rodents for ionic and nanoparticulate silver forms. Please also refer to ‘Background information used to conclude on the study design’.

- Inclusion/exclusion of developmental immunotoxicity Cohort 3:
Cohort 3 is to be proposed in the case of a particular concern relating to a potential for developmental immunotoxicity (DIT); as described in Annex IX/X; 8.7.3 (Column 2). Triggers related to structure-activity relationships (SAR), or endocrine-disrupting activity, or evidence of significant immunotoxicity in adult animals were not considered to be applicable. Whilst data from repeat dose studies (including reproductive toxicity investigations) concerning organ weight and histopathology of lymphoid organs are inconsistent. A recently conducted one-generation reproductive toxicity study in rodents (but not conforming to OECD TG 443 in respect of DIT investigations) on a soluble silver compound (silver acetate) reported shifts in several immune system parameters in offspring. Hence indicative—but not firm evidence—exists of developmental immunotoxicity potential in a rodent model. Accordingly, it is considered that this finding justifies the inclusion of Cohort 3 in the test design. Please also refer to ‘Background information used to conclude on the study design’.

- Selection of test species and route of administration
In accordance with OECD TG 443 / EU B.56, the rat is the preferred species. With due regard to the substance physical form, exposure scenarios, and also the advice provided in Section R.7.6.2.3.2. in the ECHA Guidance on information requirements and chemical safety assessment R.7a (version 6, July 2017), it is considered that the oral exposure via dietary administration is the most appropriate route to model human exposure. Exposure via incorporation in the drinking water is a possible alternative but is less preferred due to the potential for both silver adsorption on to container surfaces, and also due to experience that silver can affect the drinking water intake of experimental animals.

Specific details on test material used for the study:

- Test item identity: AgAc, AG(I) Acetate T2 HSTDP Silver(I) Acetate
- Intended use: Chemical feed stock
- Appearance: Colorless to pale-white crystalline powder
- Storage conditions: Room temperature and protexted from light
- Date manufacture: 23 October 2019
- Expiry date: 22 October 2021
- Purity: > 99.5%
- Molar mass: 166.91g/mol (of which 107.87 g/mol Ag)
- Solubility: Aqueous (room temperature): 1.02 g/100 mL or 10.2 mg/mL

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague-Dawley [Crl:CD(SD] strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females nulliparous and non-pregnant: yes
- Age of the F0 (parents) at the start of treatment: 27 to 33 days old - age of F1 at treatment PND28
- Number of animals ordered: 108 males and 108 females; unrelated (males not related to females). Spare animals were removed from the study room after treatment commenced.
- Weight range of the F0 animals at the start of treatment: males 96 to 133g; females 74 to 103 g

HOUSING:
- Cages: cages comprised of a poly-carbonated body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used throughout the study except during pairing. Grid bottomed cages, were used during pairing. These were suspended above absorbent which was changed daily.
- Cage distribution: the cages were distributed on the racking to equalize, as far as possible, environmental influence amongts the groups.
- Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
- Number of animals per cage: up to 4 per cage except during pairing period (1 animal per cage)
- Diet (e.g. ad libitum): ad libitum - diet was removed overnight before blood sampling for haematology or blood chemistry and during the period of urine collection)
- Diet supply: SDS VRF 1 Certified powdered diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent -
- Water (e.g. ad libitum): ad libitum
- Water supply: potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals
- Acclimation period: five days before commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): monitored and maintained within the range of 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12 hours light; 12 hours dark

ENVIRONMENTAL ENRICHMENT:
- Aspen wood based products: a soft white untreated wood block provided to each cage throughout the study (except F0 females during lactation, for F1 cohort 2A animals when separated into single housing overnight prior to functional observational battery testing, and when F1 Cohort 1A animals were separated into single housing overnight during urine collection) and replaced when necessary. For F0 females, chew blocks were returned on Day 21 of lactation after weaning of offspring.
- Plastic shelter: Provided to each cage throughout the study except for F0 during pairing, F0 females during lactation, F1 cohort 2A animals when separated into single housing overnight prior to functional observational battery testing and when F1 cohort 1A animals were separated into single housing overnight during urine collection ) and replaced at the same time as the cage.
For F0 females, shelters were returned on Day 21 of lactation after weaning of offspring.
- Paper shavings: from day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material was changed at the same frequency as the cage bedding.

During the acclimatization and appropriate study periods environmental enrichment in the form of Aspen wood based products (soft white untreated wood product) and a plastic shelter will be available in each home cage; except when animals are separated into single housing overnight prior to functional observational battery testing and during lactation. From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings will be provided to each cage as nesting material; this nesting material will be changed at the same
frequency as the cage bedding.

Environmental control:
Animal facility : Limited access - to minimize entry of external biological and chemical agents.
Air supply : Filtered, not recirculated
Temperature: maintained within the range of 20-24°C
Relative humidity: Maintained within the range of 40

IN-LIFE DATES: From 17 February 2021 to 10 September 2021

Route of administration:

oral: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
1. Dietary formulation: dietary concentration were adjusted weekly during the premating phase for the F0 animals, for F0 males after pairing and the selected F1 animals from nominal Day 35 of age based on expected daily food intake and mid-week body weight, to ensure achieved intake approximately to the target dose levels; calculated using male and female data to provide a mated females during gestation received the dietary concentration used during the last week of the pre-mating period.
To maintain the target dose during lactation/post weaning, dietary concentrations for littered females were adjusted to accommodate the expected increase in food intake, consistent with
the concentration adjustments reported by Sprando, 2017 and cognizant of the recommendations in Beekhuijzen, 2016. During Days 1-14 of lactation females received 50%
of the gestation phase dietary concentration and then from Day 14 of lactation females and their litters received 32% of the gestation dietary concentration up to termination of the F0
females/F1 unselected offspring. Selected F1 animals continued on 32% of the gestation dietary concentration up to nominal Day 35 of age.

2. VEHICLE : Basal Diet SDS VRF1 Certified, powdered diet. A sample (100g) of each batch of diet used was retained within Pharmacy (frozen, -10 to -30°C) until finalisation of the report.

3. DIET PREPARATION
All formulations were incorporated into the diet to provide the required concentrations by initial preparation of a premix. The amount of test item required for premix was added to an equal amount of sieved diet and stirred. An amount of sieved diet equal to the weight of the mixture was added and the mixture was stirred again until visibly homogenous. The doubling up process was repeated until approximately half the premix diet was added. At this stage the mixture was ground with a mechanical grinder. The mixture was made up to the weight of the premix with coarse diet. The premix was then mixed using a turbula mixer for 200 cycles
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): the amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formations were dispenses.
- Storage temperature of food: storage of formulation at ambient temperature (15 to 25°C)
- Justification for use and choice of vehicle (if other than water):

4. FORMULATED DIETARY CONCENTRATION (ppm): please see section "Any other information on materials and methods incl. tables"
- Stability and homogeneity: homogeneity and stability of the test item in the diet matrix was determined. The test item was determined as stable for 22 days at ambient temperature (15 to 25°C).
- Achieved concentration: Samples of each formulation prepared for administration were analysis for achieved concentration of the test item.

Details on mating procedure:

- M/F ratio per cage: yes
- Length of cohabitation: 2 weeks (or up to mating)
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how):
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved concentration: Samples of each formulation prepared for administration were analysis for achieved concentration of the test item.

Duration of treatment / exposure:

F0 animals For ten weeks before pairing until termination after litters were weaned.
F1 animals From weaning@ until termination of respective cohort.

Unselected F1 offspring: Retention of organ weights - no direct treatment, killed on
Day 22 of age

Cohort 1A : Primary assessment of effects upon reproductive systems and of general toxicity - treated from weaning to approximately:
* Groups 1 to 3: 13 weeks of age (~Day 90)
* Group 4: 10 weeks of age (~Day 70)

Cohort 1B : Spare Cohort - treated from weaning to approximately:
* Groups 1 to 3: 14 weeks of age
* Group 4: 10 weeks of age
Cohort 2A : Developmental neurotoxicity testing (neurobehavioral testing followed by neurohistopathology assessment as adults) - treated from weaning up to approximately Day 75 of age.

Cohort 2B : Developmental neurotoxicity testing - no direct treatment, assigned to neurohistopathology assessment on Day 21/22 of age

Cohort 3: Developmental immunotoxicity testing - treatment from weaning up to approximately 8 weeks of age.

Frequency of treatment:

Continuously via diet - a record of the usage of the diets was maintained on all occasions when food consumption was measured. This was performed using the initial weight of the diet container and an
on-line data check on completion of the feeding procedure to ensure that all cages were fed the correct amount of diet. No significant discrepancy was found.

Details on study schedule:

Study initiation (Study Plan signed by Study Director) 12 February 2021
Experimental start date (Animal arrival) 17 February 2021
Treatment of F0 animals commenced 23 February 2021
F0 necropsy
* Males: 05 to 08 July 2021
* Females: 23 to 29 June 2021
F1 generation commenced 25 June 2021
F1 generation necropsy
Cohort 1A Group 4: 11 August 2021
Cohort 1A Group 1-3: 31 August 2021 to 03 September 2021
Cohort 1B Group 4: 11, 12 and 17 August 2021
Cohort 1B Group 1-3: 08 to 10 September 2021
Cohort 2A: 10 to 13 August 2021
Cohort 2B: 16 to 20 June 2021
Cohort 3: 30 July 2021
Experimental completion date (Pathology) 24 December 2021

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

80 mg/kg bw/day (nominal)

Dose / conc.:

120 mg/kg bw/day (nominal)

No. of animals per sex per dose:

For F0 generation 25 animals/sex/dose
For F1 generation: cohort 1A/1B: 20/sex/dose and Cohort 2A/2B and 3: 10 animals/sex/dose

Control animals:

yes, plain diet

Details on study design:

Selection of Offspring to Form F1 Generation:
- Selection: on day 18 to 20 of age
- Number per group for F1 generation: cohort 1A/1B: 20/sex/dose and Cohort 2A/2B and 3: 10 animals/sex/dose
- Allocation - formal start of F1 generation: nominally day 28 of age (direct dose administration commenced late lactation as offspring started to consume the diet)
- Method: The offspring with the lowest within-litter identification per sex from each selected litter were selected to form the F1 generation after exclusion of grossly atypical animals.
- For F1 Cohorts 1A and 1B where possible, one male and one female were selected from each selected litter.
- For Cohorts 2A, 2B and 3 where possible: one male or one female were selected from each selected litter. Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age. Formal commencement of the F1 generation was on a nominal Day 28 of age (where possible 28+/-2days of age for selected F1 animals).
Up to two male and two females F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortality. these spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.
Following selection of F1 offspring to establish the F1 generation and to fulfil the terminal investigations required for PND22 animals, some of the spare F1 offspring from the Control group were assigned to the internal sentinel program. These animals were subject to necropsy and screening following completion of the F1 terminal investigations.

Identification of animals: unique for each F0 animals and selected F1 offspring within study. All pre-weaning offspring were numbered individually within each litter on Day 1 of age.
Method: microchip (F0 generation and selected F1 generation) - Toe tattoo (pre-weaning offspring).

Rationale for Dose Level Selection:
Target dose levels of 40, 80, 120 mg/kg bw/day were selected in conjunction with the Sponsor following examination of the data from a preliminary reproductive performance study in the rat in which dose levels up to 80 mg/kg bw/day were well tolerated but at dose levels of 160 or 320 mg/kg bw/day insufficient offspring were available for evaluation.

Parental animals: Observations and examinations:

1. Clinical Observation - F0 and F1 Generation
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as
appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

2. Clinical signs:
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
Physical examination F0 males once each week:
* F0 females weekly until mating, Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.
* F1 animals after weaning once each week.
Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior.

3. Mortality - F0 and F1 Generation:
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

4. Body Weight - F0 and F1 Generation:
The weight of animals was recorded as follows:
- F0/F1 males: Day that treatment commenced - each week and before necropsy
- F0/F1 females: Day that treatment / F1 generation commenced - each week until mating detected or scheduled termination
- F0 females: Days 0, 6, 13 and 20 after mating

5. Food consumptions (F0 and F1 generation)
F0/F1 Animals: weekly up to pairing or termination
Food consumption will not be covered for males and females during the period when paired for mating.
F0 females: Day 0-5, 6-12 and 13-19 after mating - Day 1-3, 4-6, 7-13 and 14-20 of lactation

6. Water consumption (F0 and F1 generation)
F0/F1 Animals: qualitative visual assessment will be performed during the study. If effects are suspected quantitative measurement may be included for confirmation; any requirement will be included by study plan amendment (additional cost)

7. Organ weights: for bilateral organs, left and right organs were weighed together, unless specified in the relevant pathology procedures. Requisite organs were weighed for animals killed at scheduled intervals.
For Unscheduled F1 offspring on Day 22 of age, organs were weighed from 10 animals per sex per group from as many litters as possible.

Oestrous cyclicity (parental animals):

Dry smears: For 15 days before pairing, using cotton swabs.

Wet smears: Daily after pairing until evidence of mating confirmed, using pipette lavage. For four days before scheduled termination (nominally Days 25 to 28 post partum).

For females showing no evidence of mating, following completion of the pairing period the females will be separated from the male and vaginal smearing will continue for up to five days or until the first estrus smear is seen.
- If a female shows an estrus smear during this period, the female will be killed as soon as
practically possible and subject to macroscopic examination. If necropsy is not possible on
the day of the estrus smear, smears will continue until the morning of necropsy.

- If If a female does not show an estrus smear, wet smears will re-commence on Day 22 after
separation from pairing (where day of separation = Day 0) for a period of four days with the last
smear on the morning of necropsy (Day 25 after mating)

Sperm parameters (parental animals):

Immediately after scheduled sacrifice of each F0 and F1 Cohort 1A male and collection of blood, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.

Sperm motility all groups: A sample of sperm was expressed from the left vas deferens into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 μm depth cannula by capillary action and, at least 200 sperm per animal analyzed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyzer (CASA). The results for F0 Group 3 animal 65 were excluded from group mean values as it exhibited insufficient sperm to assess 200 at motility. F1 Cohort 1A Group 2 animal 434 and Group 4
animal 470 the vas deferens was taken from the right side. Group 3 animal 457 and Group 4 animal 542 exhibited insufficient sperm to assess 200 at motility. No sample was taken from Group 2 animal 430 on the discovery the animal was a hermaphrodite.

Sperm morphology all groups: A 200 µL aliquot of the sperm/medium mixture (described above) was diluted with 800 µL of 10% neutral buffered formalin. After staining with nigrosine and eosin an
air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male. F1 Cohort 1A Group 4 animal group 2 animal 433, group 3 animal 455 and group 4 animal 542 exhibited insufficient sperm to assess 200.

Homogenization-resistant spermatid counts all groups: After removal of the tunica, the left testis of each male then homogenized for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenization-resistant spermatid count using CASA. The results for F0 Group 1 Animal 24 were excluded from group mean values following the exclusion of the testis weight at necropsy. F1 Cohort 1A Group 4 animal 470 had tissue taken from the right side.

Litter observations:

- Clinical observations: observed approximately 24 hours after birth and then daily for evidence of ill-health or reaction to treatment which includes examination for the presence of milk in the stomach whilst this can be seen through the ventral abdomen and maternal care.
On Day 1 of age, all offspring receive a qualitative assessment of body temperature, presence of milk in stomach, state of activity and reaction to handling.

- Litter size: daily on Day1-21 of lactation. Litters culled to 10 (where possible 5 males and 5 females) on Day 4 of age. All culled offspring macroscopically examined.

- Sex ratio: Day 1, 4 (before and after culling) and 21 of age.

- Individual offspring body weights: all offspring: Day 1, 4, 7, 14 and 21 of age. unselected F1: Day 22 of age

- Weaning of offspring: Day 21 of age

- Ano-genital distance: offspring on Day 1 of age, measured using digital calipers.

- Nipple count: male offspring Day 13 of age

SEXUAL MATURATION
- Males: examined daily from Day 38 of age for the completion of balano-preputial separation. Body weight recorded on day of completion of separation.

- Females: examined daily from Day 28 age until vaginal opening occurs. body weight recorded on day of vaginal opening.

Postmortem examinations (parental animals):

SACRIFICE
Male animals: All surviving animals sacrificed after at least 18 weeks of treatment and after weaning of the F1 generation, after confirmation that no further mating required.
Maternal animals:
- F0 female with viable litters - Day 28 post partum
- F0 female failing to mate, if an estrus smear was seen following completion of the pairing period animals were terminated as soon as logistically possible.
- F0 females failing to produce a viable litter: on or soon after Day 25 after mating
- F0 females with total litter loss: on or soon after the day that the last offspring dies

HISTOPATHOLOGY / ORGAN WEIGHTS
See section "Any other information on materials and methods incl. tables"

Postmortem examinations (offspring):

Sperm analysis - F0 and F1 (Cohort 1A):
- Motility : the percentages of motile and progressively motile sperm and sperm motion parameters were reported.
- Morphology: the number and percentages of normal and abnormal sperm were reported, including regions and specific types of abnormalities were reported.
- Count (testis and cauda epididymis): the sperm concentration (million/g) and total number were reported.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these
tests, whether significant or non-significant, are presented on the relevant tables. The similarity of the data was such that analyses were not considered to be necessary. All statistical analyses were carried out separately for males and females.

Please see section "Any other information on materials and methods"

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs at routine physical examination for males during treatment or for female before pairing and during gestation/lactation that could attributed to administration of test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1 male mid-dose found dead: no significant finding - factor of death undetermined
2 males high dose found dead: no significant finding - factor of death undetermined
1 female high dose found dead: significant finding - but cause of death concluded no test-item related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain for males receiving mid and high dose was low when compared with Controls, with the absolute mean weight significantly reduced at 92 and 93% of Control values at the end of the treatment period (p<0.05) ; there was no apparent dose response.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Before pairing for mating food consumption was reduced to up to 6% for males at all dose levels during Week 4 and for males at mid and high dose during Week 5. After pairing males at high dose showed food consumption reduced by up to 9% during week 12 to 16 and week 19.
Females at mid and high dose showed consumption up to 5% high than Controls during week 1 of treatment with the difference attaining statistical significant at high dose, only and 11% lower consumption at high dose during week 4 of treatment only. Females receiving high dose also showed food consumption up to 7% lower than Controls during lactation period.
Food consumption for females during gestation at all dose levels and during lactation at low and mid dose was unaffected by administration of test item.

Food efficiency:

no effects observed

Description (incidence and severity):

Efficiency of food utilization during the 10 weeks treatment period before pairing for both males and females and for females during gestation were unaffected by administration of test item.

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

During Week 10 F0 females receiving Silver Acetate showed:
* High haematocrit at all dose levels (p<0.01)
* High erythrocyte count at all dose levels(p<0.01)
* Low mean cell haemoglobin at all dose levels (p<0.01)
* Low mean cell haemoglobin concentration (not significant at 40 mg/kg/day; p<0.05 at
80 mg/kg/day; p<0.01 at 120 mg/kg/day)
* Low mean cell volume (p<0.05 at 40 mg/kg/day; p<0.01 at 80 or 120 mg/kg/day)
* Low platelet count at all dose levels(p<0.01)
* Shorter mean prothrombin clotting time at 120 mg/kg/day (p<0.01)
All other parameters measured showed no effect of treatment.

At schedule termination
* Eryrthocyte count was high for males at 80 and 120 mg/kg/day and for females at all
dose levels (p<0.01).
* Mean cell haemoglobin and mean cell volume were low for males at all dose levels
(p<0.05 at 40 mg/kg/day; p<0.01 at 80 and 120 mg/kg/day) and for females that
received 80 or 120 mg/kg/day (p<0.01).
* Red cell distribution width was high for both males and females at all dose levels
(p<0.05 for males at 40 mg/kg/day, p<0.01 for males at 80 or 120 mg/kg/day and
females at all dose levels).
* Mean cell haemoglobin concentration was low for males that received 80 or
120 mg/kg/day(p<0.01); this difference was not evident in females.
* Mean Haematocrit was low for males at 120 mg/kg/day (p<0.01); conversely females
at all dose levels had high haematocrit values (p<0.01).
* Haemoglobin concentration was low for males at 80 or 120 mg/kg/day (p<0.01) but
was high for females at all dose levels (p<0.05).
* Mean platelet count for males at 120 mg/kg/day was high (p<0.01), whilst females at
all dose levels showed low platelet counts (p<0.05).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Week 10 (F0 females only) - all low, mid and high doses
* High alanine phosphatase activity (p<0.01)
* High plasma cholesterol levels (p<0.01), dose-proportionate
* Low plasma potassium levels (p<0.01), not dose-proportionate.
In addition, females at 120 mg/kg/day had slightly high Aspartate amino-transferase activity
(p<0.01) and high plasma sodium levels (p<0.01). All other parameters measured showed no
effect of treatment.

Both males and females that received test item at all dose levels showed
* High mean plasma cholesterol levels (p<0.01) that were proportionate to dose in females but not in males.
* at high dose alkaline phosphatase activity was high for males (p<0.05) and gamma
glutamyl transpeptidase was high for females (p<0.01).
* Males showed low creatinine levels at all dose levels (p<0.05) but the reduction was not
dose-proportionate.
* Potassium levels were low for males at mid and high dose and for females at
120 mg/kg/day (p<0.05). Males at 120 mg/kg/day also showed low phosphorous (p<0.05).

All other parameters measured showed no effect of treatment.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in T4 serum concentration levels in any group or generation of males or females after dietary administration of test item at 40, 80 and 120 mg/kg bw/day when compared with controls.
There were no statistically significant in TSH serum concentration levels in any group or generation of males and females after dietary administration of test item at 40, 80 and 120 mg/kg bw/day when compared with controls.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in various organs/tissues including the brain, spleen, gastrointestinal tract, kidneys, urinary bladder, liver, pancreas, thymus, thyroids, parathyroid,
esenteric lymph node, mandibular salivary gland, and the harderian, lacrimal and preputial/clitoral glands. Most commonly, extracellular pigment was observed in various organs/tissues and was considered to represent deposition of test item at these sites. In general, pigment was more prominent in females, and there was not always an apparent dose response.
In the brain, extracellular pigment was observed focally in the area postrema and/or subfornical organ in both sexes given highest dose. These areas comprise parts of the circumventricular organ system in the brain and have in common a lack of the normal blood brain barrier, which accounts for the focal deposition of test-item in these areas.
In the gastrointestinal tract, extracellular pigment was observed in the lamina propria of the
duodenum, jejunum, ileum, cecum, colon, and rectum in both sexes given low, mid or high dose, and in the stomach in males given low, mid and high dose and in females given mid and high dose ay. This finding in the gastrointestinal tract correlated with the abnormal colour observed
macroscopically. In addition, dose related epithelial degeneration of
the glandular mucosa in the stomach was observed in females given low, mid and high dose.
In the kidneys, extracellular pigment was observed in males given high dose and in females given low, mid and high dose. In the liver, extracellular pigment was observed in portal areas in males given 80 or 120 mg/kg/day and in females given low, mid and high dose. Findings in these organs correlated with abnormally colored kidneys and livers observed macroscopically.

Extracellular pigment was also observed in the urinary bladder, pancreas, thymus (capsule),
thyroids, mandibular salivary gland, and the harderian, lacrimal and preputial/clitoral glands
in both sexes given low, mid and high doseay, and in the parathyroids in both sexes given high dose. Findings in these tissues/organs correlated with the abnormal colour observed macroscopically.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the mesenteric lymph node, increased pigmented macrophages were observed in both
sexes given low, mid and high dose , and in the liver, pigmented Kupffer cells were observed
in males given high dose and in females given mid and high dose. Pigment in these
histiocytic cells (macrophages and Kupffer cells) was considered to represent phagocytosis of
the test item. These findings correlated with abnormally coloured mesenteric lymph nodes and
liver observed macroscopically.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles and pre-coital interval were unaffected by treatment.
The majority of females in all groups showed an estrus smear prior to scheduled termination
confirming that estrous cycles had resumed after parturition.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

At mid and high dose there was a statistically significant decrease in testis weight when compared with Controls and outside of the HCD range. At low dose and high dose spermatid total count was low when compared with controls, but statistical significance was only attained at high dose; there was no dose-relationship as mean total spermatid count at mid dose was similar to control and all groups including controls, were below the HCD range.
At high dose there was a decrease in cauda epididymis sperm count (millions/g) and total million compared with concurrent Controls but the difference was not statistically significant and intra group variability was very high.
Morphologically, at high dose there was a slight increase in mean total abnormal sperm, primarily decapitate, with an associated decrease in normal sperm. This was attributed to the high number of decapitate sperm recorded for animal 83 and as it was confined to one animal, could not conclusively be associated with treatment.
The testes revealed normal progression of the spermatogenic cycle, and the expected cell
associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance and fertility were unaffected by the treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: mean achieved doses of 113 mg/kg/day for males and 127 mg/kg/day for females.

Key result

Dose descriptor:

LOAEL

Effect level:

< 40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

gross pathology

Remarks on result:

other: Degeneration in stomach mucosa in females all doses

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males at high dose level showed a higher incidence of piloerection, hunched posture and abnormal gait; these signs were also observed in a few females at high dose but at a low incidence.
The general condition of animals at low and mid dose was similar to the Controls.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Offspring (PND1 to PND21/22):
At highest dose was an increased incidence of offspring mortality and dark coloration. at highest dose the live birth and viability indices were low when compared with controls., resulting in a mean live litter size on day 1 of 13 compared to 14.5 in controls. No differences were apparent at low and mid dose.
Following litter standardization on Day 4 of age offspring survival at all dose levels was similar to Controls.
The number of implantation sites and mean litter size on PND1 were unaffected by treatment.

F1 Generation (post weaning):
treatment at high dose was associated with 9 male and 2 female mortalities.
Histopathology of those animals were examined:
Male low dose- microscopic findings included slight increased extramedullary hematopoiesis in the spleen. There were no other significant macroscopic or microscopic findings in this male and the major factor contributing to death was undetermined.
Male mid-dose was found dead on Day 28 of treatment. There were no significant macroscopic or microscopic findings in the tissues examined and the major factor contributing to death was undetermined.
Male high dose: Microscopic findings included slight increased extramedullary hematopoiesis in the spleen and slight inflammatory cell infiltrate in the heart. There were no other significant macroscopic or microscopic findings in this male and the major factor contributing to death was undetermined.
Male high dose: significant findings in the brain included minimal intramyelinic edema in the thalamus, minimal neuronal necrosis in the hippocampus and slight neuronal/glial cell necrosis in the thalamus. Other microscopic findings included minimal pigment in the lamina propria of the duodenal mucosa and minimal inflammatory cell infiltrate in the heart. The brain lesions were considered the major factor contributing to poor clinical condition and early termination of this animal.
Male high dose: significant findings in the brain included minimal intramyelinic edema in the thalamus, minimal neuronal necrosis in the hippocampus and minimal neuronal/glial cell necrosis in the thalamus. The brain lesions were considered the major factor contributing to death in this animal.
Male high dose: there were no significant macroscopic or microscopic findings in the tissues examined and the major factor contributing to death was undetermined.
Male high dose: there were no significant macroscopic or microscopic findings in the tissues examined and the major factor contributing to death was undetermined.
Male high dose: macroscopic findings were limited to a distended urinary bladder, which correlated with slight dilatation of the bladder microscopically. The major factor contributing to death was undetermined in this animal.
Male high dose: significant microscopic findings included minimal or slight intramyelinic edema of the thalamus and caudate putamen and minimal neuronal and/or glial cell necrosis in the thalamus and hippocampus. The brain lesions were considered the major factor contributing to the early termination of this animal.
2 males high dose were not examined microscopically.

1 female high dose: significant findings in the brain included minimal neuronal necrosis in the hippocampus and minimal neuronal/glial cell necrosis in the thalamus. The brain lesions were considered the major factor contributing to death in this animal.
1female high dose: microscopic findings included minimal neuronal necrosis in the hippocampus. The major factor contributing to death of this female was undetermined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

On Day 1 of age, the mean body weight for both male and female offspring of high dose was low when compared with Controls and this difference persisted up to weaning on PND21. The body weight gain for offspring at high dose was similar to control from PND1 to PND4, however from PND4 up to weaning the offspring body weight gain for both males and females was low when compared with Controls.
At low and mid dose mean offspring body weight on PND1 were similar to Controls. At low dose subsequent body weight gain was similar to controls, however, at mid-dose offspring showed low weight gain from PND7 to PND21, resulting in significantly low mean body weight on PND14 and PND21.

At weaning (PND21) selected males and females receiving mid and high dose had low mean bodyweight when compared to Controls; subsequent bodyweight gain up to PND25 was low at all target dose levels for males and females with no clear dose response.
From Day 1 of the F1 generation at nominal PND28±2 up to termination mean body weight and mean body weight gain was low for males at all target dose levels; a dose response was apparent.
During Weeks 1-2 of the F1 generation treated females showed low body weight gain, but during Weeks 2-5 weight gain was high when compared with Controls and from Week 5 body weight gain was similar to Controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males receiving test item at all dose levels showed statistically significantly low food consumption up to termination and in general there was evidence for a dose response.
During Weeks 1 and 2 of the F1 generation the mean food consumption for females receiving
test item was low when compared with Controls , with no evidence of a dose response. During Week 3 food consumption for females receiving high dose was high when compared with Controls and females at mid and high dose also showed high consumption during Weeks 5 and 6 (p<0.05). Thereafter consumption up to termination was similar to Controls.

Food efficiency:

no effects observed

Description (incidence and severity):

Overall, the efficiency of food utilization was unaffected by treatment for both males and females.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The statistical analysis of high dose data are not scientifically valid as the data was not contemporaneous with the Controls; data interpretation is limited.

A higher than control red blood cell count in both sexes were observed receiving mid dose and for females at low dose; this difference was not evident at high dose with both males and females at
his target dose level showing slightly lower red blood cell counts.

Mean haemoglobin and haematocrit for females at mid dose were high when compared
with Control; this was not evident for males at this dose level. Males at mid dose had
low mean cell haemoglobin and mean cell volume, with high mean red cell distribution width when compared with Controls.
At high dose males and females showed higher mean cell haemoglobin and mean cell haemoglobin concentration. In addition, females at high dose showed a low haematocrit and red cell distribution width and a higher mean cell volume.

Neutrophil, lymphocyte, eosinophil, basophil, monocyte and large unstained cell counts, and consequently total white blood cell count, in all treated groups of females were high when compared with Control values. These changes generally showed treatment relationship and statistical significance.
A lower than Control statistically significant platelet count was observed in females that received mid dose or high dose. A shorter prothrombin time was also seen in all treated groups of females and activated partial thromboplastin time in females that received high dose.

All other difference from control were minor, confined to one sex or lacked dose relationship, and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The statistical analysis of high dose data are not scientifically valid as the data was not contemporaneous with the Controls; data interpretation is limited.

Blood chemistry investigations of the F1 generation revealed higher than Control statistically significant alkaline phosphatase activity in all treated groups of males and females at high dose and alanine amino-transferase activity in males receiving mid dose and both sexes at 120 mg/kg/day. Aspartate amino-transferase activity was also higher than the Controls in females that received high dose.
Higher urea concentration was noted in males only at high dose in comparison with the Controls.
Higher than control statistically significant cholesterol concentration was observed in all treated groups of males and females; a dose response was apparent for the female animals.
Glucose concentration in both sexes at high dose was high when compared with the Controls.
Plasma creatinine levels were statistically significantly low when compared with Controls at
all target dose levels, but there was no evidence of a dose response.
Lower than control statistically significant albumin concentration and a lower A:G ratio was
observed in both sexes that received high dose; males at high dose also showed a higher mean globulin level.
Potassium concentration was higher than the control for both sexes at high dose and phosphorus was lower than the controls in both sexes that received high dose and females at mid dose. Sodium levels females at high dose were low when compared with Controls.
All other difference from control were minor, confined to one sex or lacked dose relationship, and were therefore attributed to normal biological variation.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis assessment was limited to Control, low and mid dose animals; assessment of high
dose animals was not performed for welfare reasons (withdrawal of food and water during urine collection was not considered appropriate).
Urinalysis investigations revealed a lower than control statistically significant total protein output and protein concentration in males at mid dose.
All other difference from control were minor, confined to one sex or lacked dose relationship,
and/or were not associated with changes in plasma and were therefore attributed to normal
biological variation.

Sexual maturation:

no effects observed

Description (incidence and severity):

The mean age at completion of balano preputial separation was essentially similar across the
groups but the mean bodyweight at completion was statistically significantly lower than
Controls at all dose levels.
The mean age at completion of vaginal opening for females receiving high dose was 1.2 days later than Controls and the mean bodyweight at completion was statistically significantly lower than Controls at all dose levels; the delay was considered slight and was considered to relate to the low bodyweight rather than a direct effect of treatment.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Ano-genital distance for both male and female animals offsrping on Day 1 of age was unaffected by administration of test item at all dose levels.

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

One male offspring from Group 2 litter at low dose was observed with nipples on day 13 of age; in the absence of any similar findings in the other males in the litter or in the intermediate and high dose groups this isolated incidence is considered not to be related to treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1. Offspring: At mid and high dose male and female offspring show low mean thymus weights with female offspring also showing low mean absolute brain weight; male at high dose showed low mean absolute brain weight.
Body weight relative thymus weights were low for both male and female offspring at mid and high dose and for female at low dose.
Body weight relative brain for female offspring at high dose was high when compared with Controls but this difference in brain weight are attributed to the body weight affected observed at mid and high dose.

2. F1 Generation:
The statistical analysis of high dose group organ weights was not scientifically valid as the data was not contemporaneous with the Controls; data interpretation is limited.
Treatment related organ weight changes were noted in the heart in both sexes, the brain in
males given high dose, and the thymus in both sexes given high dose.
Higher than control mean body weight relative heart weights were noted in both sexes given
mid and high dose. Higher than control mean body weight relative brain weight was observed in males given high dose. This change may be affected by the significantly lower than control mean
terminal body weight of this group, however, due to potential correlative microscopic findings (intramyelinic edema and neuronal/glial cell necrosis), a treatment related effect cannot be excluded. Higher than control mean absolute and body weight relative thymus weights were noted in both sexes given high dose. This change may be affected by the difference in age at
termination compared to controls, however, a treatment related effect cannot be excluded.
Differences in the organ weight parameters in the testes, epididymides, prostate and seminal
vesicles with coagulating gland in males given mid and high dose were considered to be affected by the lower than control mean terminal body weight in these groups and were not considered a direct effect of the test item.

All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small. This included the following statistically significant differences: lower than control mean absolute and/or body weight relative adrenal weights in both sexes given low, mid and high dose levels; lower than control mean absolute kidney weights in both sexes given low, mid and high dose levels, lower than control mean body weight relative kidney weights in females given low, mid and high dose levels, and higher than control mean body weight relative kidney weight in males given high dose; lower than control mean absolute pituitary weights in both sexes given low, mid and high dose levels, and lower than control mean body weight relative pituitary weight in females given high dose; higher than control mean absolute and body weight relative spleen and spleen immuno weights for females given high dose; lower than control thyroids and parathyroid weights in females given all dose levels and in males
given high dose; higher than control mean absolute and body weight relative mesenteric lymph node weight in females given high dose; lower than control mean absolute left axillary lymph node weight in males given high dose; and lower than control mean absolute uterus and cervix and oviduct weight in females given high dose.

3. Cohort 1B:
The statistical analysis of high dose group organ weight was not scientifically valid as the data was not contemporaneous with the Controls; data interpretation is limited.
Treatment related organ weight changes were noted in the heart in males.
High than control mean body weight relative heart weights were noted in males given, low, mid and high dose.
Organ weight differences noted in the prostates and seminal vesicles with coagulating gland in males given low, mid and high dose, in the testes and epididymides of males given mid and high dose were considered to be affected by the lower than controls mean terminal body weights of these groups and were not considered a direct effect of the test item.
All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small. This included the following statistically significant differences: lower than control mean absolute pituitary weights in males given low, mid and high dose mg/kg/day, and lower than control mean absolute and body weight relative pituitary weights in females given high dose; and lower than control mean absolute and body weight relative uterus and cervix and oviducts weights in females given high dose..

4. Cohort 2A:
A treatment related organ weight change was noted in the brain in males. Statistically significant higher than control mean body weight relative brain weights were noted in males given high dose.
All other differences in brain weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.

5. Cohort 2B
No organ weight changes were considered treatment related.
All other differences in brain weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.

6. Cohort 3:
A treatment related organ weight change was noted in the spleen in both sexes.
Higher than control mean absolute and body weight relative spleen weights were noted in both sexes given high dose, which attained statistical significance for body weight relative values.
All other differences in spleen weights parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.

Gross pathological findings:

no effects observed

Description (incidence and severity):

1. Gross pathology of offspring that died prior scheduled termination or culled on Day 4 of age were predominately the absence of milk in the stomach and/or autolysis.
At scheduled termination on Day 22 of age gross pathology was restricted to hair loss for 2 females at mid dose.

2. F1 Generation Cohort 1A:
Treatment-related macroscopic findings were generally observed in both sexes across all dose groups without a significant dose response and were generally limited to abnormal coloration (dark) of affected organs/tissues. This was generally observed at a lower incidence and in fewer tissues compared to F0 generation animals. Abnormal colour and/or abnormal contents in the gastrointestinal tract were observed in both sexes given low, mid or high dose. Abnormal colour of the urinary bladder and pancreas was observed in both sexes given low, mid or high dose.
abnormal colour of the mesenteric lymph node in some males and/or females given low, mid or high dose, and of the thymus in some females given mid dose was also observed.
All other macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Sprague Dawley rats of this age. Therefore, they were considered not test item related.

3. F1 Generation - Cohort 1B:
Treatment-related macroscopic findings were generally observed in both sexes across all dose groups without a significant dose response and were generally limited to abnormal coloration (dark) of affected organs/tissues. This was generally observed at a lower incidence and in fewer tissues compared to F0 generation animals, with a similar incidence and tissue distribution compared to F1 generation Cohort 1A animals. Abnormal colour and/or abnormal contents in the gastrointestinal tract were observed in both sexes given low, mid and high dose. Abnormal colour of the urinary bladder and pancreas was observed in both sexes given low, mid and high dose.
Abnormal colour of the mesenteric lymph node was also observed in some animals of both sexes given low and mid dose. All other macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls) and/or were as expected for Sprague Dawley rats of this age. Therefore, they were considered not test item related.

4. F1 Generation Cohort 2A:
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Sprague Dawley rats of this age. Therefore, they were considered not test item related.

5. F1 Generation - Cohort 2B:
No macroscopic findings were observed in F1 generation Cohort 2B animals

6. F1 Generation - Cohort 3:
Treatment-related macroscopic findings were generally limited to abnormal coloration (dark) of affected organs/tissues. This was observed at a lower incidence and in fewer tissues compared to F0 generation animals and F1 generation Cohort 1A and Cohort 1B animals. Abnormal colour and/or abnormal contents in the gastrointestinal tract were observed in a low number of males and females given high dose. Abnormal colour of the urinary bladder was observed in males given 80 or 120 mg/kg/day and in females given low, mid and high dose. Abnormal colour of the
pancreas was observed in a low number of females given high dose. Abnormal colour of the preputial/clitoral glands was observed in a low number of males and females given high dose.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

1. F1 Generation - Cohort 1A
Treatment related findings were observed in the brain, as well as the gastrointestinal tract, kidneys, urinary bladder, liver, pancreas, mesenteric lymph node and thymus.
In the brain, dose related neuronal necrosis in the hippocampus, and neuronal/glial cell necrosis in the thalamus was observed in both sexes given mid and high dose. Intramyelinic edema in the thalamus, caudate putamen and/or the corpus callosum was also observed in males given mid and high dose and in females given high dose. These findings may be correlated with the higher than control mean body weight relative brain weight observed in males given high dose. In addition, similar to findings in the F0 generation, extracellular pigment was observed focally in the area postrema in both sexes given low, mid and high dose.
In the gastrointestinal tract, extracellular pigment was observed in the lamina propria of the duodenum, jejunum, ileum, and colon in both sexes given low, mid or high dose, in the cecum in males given mid or high dose and in females given low, mid or high dose, and in the rectum in females given mid or high dose.
Extracellular pigment in the gastrointestinal tract correlated with the abnormal colour observed macroscopically. In the kidneys, extracellular pigment was observed in both sexes given low, mid or high dose. In the liver, extracellular pigment was observed in portal areas in a low number of males and females given high dose and in one male given low dose.
Extracellular pigment was also observed in the urinary bladder and pancreas in both sexes given low, mid or high dose. Findings in these tissues/organs correlated with the abnormal colour observed macroscopically.
Increased pigmented macrophages were observed in the mesenteric lymph node in a low number of males given mid dose and in females given low dose. Extracellular pigment was also observed in the capsule of the thymus in a low number of females given mid dose. Findings in these
tissues/organs correlated with the abnormal colour observed macroscopically.
Extracellular pigment observed in F1 generation Cohort 1A animals had a similar distribution and appearance to that seen in F0 generation animals and, in general, was observed at a lower incidence and severity, and in fewer tissues, compared to F0 generation animals, and similar to F0 generation animals, was slightly more prominent in females. Similar to the F0 generation, pigment in these organs/tissues was not associated with any inflammatory or degenerative changes. All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence , were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Sprague Dawley rats of this age. Therefore, they were considered not test item related. No findings were observed in the heart of animals in Control and high dose groups that were stained by immunohistochemistry for von Willebrand factor (VWF) and Collagen IV.

2. F1 Generation - Cohort 2A:
Treatment related were observed in the brain.
In the brain similar to F1 generation Cohort 1A animals, several findings were observed in both sexes given high dose. these findings included intramyelinic edema in the thalamus and caudate putamen, neuronal necrosis in the hippocampus, and neuronal/glial cell necrosis in the thalamus. this may be correlated with the higher than controls mean body weight relative brain weight observed in males given high dose. Neuronal necrosis in the hippocampus was also observed in both sexes given mid dose. In addition, similar to F0 generation and F1 generation Cohort 1A animals, extracellular pigment was observed focally in the area postrema in males given low, mid or high dose and in females given low or mid dose. Pigment in this area was not associated with any inflammatory or degenerative changes. All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was expected for Sprague Dawley rats of this age. Therefore, they were considered not test item related.

3. F1 Generation - Cohort 2B:
Treatment related findings were observed in the brain.
In the brain , similar to F0 generation and F1 generation Cohort 1A and 2A animals, extracellular pigment was observed focally in the area postrema in a low number of males and females given high dose of test item. Pigment in this area was not associated with any inflammatory or degenerative changes.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

1. Sex ratio unaffected by administration of test item at all dose levels.

2. Vaginal Opening: the interval between vaginal opening and the first oestrus was unaffected by administration of test item.

3. Oestrous cycles: the high dose group was terminated prematurely for welfare reasons at approximately 10 weeks of age and oestrous cycle assessment from nominal PND75 for Control, low and mid dose females showed no adverse effects of treatment.

4. Stage of oestrous Cycles at termination: the majority of females showed oestrus before termination, with the exception of females receiving high dose with 83% exhibiting an oestrus smear; terminal smears were not assessed for females receiving high dose because of their premature termination.

5. Ovarian Follicle Counts and Corpora Lutea:
Mean ovarian follicle and corpora lutea counts at high dose were similar to Controls and considered unaffected by administration of the test item.

6. Sperm Assessment:
Owing to the early termination of high dose, only 12 animals were examined for sperm count, motility and morphology. These animals were also approximately 10 weeks old, compared to 13-14 weeks for Controls, low and mid dose, so data are not directly comparable.
Across all treatment groups there was a notable decrease in several motion parameters, with VCL, ALH and BCF exhibiting a statistically significant decrease at high dose when compared with concurrent Control and outside of HCD range. However, there was also a statistically significant increase in progressively motile sperm, STR and LIN when compared with concurrent Control. Overall, no relationship between treatment and the mix of decreased and increased motion parameters is inferred, importantly there were increases in progressive motile sperm, STR and LIN. At mid and high dose level, there was a statistically significant decrease in cauda epididymis,
testicular weight and at all dose levels testicular and cauda epididymal total counts millions were low when compared with concurrent Control.
At low dose, testes spermatid count (millions/g) were low when compared with Controls (p<0.01); the mean value was within HCD range and this difference was not observed at mid and high dose levels.
Morphologically at high dose there was an increase in total abnormal sperm and decapitates (with an associated decrease in normal sperm) when compared to concurrent control and outside of HCD range, but this was not statistically significant, and was primarily attributed to one animal (475) with a 29% incidence of decapitate sperm.
Morphologically at mid dose there was a non-statistically significant increase in total abnormal sperm % with an associated decrease in normal sperm % compared with concurrent Control, however this was attributed to animal no. 457 exhibiting atypical results. (ref group
mean tables with 457 exclusion for comparison).

7. Cardiac Troponin:
The statistical analysis of high dose data are not scientifically valid as the data was not contemporaneous with the Controls; data interpretation is limited.
Troponin investigations conducted on the F1 generation revealed higher levels in treated males; individual differences also showed a high degree of intra group variability and mean levels were greater at low and mid dose . Mean levels at high dose were slightly high compared with Controls but much lower than the mean values at low and mid dose.
Cardiac troponin levels were unaffected in females at all dose levels.

.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

1. Auditory Startle Response Habituation
Group mean auditory startle latency to peak values for all treated males were generally similar to Controls for each block of 10 trials, with the exception of males at high dose during trials 21-30 which was marginally high when compared with Controls. This difference attained statistical
significance, however, values throughout the rest of the trials and the overall value for all 50 trials were generally similar to the Control group. The majority of latency to peak values for all groups of treated females were low compared with Controls and statistical significance was achieved for females at high dose during trials 31-40; however, when compared with HCD, values were in range or slightly above.
The majority of group mean auditory startle peak amplitude values for males at all dose levels were low when compared with Controls with males at high dose showing the least habituation with a value of -4.6. However, all but one value was within the HCD range.
Most peak amplitude values for females at high dose were low, showing the lowest habituation of -3.3.
Neither male or female habituation values attained statistical significance, however, the
values for females at low and high dose were below the HCD range values at mid dose were within the HCD range.
In conclusion, there was a suggestion of an effect on startle latency to peak response and
habituation at high dose level.

2. Motor Activity
The majority of group mean high (rearing) and low (ambulatory) beam activity scores for all groups of treated males were low when compared with Controls with statistical significance was achieved at many of the 6-minute interval, and total high and low beam scores for males in at mid and high dose also achieved statistical significance. Most high and low beam scores for males at high dose and some mid dose, for the first 24-minute s were below the HCD minimum. The first half of the 1-hour recording period is when the majority of exploratory behaviour occurs indicating that activity levels were low from the start of assessment. A dose-relationship was also evident although only the high beam total score fell below the HCD minimum. The low beam total score
as with the HCD range. The majority of high and low beam scores for females at high dose and to a lesser extent, at mid dose were also low when compared with Controls, again with statistical
significances attained at many of the 6-minute intervals and the total score (high beam at high dose only). Again, many of the 6-minute interval scores and the total scores for females at mid and high dose were below the HCD minimum and these continued throughout the duration of the 1-hour recording period. All total high and low beam scores for males and females at low dose were within the HCD range so no effect of treatment was inferred.

3. In Cage Observations:
During the in cage observations two males at mid dose and two males at high dose showed abnormal motor movements which included chewing mouth movements and licking around mouth and two females at 120 mg/kg/day were observed with their eyelids completely closed; ne of these females was recorded as being asleep but the other was recorded as being awake which is not normally an observation when the eyelids are completely closed.

4. In Hand Observations:
During the in hand assessment, there were 3 males at high dose that were slightly awkward to handle compare to non in the Control group.

5. Arena Observations:
During the arena assessment there were a number of findings predominately in males and females at high dose and a few animals at mid dose.
- Palpebral closure was observed to varying degrees in three out of nine males and in one female at high dose.
- An elevated gait was noted in all groups including the Controls but there were other gait observations noted in the treated groups that were not seen in the Control group such as flattened, staggering or unsteady.
- Tremor was seen in both males and females at high dose although it was also noted in one male in the Control group.
- Abnormal motor movements were observed in males and females at mid or high dose such as licking around mouth and chewing mouth movements although there was also one male in the Control group who also showed licking around mouth.
- Both activity and rearing counts were low in all treated males and females in all treated groups with statistical significance achieved for males at mid and high dose and females at all dose levels. All activity and rearing count values for males in all treated groups were below the HCD minimum as were the activity count values for females in all treated groups; however activity counts in control females were above the HCD range. the only rearing count value that was below the HCD minimum was in the females at high dose; but in general, these low levels of activity mimic what was seen in the automated activity system although the activity and rearing counts in the arena suggest effects at mid and high dose. At low dose, no statistically significant effects on activity or rearing counts were seen in males, the rearing count in females was within the HCD range and the control rearing and activity counts were higher than in the HCD range, therefore, as there was no evidence of an effect on activity on the automated recording system, no effect of treatment was inferred in the arena.

6. Reactivity Investigations:
During the reactivity investigation, 4 males at high dose showed a reduced approach response, showing no reaction the probe, compared to one in the Control group and 2 females showed a weak response to the startle reflect, compared to one in the Control group.
3 females in each of the groups receiving mid and high dose failed the pupil closure reflex response with pupils that failed to dilate; 2 males at high dose also failed pupil closure however 2 Control males also failed.
1 male and 1 female at high dose showed a slow or partial response when assessed for proprioception.
Group mean body temperature was very slightly low in all groups of treated females when compared with Controls; with the differences attaining statistical significance. however, when compared to HCD, all values were within the HCD range and the value for Controls was slightly above the HCD range.
Group mean bodyweights for males at all dose levels and females at high dose were low when compared with Controls; with values for males at mid and high dose attaining statistical significance. These values were also below the HCD range.
Group landing footsplay values for males at mid and high dose were high when compared with Controls with the value for males at high dose attaining statistical significance; both values were above HCD range. An increase in landing footsplay is a classic sign of neurotoxicity. the same pattern in landing footsplay was not seen in the females; although the value for female mid dose was high compared with Controls, the value for females at high dose was low when compared with Controls.
Group mean forelimb grip strength values for males and females high dose group were low when compared with Controls with the value for females attaining statistical significance. in addition, hindlimb grip strength values for all treated males and females were low when compared with Controls with the values for males at high and mid dose and for females at high dose attaining statistical significance. All values were within the HCD range or slightly above, however, the low value at high dose are considered to be treatment related.

7. Brain morphometry:
7.1. F1 Generation - Cohort 2A:
A statistically significant lower than control mean hippocampus measurement was noted in males given mid and high dose. As this finding was observed in a single sex, the relevance is unclear, however, a treatment related effect cannot be excluded. All other differences in brain morphometry parameters were considered with normal variation and considered incidental.

7.2. F1 Generation - Cohort 2B:
All differences in brain morphometry parameters were consistent with normal variation and considered incidental.

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

Overall modest effects were observed on the immunophenotyping parameters measured in Sprague Dawley rat spleen leukocytes as a result of the dietary administration of test item, however a statistically significant increase in the percentage of B cells in females was observed in animals receiving low, mid and high dose when compared with Controls.
This immunophenotyping difference may have been related to the dietary administration of test item however, the increase in the percentage of B cells in females did not translate into an increase in IgM production as no significant differences were found between the Control Group and the treated groups in the anti-keyhole limpet hemocyanin (KLH) IgM analyses (TDAR).

Dose descriptor:

NOAEL

Generation:

F1 (cohort 2B)

Effect level:

>= 120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental immunotoxicity

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

<= 80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 2A)

Effect level:

<= 40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

neuropathology

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

80 mg/kg bw/day (nominal)

System:

central nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Reproductive effects observed:

yes

Lowest effective dose / conc.:

80 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Diagnostic Veterinary Screening
In addition to the Sentinel Monitoring programme, fecal samples were taken
from 5 male and 5 female study cages for the purpose of screening for Toolan’s H1 and Rat Minute Virus by PCR analysis, at the several occasions 
The faecal samples obtained from study cages for the purpose of screening for Toolan’s H1 and Rat Minute Virus by PCR analysis were all negative.
The F0 sentinel samples were negative however the F1 sentinel samples showed 5/5 positive for RMV (rat minute virus) and 1/5 positive for H-1 (Toolan’s H1 virus).
Rat Minute Virus (RMV) and Toolan’s H-1 virus are not associated with clinical disease in natural/subclinical infections and, in addition, are not associated with any pathological
lesions. Based on the the histopathological examination in this study, there were no findings could be attributed to RMV or Toolan’s H-1 viral infections. Therefore, these infections are considered to have no impact on the interpretation of the study pathology data. In addition Control data for F0-F1 inlife parameters up to sexual maturation of the selected F1 animals
were compared with Control data from a comparable OECD443 dietary study in the SD rat that was conducted in 2020-2021. This was reviewed by a veterinary surgeon and it was concluded that no significant findings/differences were observed particularly as regards offspring morbidity, mortality, growth or development.

 

Deviation from Study Plan

Category	Deviations
Animal Data Collection	It was a study plan requirement that on Day 22 a body weight and clinical sign was recorded. In error pup F8 from Sam 1F224 did not have these recorded prior to dispatch
Animal Husbandry	It was a study plan requirement that the animals had access to the treated diet until the date of dispatch when it was replaced by pelleted diet. When the unselected pups from Dams 1F 213,214,220 3F 262,264 4F 285 were weaned from mum they were given pelleted diet in error. These animals were then dispatched on the 18 Jun. Group 3 Litter 262 and 264 and Group 4 litter 285 underwent biosampling for Silver analysis on PND22; PI was informed of the error. This did not impact on study integrity.
Samples/Reagents/Test Materials - Handling	Contrary to protocol, the serum taken for Copper/Selenium analysis for the F0 females was placed into a -80°C freezer on the 25 June 2021. The error was noticed, and the samples transferred to -10 to -30°C storage on the 29 June 2021. PI advised of deviation and from their perspective this had no impact on the integrity of the study samples. "Acceptable stability at -20°C infers stability at -80°C (Arrhenius equation). Furthermore, while we have not conducted previous studies for selenium, we have previously conducted numerous copper studies in which we have assessed stability in matrix at -80°C out as far as 1 year."
Sampling Error	Fecal samples for animals 4M 467 and 4F 768 were not taken from the rectum before it was immersed in neutral buffered formalin. After approximately 30 minutes the rectums were removed from the fixative, rinsed and then the fecal samples were taken. This equates to 2/9 decedents; the majority of samples had been taken and were suitable for screening.
Incorrect/Missed sample/tissue collection	Contrary to protocol F2A animal 4M 576 was only subject to a macro with abnormality's retained instead of a full list retention which was confirmed by Mark Blee who had contacted Ruth Renaut. This was a full Cohort OECD443 and as such retention of abnormalities only for this decedent animals had no impact on the study integrity or validity.
Sampling Processing	Contrary to protocol, no valid organ weight was taken for the prostate of 1M 483, as the value recorded was found to be erroneous at data check. The organ was re-weighed post-fixation. This deviation for a Control male in Cohort 1B had no impact on study integrity.
Data recording/documentation error	Contrary to protocol, no valid organ weight was taken for the left testis of animal 24 or the liver of animal 61, as the weights recorded were found to be erroneous at data check and were consequently excluded. These exclusions had no impact on study integrity
Sample collection	It was a study plan requirement that urine samples were obtained prior to termination. No sample was obtained for 3F 742 as the collection pot was postitioned incorrectly under the funllet resulting in the uring missing the pot. The lack of sample for one animal had no impact on the study integrity
Sample collection	Immuno spleen for animal 1M 418 placed into formalin in error, sample taken out straight away and rinsed saline and placed in HBSS. Analytical department informed, impact was not known until data analysis and data review was complete.
Missed/Extra procedure	Contrary to protocol, no heart sections for transmission electron microscopy (TEM) were taken for animals 1F 711+712 The omission of two Control samples had no impact on study integrity
These study deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study

Conclusions:

Dietary administration of test item at target dose levels of 40, 80 and 120 mg/kg/day (low, mid and high dose) was associated with a number of effects including: F1 mortality at high dose level; F0/F1 red blood cell parameters at all dose levels; F1 offspring survival at high dose; F1 offspring
body weight at 80 or 120 mg/kg/day; F1 neurobehaviour/sensory function at mid or high dose; motor activity for F1 males and females at mid or high dose; Sperm counts at high dose for F0 males and all dose levels for F1 males; F1 sperm morphology at high dose; Epithelial degeneration of the glandular mucosa of the stomach in F0 females at all dose levels; F1 histopathological findings of intramyelinic edema and neuronal and/or glial cell necrosis at 80 or 120 mg/kg/day and F1 brain morphometry (low mean hippocampus) at mid or high dose. The minor reductions recorded in F0 and F1 male body weight gain up to 8% and 14% lower than Controls respectively were judged non adverse.
It was therefore concluded that the various no observed adverse effect levels (NOAELs) on this study were:
• Systemic toxicity in F0 adults : not established (due to degeneration in stomach
mucosa in females at all doses).
• Systemic toxicity in F1 adults: 40 mg/kg/day (due to the following effects of treatment at mid or high dose: reduced activity and rearing of males and females in the arena, reduced reactivity, abnormal motor movement/gait, intramyelinic edema and neuronal and/or glial cell necrosis and F1 brain morphometry (low mean hippocampus) – mean achieved doses of 36 mg/kg/day in males and 40 mg/kg/day in females.
• F0 mating performance and fertility: 120 mg/kg/day - mean achieved doses of
113 mg/kg/day for males and 127 mg/kg/day for females.
• F1 offspring survival and growth up to weaning: 80 mg/kg/day (due to reduced
offspring survival and reduced growth at 120 mg/kg/day) – mean achieved doses of
100-107 mg/kg/day.
• Developmental neurotoxicity in selected F1 animals: 40 mg/kg/day (due to the following effects of treatment at 80 or 120 mg/kg/day: reduced activity and rearing of males and females in the arena, reduced reactivity, abnormal motor movement/gait, intramyelinic edema and neuronal and/or glial cell necrosis and F1 brain morphometry (low mean hippocampus) – mean achieved doses of 36 mg/kg/day in males and 40 mg/kg/day in females.
• Developmental immunotoxicity in selected F1 animals – 120 mg/kg/day - mean achieved doses of 110 mg/kg/day in males and 116 mg/kg/day in females.

Executive summary:

The purpose of this study was to assess the influence of Silver Acetate on reproductive performance when administered continuously via the diet to Sprague Dawley rats in anOECD 443 Extended One Generation Reproductive Toxicity Study. Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on sexual maturation and estrus cycles, developmental neurotoxicity and developmental
immunotoxicity.
In the F0 generation, three groups of 25 male and 25 female rats received Silver Acetate at target dose levels of 40, 80 or 120 mg/kg bw/day orally, via the diet. Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for
ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. The F1 generation was treated from weaning to their scheduled termination (relevant to each cohort) at the same dose levels and volume-dose as the F0 generation, with exception of animals at 120 mg/kg bw/day in Cohorts 1A and 1B which were terminated prematurely on welfare grounds at approximately 10 weeks of age rather than 13-14 weeks of age. 

A similarly constituted Control group received untreated basal diet, at the same volume dose and throughout the same relevant period as the treated groups. For the F0 generation data were recorded on clinical observations, body weight, food consumption, water consumption (by visual assessment), estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance. Clinical pathology (hematology and blood chemistry) and thyroid hormone analysis, blood silver analysis, serum copper/selenium analysis, sperm assessment, organ weight and macroscopic pathology and microscopic pathology investigations were performed.

For F1 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, organ weights and macropathology were assessed. Nipple counts were performed on male offspring on Day 13 of age. Serum samples that were collected from litters on Day 4 of age were analyzed for serum copper/selenium levels and blood samples were analysed for silver levels. Serum samples from selected offspring on Day 22 of age were analyzed for thyroid-related hormones and serum copper/selenium levels, and blood samples were analysed for silver levels.

At weaning the F1 generation was split into five cohorts:

• For F1 Cohort 1A, data were recorded on clinical condition, body weight, food consumption, sexual maturation and estrous cycles. Clinical pathology (hematology, blood chemistry, cardiac troponin, and urinalysis) and thyroid-related hormones, silver blood levels, serum copper/selenium levels sperm assessment, ovarian follicle and corpora lutea counts, organ weight, macroscopic pathology, full microscopic pathology and spleen immunophenotyping investigations were performed.
• For F1 Cohort 1B, data was recorded on clinical condition, body weight, food consumption, sexual maturation and estrous cycles. Silver blood levels and copper/selenium serum levels were assessed. Organ weight and macroscopic pathology investigations were performed.
• For F1 Cohort 2A, data was recorded on clinical condition, body weight, food consumption and sexual maturation. Neurobehavioral screening, brain weight, brain morphometry, macroscopic pathology and microscopic pathology investigations were performed.
• For F1 Cohort 2B, animals received no direct treatment, and no specific in-life investigations were performed. Animals were dispatched to necropsy at weaning for microscopic pathology investigations of the brain including brain morphometry. For F1 Cohort 3, data was recorded on clinical signs, body weight, food consumption and sexual maturation. Spleen weight, macroscopic pathology and T-cell dependent antibody response investigations were performed.

Results
There was no evidence for silver in the blood from Control animals. Blood silver content increased with target dose however the increases were not dose proportionate and values in F1 PND 22 pups were significantly higher than adults.

In general serum copper and selenium levels decreased as the target dose increased, the decreases in serum levels were not dose proportionate but tended to be greater for copper and for adult animals when compared with offspring on PND22.

There were no statistically significant differences in thyroid stimulating hormone or thyroxine serum concentrations levels in any group or generation of males or females after dietary administration of Silver Acetate at 40, 80 and 120 mg/kg/day when compared with Controls.

Overall modest effects were observed on the immunophenotyping parameters measured in Sprague Dawley rat spleen leukocytes as a result of the dietary administration of Silver Acetate, with a statistically significant increase (p<0.05) in the percentage of B cells in females at 40, 80 and 120 mg/kg/day however, the increase in the percentage of B cells in females did not translate into an increase in IgM production as no significant differences were found between the Control Group and the treated groups in the anti-keyhole limpet hemocyanin (KLH) IgM analyses (TDAR).

F0 responses
The mean achieved dose levels for the pre-pairing treatment period were within 10% of target dose levels of 40, 80 or 120 mg/kg/day. During gestation and lactation, the mean achieved dose levels for females were more variable, but all exceeded the target dose.

One male at 80 mg/kg/day and two males at 120 mg/kg/day died but the cause of death was undetermined. One female at 120 mg/kg/day was killed owing to a mammary lesion which was unrelated to treatment.

Body weight gain for males at 80 and 120 was significantly reduced at termination (p<0.05); female body weight was unaffected by treatment.
Intermittent, transient effects on food intake were observed for males at 40, 80 or 120 mg/kg/day and females at 120 mg/kg/day in Week 4 and during Days 4 to 13 of lactation.

The efficiency of food utilization for animals before pairing and for females during gestation was unaffected by treatment.

There was no effect of treatment on estrous cycles, pre-coital interval, mating performance or fertility and the gestation index (number of litters born/number of mated females) was 100% in all groups. A marginal shift toward a longer gestation length was observed at 120 mg/kg/day.

Haematology at Week 10 for females and termination for males and females showed differences in erythrocyte count, mean cell haemoglobin and mean cell volume, red cell distribution width, mean cell heamoglobin, mean haemocrit, haemoglobin concentration, platelet count and white blood cell count with variable dose response and with males more affected than females. Females at 120 mg/kg/day showed high counts for eosinophils, monocytes and large unstained cells. All other parameters measured showed no effect of treatment.

Blood chemistry for females in Week 10 of treatment and for both sexes at termination showed, principally, high alkaline phosphatase activity, high plasma cholesterol and low potassium levels but differences were not always dose proportionate.

There was a treatment related effect on testis weight and spermatid total millions, cauda epididymis sperm count and total millions following treatment with silver acetate at 120 mg/kg/day; however motile, progressive, motion and morphology were unaffected.

Body weight-relative heart weight was high for both males and females at all dose levels and all treated groups of males had low mean absolute pituitary weights and absolute/body weight-relative mean prostate weights. Low mean absolute testes weight was also observed at 80 and 120 mg/kg/day and high mean body weight-relative spleen weight was observed at 120 mg/kg/day. Females that received 120 mg/kg/day showed low absolute and body weight-relative mean thymus weight.

The majority of macroscopic findings at scheduled termination comprised abnormal colouration of tissues, including the gastrointestinal tract, kidneys, lacrimal glands, liver, harderian glands, mesenteric lymph nodes, pancreas, preputial/clitoral glands, salivary glands, thymus, thyroids, urinary bladder and uterus.

At histopathology, extracellular pigment was observed in various organs/tissues and was considered to represent deposition of test item at these sites. In general, pigment was more prominent in females, and there was not always an apparent dose response. Pigment was not associated with any inflammatory or degenerative changes.

Other histopathological findings included increased extramedullary hematopoiesis observed in the spleen of males at 80 or 120 mg/kg/day and epithelial degeneration of the glandular mucosa of the stomach in females that received 40, 80 or 120 mg/kg/day.

F1 litter responses
At 120 mg/kg/day was an increased incidence of offspring mortality and dark coloration; clinical condition of offspring at 40 or 80 mg/kg/day was considered unaffected by administration of Silver Acetate.

There was no effect of treatment on the number of implantation sites, mean litter size or sex ratio on postnatal Day 1.

At 120 mg/kg/day the live birth and viability indices were low when compared with Controls, resulting in a mean live litter size on Day 1 of 13.0 compared to 14.5 in Controls and 11.7 on Day 4 of compared to 14.4 in Controls; no differences were apparent at 40 or 80 mg/kg/day.  Following litter standardization on Day 4 of age offspring survival at all dose levels was similar to Controls.

Mean body weight for male and female offspring at 120 mg/kg/day was low when compared with Controls on Day 1 of age and remained low until weaning. At 80 mg/kg/day mean offspring body weight on Day 1 was similar to Controls but lower than Controls on Days 14 and 21. There was no effect on offspring weight or weight gain at 40 mg/kg/day.

There was no effect of treatment on anogenital distance in male or female offspring and no effect on nipple count in males.

Macroscopic findings for offspring that died or were culled on Day 4 of age were predominantly absence of milk in the stomach and/or autolysis.
There were no significant macroscopic findings in offspring terminated on Day 22 of age. At 80 or 120 mg/kg/day low mean absolute and body weight-relative thymus weights (p<0.01) were observed in both sexes, and females at 40 mg/kg/day showed low relative weights.

On PND22 mean absolute brain weight was low in females at 80 and 120 mg/kg/day.

F1 responses
The mean achieved dose levels for selected F1 animals were 36, 72 or 110 mg/kg/day for males and 40, 81 or 116 mg/kg/day for females and were no more than 10% below target dose levels of 40, 80 or 120 mg/kg /day. 

Treatment at 120 mg/kg/day was associated with nine male and two female mortalities between Day 19 and Day 47 of the F1 generation. Of these seven decedents were subject to full microscopic examination which revealed five with brain lesions which were considered the major factor contributing to these deaths.

Following the high incidence of mortality at 120 mg/kg/day the high dose group was terminated prematurely at approximately 10 weeks of age.

Males at the target dose of 120 mg/kg/day showed a higher incidence of piloerection, hunched posture and abnormal gait; these signs were also observed in a few females at 120 mg/kg/day but at a low incidence.

At weaning on PND21 selected males and females receiving 80 or 120 mg/kg/day had low mean bodyweight and subsequent bodyweight gain up to PND25 was low at all target dose levels (p<0.05/0.01) with no clear dose response.

From Day 1 of the F1 generation (PND28±2) up to termination mean body weight, mean body weight gain and food consumption was low for males at all target dose levels; a dose response was apparent.

During Weeks 1-2 of the F1 generation treated females showed low body weight gain and food consumption. During Weeks 2-5 weight gain was high for treated females and from Week 5 body weight gain was similar to Controls. Periods of high food consumption were recorded at 80 and 120 mg/kg/day from Week 3-6; subsequent consumption for treated females was similar to Controls.

Overall, the efficiency of food utilization was unaffected by treatment for both males and females.

The mean age at completion of balano preputial separation was essentially similar across the groups but the mean bodyweight at completion was statistically significantly lower than Controls at all dose levels (p<0.01). The mean age at completion of vaginal opening for females receiving 120 mg/kg/day was 1.2 days later than Controls (p<0.01) and the mean bodyweight at completion was statistically significantly lower than Controls at all dose levels (p<0.05/0.01).

The interval between vaginal opening and the first estrus and estrous cycles were unaffected by administration of Silver Acetate.

Hematology at scheduled termination (approximately 13 weeks of age) of males at 80 mg/kg/day and females at 40 or 80 mg/kg/day showed high mean erythrocyte counts. Males at 80 mg/kg/day also showed low mean cell hemoglobin, low mean cell volume and high red cell distribution width and females at 80 mg/kg/day showed high hemoglobin and high hematocrit. Conversely males and females at 120 mg/kg/day that were terminated prematurely at approximately ten weeks of age showed low erythrocyte counts, high mean cell hemoglobin and high mean cell hemoglobin concentration. In addition, females at 120 mg/kg/day had a low hematocrit, low red cell distribution width and high mean cell
volume.

White blood cell parameters at either scheduled or premature termination were high for females that received Silver Acetate.

Mean platelet counts were low for females at 80 or 120 mg/kg/day, prothrombin times were shorter in all treated groups of females and activated partial thromboplastin time was shorter for females at 120 mg/kg/day.

At premature termination blood chemistry for animals at 120 mg/kg/day principally showed high alkaline phosphatase activity, high alanine amino-transferase activity, high aspartate amino-transferase activity (females only), high cholesterol, high glucose, high urea (males), low creatinine, low albumin/A:G ratio, high potassium, low phosphorous levels and low sodium(females).

At scheduled termination of animals at 40 or 80 mg/kg/day blood chemistry investigations revealed high alkaline phosphatase activity (males), high alanine amino-transferase activity (males), high cholesterol and low creatinine.

Troponin investigations conducted on the F1 generation revealed higher levels in treated males, with mean levels at 120 mg/kg/day were slightly high but much lower than the mean values at 40 or 80 mg/kg/day (no historical control data were available for comparison). Cardiac troponin levels were unaffected in females at all dose levels.

Urinalysis investigations of the F1 generation revealed a low total protein output and protein concentration in males at 80 mg/kg/day (Urinalysis assessment was limited to Control, low and mid dose animals).

For auditory startle response the majority of latency to peak values for all groups of treated females were low compared with Controls and statistical significance was achieved for females at 120 mg/kg/day during trials 31-40. Group mean auditory startle latency to peak values for males at 120 mg/kg/day was high during trials 21-30.

The majority of group mean auditory startle peak amplitude values for males at all dose levels and females at 120 mg/kg/day were low when compared with Controls, with animals at 120 mg/kg/day showing the least habituation.

The majority of group mean high (rearing) and low (ambulatory) beam activity scores were low for males and females at 80 or 120 mg/kg/day.

In cage, in hand and arena observations showed a number of findings predominately at 120 mg/kg/day but also in a few animals at 80 mg/kg/day including, but not restricted to, abnormal motor movements, gait and activity.

Reactivity investigations showed a number of observations predominately at 120 mg/kg/day but also some were observed at 80 mg/kg day including reduced approach response, a weak startle response, failed pupil closure, high landing foot splay, reduced forelimb/hindlimb grip strength. In addition, females at all dose levels showed slightly low body temperatures

Mean ovarian follicle and corpora lutea counts at 120 mg/kg/day were similar to Controls and considered unaffected by administration of Silver Acetate.

Only 12 F1 males were available for assessment of sperm parameters and these males were terminated approximately 3 weeks earlier than other groups. There was an effect on cauda epididymal spermatid counts and an increased incidence of abnormal morphology (particularly decapitates, mainly in one animal) at 120 mg/kg/day.

For F1 Cohort 1A animals treatment related organ weight changes were noted in the heart in both sexes, the brain in males given 120 mg/kg/day, and the thymus in both sexes at 120 mg/kg/day. F1 Cohort 1B males also showed treatment related organ weight changes in the heart.

For F1 Cohort 2A males at 120 mg/kg/day had high body weight relative brain weight; this was not evident in Cohort 2B.

For F1 Cohort 3 higher than control mean absolute and body weight relative spleen weights were noted in both sexes at 120 mg/kg/day.

Treatment-related macroscopic findings in F1 Cohorts 1A, 1B, 2A and 3 were generally observed in both sexes across all dose groups without a significant dose response and were generally limited to abnormal coloration (dark) of affected organs/tissues. This was generally observed at a lower incidence and in fewer tissues compared to F0 generation animals. This abnormal coloration was no apparent in F1 Cohort 2B animals that were necropsied at weaning.

In the brain, dose related neuronal necrosis in the hippocampus, and neuronal/glial cell necrosis in the thalamus were observed in both sexes given 80 or 120 mg/kg/day. Intramyelinic edema in the thalamus, caudate putamen and/or the corpus callosum was also observed in males given 80 or 120 mg/kg/day and in females given 120 mg/k/day Brain morphometry measurements revealed statistically significantly low hippocampus measurement for males that received 80 or 120 mg/kg/day.

Conclusion
Dietary administration of Silver Acetate at target dose levels of 40, 80 and 120 mg/kg/day was associated with a number of effects including: F1 mortality at 120 mg/kg/day; F0/F1 red blood cell parameters at all dose levels; F1 offspring survival at 120 mg/kg/day; F1 offspring body weight at 80 or 120 mg/kg/day; F1 neurobehaviour/sensory function at 80 or 120 mg/kg/day; motor activity for F1 males and females at 80 or 120 mg/kg/day; Sperm counts at 120 mg/kg/day for F0 males and all dose levels for F1 males; F1 sperm morphology at 120 mg/kg/day; Epithelial degeneration of the glandular mucosa of the stomach in F0 females at all dose levels; F1 histopathological findings of intramyelinic edema and neuronal and/or glial cell necrosis at 80 or 120 mg/kg/day and F1 brain morphometry (low mean hippocampus) at 80 or 120 mg/kg/day. The minor reductions recorded in F0 and F1 male body weight gain up to 8% and 14% lower than Controls respectively were judged non adverse.

It was therefore concluded that the various no observed adverse effect levels (NOAELs) on this study were:

• Systemic toxicity in F0 adults : not established (due to degeneration in stomach mucosa in females at all doses).
• Systemic toxicity in F1 adults: 40 mg/kg/day (due to the following effects of treatment at 80 or 120 mg/kg/day: reduced activity and rearing of males and females in the arena, reduced reactivity, abnormal motor movement/gait, intramyelinic edema and neuronal and/or glial cell necrosis and F1 brain morphometry (low mean hippocampus) – mean achieved doses of 36 mg/kg/day in males and 40 mg/kg/day in females.
• F0 mating performance and fertility: 120 mg/kg/day - mean achieved doses of 113 mg/kg/day for males and 127 mg/kg/day for females.
• F1 offspring survival and growth up to weaning: 80 mg/kg/day (due to reduced offspring survival and reduced growth at 120 mg/kg/day) – mean achieved doses of 100-107 mg/kg/day.
• Developmental neurotoxicity in selected F1 animals: 40 mg/kg/day (due to the following effects of treatment at 80 or 120 mg/kg/day: reduced activity and rearing of males and females in the arena, reduced reactivity, abnormal motor movement/gait, intramyelinic edema and neuronal and/or glial cell necrosis and F1 brain morphometry (low mean hippocampus) – mean achieved doses of 36 mg/kg/day in males and
  40 mg/kg/day in females.
• Developmental immunotoxicity in selected F1 animals – 120 mg/kg/day - mean achieved doses of 110 mg/kg/day in males and 116 mg/kg/day in females.

Reference 2

Endpoint:

one-generation reproductive toxicity

Remarks:

To assist in dose level selection for a main extended one generation reproductive toxicity study (EOGRTS)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 July 2020 - 18 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Weight of evidence approach described in 'Silver metal (massive and powder): Weight of Evidence' (document attached in IUCLID section 13 - "CSR Annex 11 - Weight of Evidence Justification for Silver metal - human health endpoints).
The study design was recommended by the CRO as a dose-range finding study for the OECD 443 Extended One Generation Reproductive Toxicity Study.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was not designed to meet any particular regulatory requirements.

GLP compliance:

no

Remarks:

No claim for compliance with Good Laboratory Practice was made, although the work performed generally followed Good Laboratory Practice principles

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature and protected from light
- Stability of formulation: 22 days at ambient temperature (15 to 25°C)

- Appearance: colourless to pale-white crystalline powder
- Storage conditions: room temperature and protected from light
- Molar mass: 166.91g/mol
- Solubility: aqueous (room temperature): 1.02g/mol

Species:

rat

Strain:

Sprague-Dawley

Remarks:

.

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by
regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the
historical control data available at this laboratory

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain/Species: Crl:CD(SD) rat
- Supplier: Charles River (UK)
- Number of animals ordered: Group 1 to 5 : 64 males and 64 females - Group 7: 14 males and 14 females. Spare animals were removed from the study room after treatment commenced.
- Duration of acclimatization: Group 1 to 5: 12 days before commencement of treatment and Group 7: five days before commencement of treatment.
- Age of the F0 animals at the start of treatment: Group 1 to 5: males 75 to 81 days old and females 75 to 95 days old. The group : 75 to 81 days old.
- Weight range of the F0: Group 1 to 5: male 372 to 473 g and 204 to 290 g for females. For Group 7: males 377 to 416 g and females 262 to 288g.

ALLOCATION TO TREATMENT GROUP (F0 GENERATION):
On arrive by non-selective allocation to cages. On Day 1 of the study the body weight of animals were reviewed by Study Management to reduce inter/intra group variation.

SELECTION OF OFFSRPING TO FORM F1 GENERATION (Group 1 to 3 only):
- Selection: prior to Day 21 of age
- Number per group: then males and ten females
- Method: The offspring with the lowest within-litter identification per sex from each selected litter were selected to form the F1 generation, after exclusion of grossly atypical animals. Where possible, one male and one female were selected from each selected litter (if more were required, up to two males and two females were selected from each selected litter). Formal commencement of the F1 generation was on a nominal Day 28 of age (where possible 28+/-2 days of age for selected F1 animals).
Selected animals were microchipped on Day 28-20 of age and separated from littermates on Day 21 of age. Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.

IDENTIFICATION OF ANIMALS:
- Identification animals: Each F0 animals and selected F1 animals was assigned a number and identified uniquely within the study using a microchip. All F1 offspring were numbered individually within each litter on Day 1 of age by a toe tattoo.
- Identification of cages: Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identify to the occupants.

ANIMAL REPLACEMENT:
before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weights from the same batch. Replacement before allocation: Based on body weight range extremes - two males and one female.

ANIMAL CARE AND HUSBANDRY
- Environmental Control - animal facility: limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Temperature and relative humidity: Monitored and maintained within the range of 20-24°C and 40-70%. Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
- Lighting: artificial lighting, 12 hours light: 12 hours dark.
- Electricity supply: public supply with automatic stand-by generators.

ANIMAL ACCOMMODATION
- Cages: cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used throughout the study except during pairing. Grid bottomed cages were used during pairing. These were suspended above absorbed paper which was changed daily.
- Cage distribution: the cage were distributed on the racking to equalize, as far as possible environmental influences amongst the groups.
- Bedding: solid bottom cages contained softwood based bark-free fibre bedding which was changed at appropriate intervals each week.

ENVIRONMENT ENRICHMENT
- Aspen chew block: as soft white untreated wood block; provided to each cage throughout the study (except from late gestation and during lactation) and replaced when necessary.
- Plastic shelter: provided to each cage throughout the study (except during pairing, from late gestation and during lactation) and replaced at the same time as the cages.
- Paper shaving: From Day 20 after mating and throughout lactation, approximately two handfuls of paper shakings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

DIET SUPPLY
- Diet: SDS VRF1 Certified powdered diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Availability: Non-restricted

WATER SUPPLY:
- Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Availability: Non-restricted

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

Untreated based diet of the same batch

Details on exposure:

FORMULATION:
Dietary concentration were adjusted weekly during the premating period for the F0 animals, for F0 males after pairing and for the selected F1 animals nominal Day 35 of age based on estimated daily food intake and mid-week body weight, to ensure achieved intake approximated to the target dose levels. Males and females during the pairing prior and mated females during gestation received the dietary concentration used the last week of the pre-mating period. To maintain the target dose during lactation, dietary concentrations for littered females were adjusted to accommodate the expected increase in food intake, consistent with the concentration adjustments reported by Sprando et al. 2017 and cognizant of the recommendations in Beekhuijzen 2016. Selected F1 offspring in Group 2 and 3 continued on the reduced lactation dietary concentration up to nominal Day 35 of age.

- Method of preparation: All formulation were prepared under yellow lights. The test item was incorporated into the diet to provide the required concentrations by initial preparation of a premix. The amount of test item required for the premix was added to an equal amount of sieved diet and stirred. An amount of sieved diet equal to the weight of the mixture was added and the mixture was stirred again until visibly homogenous. The doubling up process was repeated until approximately half the premix diet was added. At this stage the mixtures was ground with a mechanical grinder. The mixture was made up to the weight of the premix with coarse diet. The premix was then mixed using a turbula mixer for 200 cycles.

- Frequency of preparation: weekly
- Storage of formulation: 22 days at ambient temperature (15 to 25°C)

ADMINISTRATION:
- Route: oral, via the diet
- Control (group 1): Untreated basal diet of the same batch
- Frequency: continuously via diet
- Diet: a record of the usage of the diets was maintained on all occasions when food consumption was measured. this was performed using the initial weight of the diet container and an on-line data check on completion of the feeding procedure to ensure that all cages were fed the correct amount of diet. No significant discrepancy was found.

Details on mating procedure:

- F0 pairing commenced: after four weeks of treatment
- Male/female ratio: 1:1 from within the same treatment gruoups (sibling pairing was not permitted).
- Duration of pairing: Up to two weeks.
- Daily checked for evidence of mating: Ejected copulation plug in cage tray and sperm in the vaginal smear.
- Day 0 of gestation: When positive evidence was detected.
- Male/female separation: day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Remarks:

Samples of each formulation prepared for administration in were analysed for achieved concentration of the test item.

Details on analytical verification of doses or concentrations:

All specimens were analysed within the documented ambient stability period of 22 days at ambient temperature using a fully validated bioanalytical method for silver in solid rodent diet formulation: with calibration and QC samples were analysed within the documented ambient stability period of 67 days at ambient temperature. The lower limit of quantification for the assay (LLOQ) was 1.00 μg/mL for silver and μg/mL.

The formulation samples were digested in 4 x mass to volume of concentrated (65-75%) nitric acid to yield a digest for analysis. Following digestion, 9.98 mL of rhodium internal standard solution was combined with 25 μL of digested solid rodent diet formulation in a 15 mL polypropylene tube, capped and rotary mixed for at least 30 minutes. An aliquot of each processed sample was then analysed by ICP-MS together with quality control samples and a multilevel calibration.

All solid rodent diet formulation samples were analysed in accordance with the validated method and within the documented ambient storage period. There was no evidence of silver in any of the
control group samples. On every occasion, the dose formulation samples were within the predefined acceptance limits for this study.

Duration of treatment / exposure:

F0 animals: For a minimum of 29 days prior to pairing until scheduled termination after at
least 65 days of treatment for males and on Day 21 post-partum for females.

F1 animals: Groups 1 to 3 (0. 4 , 40 mg/kg bw/day): From when offspring start to consume diet until completion of sexual maturation (approximately eight weeks of age).

Frequency of treatment:

Administration continuously via the diet.

Dose / conc.:

80 mg/kg bw/day (nominal)

Remarks:

Group 7

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

4 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

160 mg/kg bw/day (nominal)

Remarks:

Group 4

Dose / conc.:

320 mg/kg bw/day (nominal)

Remarks:

Group 5

No. of animals per sex per dose:

12 animals per sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels was based on the outcome of a 2-week palatability study in male and female rats, and three published studies.

In a published study on reproduction and postnatal development (Sprando 2017), target concentrations of 0, 0.4, 4.0 and 40 mg AgAc /kg bw/day of test item in the drinking water reported no significant differences in body weight or food intake during a 10 week pre-mating administration period or during gestation or lactation, although mean weight gain in the high dose group was about 90% of the control value.

A recent 10 week rat dietary study at 4, 40 and 400 mg AgAc/kg of food, calculated to achieve target dose levels of 0.4, 40 and 40 mg AgAc/kg bw/day reported actual dose levels of 0.3, 3.1 and 27.8 mg/kg bw/day in females and 0.3, 2.8 and 25.4 in males (van den Brule 2019). No significant effect of treatment on body weight or food intake was reported in either sex at any dose level. In a rat embryo-foetal development study, pregnant female rats receiving 0, 10, 30 or 100 mg Ag Ac /kg bw/daily from Days 6 to 19 of gestation by gavage reported few clinical signs, mainly weight loss, rooting after dosing and piloerection at 30 and 100 mg AgAc/kg bw/day. No dose-related trends were reported for body weight, adjusted (for gravid uterine weight) body weight or food intake but one high dose morbid female was euthanized on Day 12 of gestation (Price 2002).

A previous screening study at the same laboratory, with 15 time-mated females per group investigated dose levels of 0, 8, 40, 80, 160 or 290 mg AgAc/kg bw daily from Day 6 to 19 of gestation. At 160 or 290 mg AgAc/kg bw/day all animals were terminated before the end of treatment owing to clinical signs include weight loss, rooting in the bedding (indicating aversion to treatment), respiratory distress, chromodacryorrhea, piloerection and lethargy. Maternal body weight was not affected at doses up to 80 mg AgAc/kg bw/day, but food and water intake were reduced from Day 6 or 9 onwards.

Based on the outcome of the palatability study where dietary concentrations were tolerated but did not achieve the target dose level of 320 mg/kg/day in males, the initial dietary concentration for the high dose level for this study was selected as 7000 ppm. F0 females and litters from Group 4 and 5 showed high offspring mortality during early lactation and were terminated prematurely; subsequently the F1 generation was derived from Groups 1 to 3. As Group 3 animals/offspring (target dose of 40 mg/kg/day) have not shown a significant response to treatment it has been decided that a further dose level between 40 and 160 mg/kg/day should be assessed to enable dose selection for the subsequent extended one generation study. A second phase will therefore be conducted to assess a target dose of 80 mg/kg/day. An additional group (Group 7) comprising of 12 male and 12 female animals will be allocated to study and treated for four weeks before pairing, during gestation and up
to weaning on Day 21 of lactation; this group will terminate at weaning without selection of a subsequent generation.

- Haematology, Peripheral Blood: Blood samples were collected after overnight withdrawal of food. Samples were collected at termination (up to 12 animals per dose).
- Blood Chemistry: Blood samples were collected after overnight withdrawal of food. samples were collected at termination (up to 12 animals per dose).
- Exposure assessment and biomarkers: F0 and F1 adult animals: blood samples were obtained from study animals, the sampling occasions were detailed below, with sampling commencing at approximately 09:00 hours on each occasion: females: GD6, GD17 - at termination (LD21) - males at termination. For F1aniamls: offspring PND21 and termination.

- Blood sample site: F0/F1 adult animals; jugular vein during study/ sub-lingual vein or orbital sinus at termination (no recovery from anesthesia). Anesthesia: Not required for jugular vein samples. Isofurane for sub-lingual or orbital sinus samples.

Contingency Serum Samples:
- Blood tubes: greiner minicollect with clotting activator.
- Anticoagulant: none
- Blood volume: 1.0 mL
- Treatment of samples: samples were kept at ambient temperature for a minimum of 30 minutes prior to centrifugation.
- Centrifugation conditions: centrifugation at 4°C for ten minutes at 2000g.
- Analytical matrix: Serum
- Volume required: 0.4 mL (all available collected)
- Temporary storage conditions: dry ice
- Final storage conditions: -60°C to -90°C
- Contingency serum samples were retained at Covance pending possible future analysis.

F1 offspring - Day 4 of age:
On Day 4 of age following litter standardization all surplus offspring were subject to biosampling at necropsy, with the aim to provide at least eight samples.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Animals were inspected visually at least twice daily for evidence of ill-health or reaction to
treatment.
- Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s).
- During the acclimatization period, observations of the animals and their cages were recorded
at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
A detailed physical examination was performed on each animal to monitor general health according to the following schedule for physical examination:
- F0 males once each week
- F0 females weekly until mating, Day 0. 5 . 12. 18 and 20 after mating and Days 1, 7, 14 and 21 of lactations.
- F1 animals after weaning once each week - Group 1 to 3 only.

MORTALITY:
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. A complete necropsy was performed in all cases .

BODY WEIGHT: Yes
- Time schedule for examinations:
- F0 males - twice weekly and before necropsy
- F0 females: twice weekly until mating detected - Days 0, 3, 6, 10, 14, 17 and 20 after mating - Days 1, 4, 7, 11, 14, 18 and 21 of lactation.
- F1 selected animals Group 1 to 3 only: Days 21 and 25 of age. Twice weekly from nominal Week 4 (Day 28) of age until terminal examination and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as: F0 males and females: twice weekly - food consumption was not recorded for males and females during the period when paired for mating (Day 29 to 36), but recommenced for males from Day 36. F0 females after mating: Days 0-2, 3-5, 6-9, 10-13, 14-16 and 17-19 after mating. Days 1-3, 4-6, 7-10, 11-13, 14-17 and 18-20 of lactation. F1 selected animals - groups 1 to 3 only: twice weekly from nominal Week 4 of age until termination.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. From these record the mean weekly or daily consumption per animal (g/animal/day) was calculated for each relevant phase.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Fluid intake assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.

PARTURATION OBSERVATION AND GESTATION LENGTH (P0 GENERATION):
- Duration of gestation: Time that elapsed between mating and commencement of parturition.
- Parturition observation: From Day 20 after mating animals were checked three times daily for evidence of parturition. The progress and completion of parturition was monitored: numbers of live and dead offspring were recorded, and any difficulties observed were noted.

Oestrous cyclicity (parental animals):

Dry and wet smears were taken:
- Dry smears: for 15 days before pairing suing cotton swabs.
- Wet smears: using pipette lavage after pairing until evidence of mating confirmed.

Litter observations:

- CLINICAL OBSERVATIONS: observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment.
- LITTER SIZE: Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age. On Day 4 of age, litters containing more than then offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter. Culled offspring were subject to biosampling at necropsy. Culled offspring with clinical signs on Day 4 of age were individually identified for subsequent macroscopic examination.
- SEX RATIO OF EACH LITTER: Recorded on Days 1, 4 and Day 21 of age.
- INDIVIDUAL OFFSPRING BODY WEIGHTS: group 1 to 5: Days 1, 4, 7, 11, 14, 18 and 21 of age. Group 7: Day 25 of age for selected F1 offspring and spares only.
- WEANING OF OFFSPRING: The dam was removed from the litter cage and offspring were weaned on Day 12 of age.
- ANO-GENITAL DISTANCE: Day 1 of age - all offspring
- NIPPLE/AREOLAE COUNT: Day 13 of age - male offspring

- SEXUAL MATURATION - F1 SELECTED OFFSPRING:
Males: Sexual maturation was assessed by daily examination from Day 38 of age until balano-preputial separation occurred. Body weight was recorded on the day of completion of separation.
Females: Sexual maturation was assessed by daily examination from Day 28 of age until vaginal opening. Body weight was recorded on the day of vaginal opening.

- BIOMARKER ANALYSYS:
On day 4 of age following litter standardisation all surplus offspring were subject to biosampling at necropsy, with the aim to provide at least eight samples. of whole blood per group for analysis of silver and serum samples for copper and selenium, ceruloplasmin activity and where possible, Glutathione peroxidase activity.

Postmortem examinations (parental animals):

TIME OF NECROPSY:
- F0 males: after 65 days of treatment successful littering by F0 females
- F0 females: scheduled kill on Day 21 of lactation. Failing to produce a viable litter: Day 25 after mating. Total litter loss, on the day the last offspring died.
- Unselected offspring: Culls - Day 4 of age - scheduled kill (Day 21 of age) - scheduled spares, after establishment of formal F1 generation.

PATHOLOGY PROCEDURES FOR F0 ANIMALS:
- Weight and necropsy: abnormalities: brain, bone marrow removed from femur, kidney, liver (+ histology and pathology light microscopy), mammary, small intestine, spleen, ovaries, testes, thyroid with parathyroids, uterus.

MACROSCOPIC PATHOLOGY :
Detailed macroscopic examination was performed in an attempt to determine cause of ill-health/death. Tissues retained, including collection of caecal faecal samples and a small section of the small intestine (preferably ileum) retained in TriPure medium or RNAlater medium. Any
abnormal tissues were retained and were weighed at the discretion of necropsy
staff.

SCHEDULED SACRIFICE:
All adult F0 animals were subject to a complete macroscopic examination. Any abnormal tissues were retained at the discretion of necropsy staff. in addition, the following was recorded for all F0 females:
- Uterus: number of implantation sites
- Females which are not pregnant: number of implantation sites checked after appropriate staining technique
- Female whose litter dies before Day 21 of lactation: mammary tissues appearance.

ORGAN WEIGHT:
For bilaterial organs, left and right organs were sighted together, unless specified in the relevant pathology procedures table. Requisite organs were weighted for animals killed at scheduled intervals.

HISTOLOGY:
- Processing: Tissues samples were dehydrated, embedded in paraffin was and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
- Mammary tissues retained in PFA: All group 4 F0 females.
- Liver retained in PFA: F0 male from Groups 1, 4 and 5
- Routine staining: section were stained with hematoxylin and eosin.

LIGHT MICROSCOPY:
- Premature termination - all group 4 females - mammary tissues.
- Scheduled kill - males in groups 1, 4 and 5 - liver

Postmortem examinations (offspring):

TIME OF NECROPSY:
- F1 males and females - group 1 to 3 only: Scheduled kill: from nominal eight weeks of age.

PATHOLOGY PROCEDURE FOR F1 OFFSPRING AT DAY 21 OF AGE:
- Abnormalities: Brain (1M + 1F from ten litters/group ) - 5 coronal sections: Olfactory lobes, Forebrain, Cerebrum (hippocampus, thalamus, hypothalamus), Cerebrum (tectum, tegmentum),
Medulla oblongata, Mid-sagittal: Cerebellum, Pons Brain (1M + 1F from eight litters/group) Brain (1M + 1F from three litters/group)

PATHOLOGY PROCEDURES FOR ALL F1 ADULTS ANIMALS:
- Abnormalities: brain, ovaries, testes and uterus.

HISTOLOGY:
- Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a
nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

LIGHT MICROSCOPY:
- Schedule kill - all animals of group 1 and 7 -

Statistics:

This report contains serial observations pertaining to all days of treatment completed, together with signs data collected during the necropsy period. No serial observations relating to the
acclimatization period are included in this report.
Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data. The summary statistics and the individual data were stored in the computer to a certain number of decimal places, different for each parameter. For presentation purposes, however, they were usually rounded to fewer places. It is, therefore, not generally possible to reproduce the presented means and standard deviations exactly using the presented individual data.

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For precoital interval, mating performance and fertility and gestation index the similarity of the data was such that analyses were not considered to be necessary. All statistical analyses were carried out separately for males and females. Data relating to food consumption were analysed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/foetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population. The following data types were analysed at each timepoint separately: Body weight, using absolute weights and gains over appropriate study periods - Food consumption, over appropriate study periods.

For further details, please see section "Any other information on materials and methods incl. tables"

Reproductive indices:

- Pre-coital interval: individual intervals were tabulated for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations.
- Mating performance and fertility: percentage mating(%), conception rate (%), Fertility index (%).
- Gestation length and gestation Index: Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 (day of mating for calculation purpose). Where parturition had started overnight, this value was adjusted by subtracting half of one day.
- Lactation Index (%)
- Sex Ratio: The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Day 1, 4 and 21 of age.
- Offspring examinations: Ano-genital distance were presented both as absolute/unadjusted and adjusted for body weight, using the weight recorded on Day 1 of age. A check was performed to assess for the presence or absence of nipple/areolae for the male
offspring on Day 13 of age. As no nipples were present, no data are included.

Offspring viability indices:

- Sexual maturation: individual values were tabulated for age and body weight at completion. Group mean values were calculated from individual values presented.
- Individual litter values were tabulated for the number of implantation sites, total at Day 1 and
live at Days 1, 4 (before and after culling), 7, 11, 14, 18 and 21 of age. Group mean litter size
and SD were calculated from the individual litter values.
- Survival Index: post-implantation survival index (%) - live birth Index (%) - Viability Index (%) -

Clinical signs:

no effects observed

Description (incidence and severity):

Routine physical examination of males from the start of treatment up to termination and for females before pairing, during gestation and lactation did not reveal any findings that could be attributed to administration of Silver Acetate at any dose levels; assessment of females during lactation was restricted to females at target dose levels of 4, 40 and 80 mg/kg bw/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

Between completion of parturition and Day 2 of lactation four females at 320mg/kg bw/day and two at 160 mg/kg bw/day were killed following total litter loss with offspring either found dead, missing (presumed cannibalized) or killed for welfare reasons. As littering progressed it became apparent that these target dose levels were not tolerated and the remaining females in these dose groups were terminated on welfare grounds; at the time of termination the females were between Gestation Day 20 and Lactation Day 4.

Macroscopic examination of the F0 females at 160 and 320 mg/kg bw/day revealed a number of abnormalities including abnormal colour/content of the GI tract and abnormal colouration of the liver, pancreas, spleen and mesenteric lymph nodes; these findings were more frequent at 320 mg/kg bw/day.

At macroscopic examination the mammary tissue appeared pale and inactive for two females that received 160 mg/kg bw/day (no. 140, total litter loss on Day 1 of lactation, and no. 147, killed for welfare reasons on Day 1 of lactation, with 12 live offspring, three dead offspring and three missing) and for four females that received 320 mg/kg bw/day (nos.151, 154 and 157 with total litter loss on Days 1/2 of lactation and no.159 killed for welfare reasons on Day 1 of lactation with three live and one dead offspring).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the first three days of treatment males at 320 mg/kg bw/day showed a mean body weight loss of 15g and when compared with Controls the overall the mean body weight gain (Days 1-64) for males at this target dose level was significantly low at approximately 20% of Control gain (p<0.01), resulting in low mean body weight at the end of the treatment period (p<0.01; 445g compared with 557g in the Control group).

Overall body weight gain (Days 1-64) for males at 40 or 160 mg/kg bw/day was approximately 88% and 89% of the Controls, respectively; these differences did not attain statistical significance. Body weight gain at 4 or 80 mg/kg bw/day showed no adverse effects of treatment.

Body weight gain of F0 females over the pre-pairing treatment period at 320 mg/kg bw/day was significantly reduced, at 38% of Controls (p<0.01); an initial weight loss was followed by periods of low weight gain. During gestation, body weight gain for females at 320 mg/kg bw/day was significantly lower from GD3 onwards, resulting in an overall body weight gain (GD0-20) of 42% of Controls (p<0.01) and a reduced mean body weight on GD20 of 315g (p<0.01) compared with a
Control mean of 410g. Gestation body weight gains at dose levels up to and including 160 mg/kg/day did not show any adverse effects of treatment.

There was no effect of treatment on body weight gain during lactation at 4 or 40 mg/kg/day.
At 80 mg/kg bw/day body weight gain for Days 4-7 was 15% of Controls and weight loss was apparent from Days 11-14, resulting in an overall weight loss of 3 g for Days 1-21 post partum compared with a control gain of 23 g.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the first week of treatment, food consumption of F0 males at 320 mg/kg/day was 65% of Control values and mean intake remained significantly low for the majority of recorded intervals until termination; 19-21g/day compared with Control values of 22-34g (p<0.05, p<0.01).

Females at 320 mg/kg/day showed food consumption that was significantly lower than Controls for Days 4-7, 18-21 and 25-28 of the pre-pairing period (p<0.01, p<0.05 and p<0.01) and throughout
gestation (p<0.01), with mean values of 13-15g/day compared with Control values of 19-25 g/day.

At dose levels up to and including 160 mg/kg bw/day food consumption for males and females before pairing and during gestation was unaffected by administration of Silver Acetate.

Food consumption during lactation showed no adverse effects of treatment at 4, 40 or 80 mg/kg bw/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At scheduled termination males at 160 or 320 mg/kg bw/day showed statistically significant reductions in hematocrit, hemoglobin level, mean cell haemoglobin, mean cell haemoglobin concentration and mean cell volume (p<0.01) when compared with Controls, but there was no evidence of a dose response. Males at 40 mg/kg bw/day also showed low mean cell haemoglobin (p<0.05). Platelet counts for males that received 40, 160 or 320 mg/kg bw/day were slightly higher than Controls (p<0.05, p<0.01 and p<0.01, respectively) but the increase was not dose proportionate. Males at 4 mg/kg/day and surviving females at 4 or 40 mg/kg bw/day did not show any haemotological differences that could be attributed to administration of Silver Acetate.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Alkaline phosphatase activity was significantly increased (p<0.01) in males at 40, 160 or 320 mg/kg bw/day but there was no dose-relationship. Cholesterol levels for males increased with dose and all treated groups were significantly high when compared with Controls (p<0.05 at 4 mg/kg bw/day and p<0.01 at 40, 160 or 320 mg/kg bw/day). Males at 320 mg/kg bw/day also showed significant increases in (p<0.01) alanine aminotransferase activity (98%) and calcium levels (5%) compared to Controls, and reductions in (p<0.01) creatinine (17%) and chloride (3%).
In females, there were significant increases in alkaline phosphatase activity (40%, p<0.01), phosphorous level (80%, p<0.05) and albumin/globulin ratio (13%, p<0.05) at 40 mg/kg/day, compared to Control and minor increases in cholesterol level (16%) and gamma-glutamyl transpeptidase activity. There was no effect on any measured parameter at 4 mg/kg bw/day.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination of the liver of males that received 160 or 320 mg/kg bw/day identified treatment-related microscopic findings. Pigment was observed in Kupffer cells and to a lesser extent in hepatocytes and in the vascular walls of the liver. Pigment accumulation was considered adverse since it was of up to moderate severity, correlated with macroscopic findings and with increases in enzymes Alkaline phosphatase and Alanine Aminotransferase observed in thrse males, suggesting impaired hepatic function. Centrilobular hepatocellular hypertrophy was also noted in males administered 160 and 320 mg/kg/day and correlated with increased liver weight. Centrilobular hepatocellular hypertrophy is generally considered an adaptive change and may have indicated induction of enzyme gene expression (Maronpot et al. 2010) and was
considered non-adverse in this study. No Silver Acetate-related findings were observed in the mammary glands of females administered 160 mg/kg/day, but no Control samples were available for comparison.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Mating performance:
Mating performance and fertility was unaffected by treatment at dose levels up to and including 320 mg/kg/day. All males and females mated and no more than one female in any of the treated groups did not conceive.

Gestation Length and Gestation Index:
Assessment of gestation length and gestation index at 4, 40 and 80 mg/kg bw/day did not show any clear effects of treatment: All females littered between Day 21 and 23 of gestation and the gestation index was higher than Control in the treated groups. Assessment of the gestation length at 160 mg/kg bw/day and 320 mg/kg/day was limited to females that littered before these groups were terminated. Of the eight females at 160 mg/kg bw/day which littered, seven (88%) had littered by Day 23 of gestation (compared with 11/11 Control females) and one littered on Day 24. At 320 mg/kg bw/day, four females littered by Day 23 and 2/6 littered on Day 23.5, indicating a slightly longer duration of gestation in these groups.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle assessment, including the number of females with 4 or 5 day cycles, irregular cycles/acyclic or extended estrus did not show any effect of treatment.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The majority of animals mated within four days of pairing at the first oestrus; pre-coital interval was therefore unaffected by treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Clinical signs:

no effects observed

Description (incidence and severity):

There was no clear effect of treatment on the clinical condition of offspring at 4, 40 or 80 mg/kg bw/day; one litter at 80 mg/kg bw/day, no. 174, was observed with whole body pallor from Day 7 to Day 14 of age and patchy coat on Days 20/21 of age but as only this litter was affected this could not conclusively be related to treatment.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Group 4 (160 mg/kg bw/day) and Group 5 (320 mg/kg bw/day) were terminated for welfare
reasons when females were between Day 20 of gestation and Day 4 of lactation after four
total litter losses at 320 mg/kg bw/day and two at 160 mg/kg/day.

Offspring body weight for those assessed on Day 1 of age was lower than Controls and
macroscopic examination of offspring revealed an absence of milk in the stomach in the
majority, but no other abnormalities.

Owing to the termination of Groups 4 and 5, full evaluation of litter data was restricted to
those litters from females treated at 4, 40 or 80 mg/kg bw/day.

There was no clear effect of treatment on the clinical condition of offspring at 4, 40 or 80 mg/kg bw/day; one litter at 80 mg/kg bw/day, no. 174, was observed with whole body pallor from Day 7 to Day 14 of age and patchy coat on Days 20/21 of age but as only this litter was affected this could not conclusively be related to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Offspring body weight on Day 1 of age and subsequent body weight gain up to Day 7 of age was unaffected by parental treatment at 4, 40 or 80 mg/kg bw/day. At 4 and 40 mg/kg/day offspring bodyweight gain from Days 7-11 of age was slightly lower than Controls with the difference attaining statistical significance for males and females at 40 mg/kg/day and for female offspring at 4 mg/kg bw/day (p<0.05) but overall weight gain was generally similar.
Offspring body weight at Day 21 of age for the 80 mg/kg bw/day males and females was
lower than all other groups and overall weight gain (Days 1-21 post-partum) was also lower.
Although mean pup weights on Day 1 of age were similar to Controls, these differences could
not be conclusively related to treatment as the Control group was not directly concurrent.

F1 Generation:
At weaning on Day 21 of age mean body weight was 8 and 6% lower than Controls for males
at 4 and 40 mg/kg bw/day and 8% lower in both treated female groups. The mean body weight on Day 1 for males selected for the F1 generation (at nominal Day 28 of age) was 10 and 6% lower than Controls at 4 and 40 mg/kg bw/day and selected females were 6 and 10% lower. These differences became statistically significant in males (p<0.05-p<0.01) from Day 12 of the F1 generation onwards, although not dose-related, and overall gain from Day 1 to Day 29 of the F1 was 11% lower than Controls at 4 mg/kg/day and 5% at 40 mg/kg/day (p<0.01). In females, the effect was dose-related as mean body weight remained significantly low when compared with Controls (p<0.05, p<0.01) at 40 mg/kg bw/day until Day 29 (p<0.01) and at 4 mg/kg bw/day mean weights were significantly low on Days 12, 15 and 22 (p<0.05) Overall body weight gain (Days 1-29) for females at 4 and 40 mg/kg bw/day was slightly low, at 93 and 90% of Control values, but the differences were not statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for selected F1 animals from Days 1 to 29 of the F1 generation was unaffected by administration of Silver Acetate at 4 or 40 mg/kg/day. Group mean values reported as g/animal/day were generally comparable.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The mean age at completion of sexual maturation for both males and females were comparable to Controls at 4 and 40 mg/kg bw/day. The mean body weight on the day of completion of preputial separation was 9 and 7% lower than Controls for males receiving 4 or 40 mg/kg bw/day (p<0.01). Females at 40 mg/kg bw/day also showed 9% lower mean body weight at vaginal opening but this difference did not attain statistical significance.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Ano-genital distance of offspring on Day 1 of age was unaffected by parental treatment at 4, 40 or 80 mg/kg bw/day as all measured and adjusted (for body weight) mean values were similar for each group/sex.

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation:
There was no effect on mean absolute brain or testis weights in F1 males at 4 or 40 mg/kg bw/day, but relative (to body weight) brain weights were significantly higher than Controls (p<0.05), reflecting significant (but not dose-related) reductions in terminal body weight (p<0.01).

The mean absolute brain weight for F1 females at 40 mg/kg bw/day was 5% lower than Controls (p<0.05) but relative brain weight was slightly higher, reflecting the 10% lower terminal body weight ( p<0.01). There was no effect on absolute or relative brain weight in females at 4 mg/kg bw/day or on absolute or relative ovary or uterus weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Offspring:
Macroscopic examination of offspring that died prior to scheduled termination did not reveal any findings at 4, 40 or 80 mg/kg/day that could be attributed to administration of Silver Acetate.
At scheduled termination of unselected offspring on Day 21 of age macroscopic findings were restricted to one litter at 80 mg/kg/day (litter no. 174) where all offspring exhibited a pale liver and generalized hairloss (consistent with clinical signs reported in 6.6.1), one offspring had a
diaphragmatic hernia and one offspring had a liver mass on the median liver lobe.

F1 generation:
Macroscopic examination of F1 animals at scheduled termination following attainment of sexual maturation (Day 29 of the F1 generation) did not reveal any abnormality.

Histopathological findings:

no effects observed

Description (incidence and severity):

Detailed brain neurohistopathology of F1 offspring at Day 21 of age did not reveal any
pathological changes or developmental abnormalities.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

An adverse effect on post-implantation survival, live birth index and litter size was apparent
at 160 and 320 mg/kg bw/day.
Litter size at birth, number of implantations, post-implantation survival index, live birth
index, viability indices, lactation index and litter sex ratio showed no adverse effects of
treatment at 4, 40 or 80 mg/kg bw/day.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Brain weight for offspring on Day 21 of age showed no adverse effects of treatment.

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

160 mg/kg bw/day (nominal)

Treatment related:

not specified

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Deviations from study plan:

The following deviations from study plan occurred:
1. Day 25 Gestation Dispatch (Failed to Litter):
On Day 25 after mating after the 09:00 parturition check animal Group 2 female no. 121 and Group 3 female no 134 were due to be dispatched to necropsy as Failed to Litter as per study plan. These animals were not dispatched until Day 26 after mating in error. No adverse signs were documented against these animals throughout this time period. Impact analysis: Both females were not pregnant, and no loss of data occurred; therefore this study plan deviation had no impact on either animal welfare or the integrity of the study.

2. Dispatch:
The selection file stated that for Group 1 litter no. 111, offspring M2 and F9 were to be retained on study as spare animals; however these animals were despatched to necropsy on Day 21 of age in error. Impact analysis: The study plan states that two male and two female animals per group will be selected as ‘spare’ animals, this deviation from study plan results in Control ‘spare’ animals being limited to one male and one female. This deviation from study plan had no impact on the integrity of this preliminary study as the ‘spare’ animals were not needed.

3. Visual Water Consumption:
The Study Plan required qualitative (visual) assessment of water consumption. This was not scheduled on the Pristima data collection system and was not performed up to 28 July 2020 and commenced in Week 5 of study. Impact analysis: Although formal observations/recordings were not performed the
animals/cages were observed twice daily until Week 5 and no significant difference in water consumption was observed.

Liver weight not taken
Contrary to study plan, the liver weight for Group 7 female 169 was not taken in error. Impact analysis: The omission of a single liver weight in this preliminary study has no impact on study integrity.

These deviations were considered to have not affected the integrity or validity of the study.

Conclusions:

Based on the findings in this study, dose levels of 160 and 320 mg/kg bw/day were not tolerated and were considered unsuitable for the subsequent OECD 443 study. As no significant adverse effects were observed in parental animals or in F1 pups (to weaning) at 80 mg/kg bw/day, the
ptimal high dose level for the OECD 443 study is considered to be between these levels, at 120 mg/kg bw/day.

Executive summary:

The purpose of this study was to assess the influence of Silver Acetate (chemical feed stock) on reproductive performance in the Sprague Dawley rat to assist in dose level selection for a Extended One Generation Reproductive Toxicity Study (EOGRTS) according to OECD443. A pre-mating period of four weeks for males and females was selected to optimize assessment of any potential effects on reproductive outcome.

In the F0 generation, four groups of 12 male and 12 female rats received Silver Acetate (AgAc) orally, via the diet, at target dose levels of 4, 40, 80, 160 and 320 mg AgAc/kg bw/day. Males were treated for 29 days before pairing and until necropsy after litters had weaned (after at least 65 days of treatment). Females were treated for 29 days before pairing, throughout pairing, gestation and until necropsy on Day 21 of lactation. In the F1 generation, three groups of 10 males and 10 females were treated at target dose levels of 4 or 40 mg/kg bw/day from weaning during late lactation to their scheduled termination on Days 31-33 of the F1 generation [at nominal Day 59-61 (+/- 2days) of age] following completion of sexual maturation. A similarly constituted Control group received the vehicle, (untreated diet) for the same duration for each generation.

During the F0 generation, clinical condition, body weight, food consumption, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, exposure assessment and biomarkers, organ weights and macroscopic and microscopy pathology investigations were undertaken. In addition, haematology and blood chemistry investigation s were undertaken for males at 4, 40, 160 and 320 mg/kg bw/day and females at 4 and 40 mg/kg bw/day.

The clinical condition, litter size and survival, sex ratio, body weight, exposure assessment
and biomarkers, organ weights and macropathology for all offspring were also assessed.

For the selected F1 generation at 4 and 40 mg/kg bw/day clinical condition, body weight,
food consumption, sexual maturation, exposure assessment and biomarkers, organ weights
and macroscopic pathology investigations were undertaken.

Results
Biomarkers
Ceruloplasmin activity was observed to decrease with increasing doses of Silver Acetate at 4 , 40, 160 and 320 mg/kg bw/day for F0 male and female animals on Day 12, F0 males on Day 43 and female animals on Gestation Day (GD) 6. F1 Male and female animals followed the same trend on Day 21 at 4 mg/kg bw/day and 40 mg/kg bw/day; male and female animals dosed at 80 mg/kg bw/day showed results inconsistent with this trend. There was little impact on Glutathione Peroxidase (GPx) activity observed following administration of Silver Acetate at dose levels up to and including 320 mg/kg bw/day in male rats. At 320 mg/kg bw/day, female rats showed a decrease (37-42% lower than in Controls) in activity at Day 12 and GD6. Both male and female animals at 80 mg/kg bw/day showed
low GPx activity. Blood levels of silver generally increased with increasing target dose levels; no detectable levels of silver were found in the Control samples.

Silver tissue concentrations were below detectable levels in the brain samples obtained from Control offspring on both Day 4 and Day 21 of age. Silver levels in the rat tissue samples increased with increasing target dose levels, with the highest concentrations detected in the milk pellets obtained from offspring on Day 4 of age.

Serum levels of copper and selenium tended to decrease as the target dose level increased; the observed effect was more marked for copper. There was evidence for a dose-related decrease in copper levels in the testes for F0 males and a non-dose related increase in selenium levels in the ovaries for F0 females. There was no clear effect of treatment on the ovarian copper levels or the testis selenium levels.

F0 Mortality
Between completion of parturition and Day 2 of lactation four females at 320mg/kg bw/day and two at 160 mg/kg bw/day were killed following total litter loss with offspring either found dead, missing (presumed cannibalized) or killed for welfare reasons. As littering progressed it became apparent that these dose levels were not tolerated and the remaining females in these dose groups were terminated on welfare grounds; at the time of termination the females were between GD20 and LD4.

Macroscopic examination of the F0 females at 160 and 320 mg/kg/day revealed a number of abnormalities including abnormal colour/content of the GI tract, abnormal coloured pancreas and mesenteric lymph nodes. At macroscopic examination the mammary tissue appeared pale and inactive for two females that received 160 mg/kg/day (no. 140 total litter loss on Day 1 of lactation, and no. 147 killed on Day 1 of lactation) and for four females that received 320 mg/kg bw/day (nos.151,154 and 157 with total litter loss on Days 1/2 of lactation and no.159 killed on Day 1 of lactation).

Post-implantation survival and the live birth index were low at 160 and 320 mg/kg/day resulting in low litter sizes at this dose levels.

The offspring body weight for those assessed on Day 1 of age was low when compared with Controls and examination of the decedent offspring predominately revealed an absence of milk in the stomach.

F0 Generation
The target dose levels on this study were 4, 40, 80, 160 and 320 mg/kg/day and the mean
achieved dose levels were as follows:

• F0 males: 3.90, 38.7, 76.0, 152, and 308mg/kg bw/day (95-98% of intended)
• F0 females before pairing: 4.52, 44.6, 94.6, 177 and 378 mg/kg bw/day (111-118% of
  intended)
• F0 females during gestation: 4.97, 50.4, 90.6, 198 and 338 mg/kg bw/day (106-126% of
  intended)
• F0 females during lactation: 5.74, 54.0 and 92.1 mg/kg/day (115-144% of intended) at 4,
  40 and 80 mg/kg bw/day, respectively.

Routine physical examination of males from the start of treatment up to termination and for females before pairing, after mating and during lactation did not reveal any findings that could be attributed to administration of Silver Acetate Overall the mean body weight gain for males at 320 mg/kg bw/day was markedly low at approximately 20% of Controls, resulting in low mean body weight at the end of the treatment period.

The overall body weight gain of F0 females during the four-week pre-pairing treatment period was low at the target dose of 320 mg/kg bw/day at approximately 38% of Controls. From Day 3 of gestation body weight gain for females at 320 mg/kg bw/day was low resulting an overall body weight gain that was approximately 42% of Controls and a low mean body weight on GD20. Assessment of body weight gain during lactation which was restricted to females at the target dose levels of 4, 40 and 80 mg/kg bw/day showed poor body weight performance during Days 4-7 and Days 11-14 for females receiving 80 mg/kg bw/day. For the majority of food consumption phases F0 males at 320 mg/kg bw/day showed statistically significantly low mean values when compared with Controls (p<0.01).
Before pairing females at 320 mg/kg bw/day showed periods of low food consumption and subsequently females at this target dose level showed consistently low food consumption throughout gestation. Assessment of food consumption during lactation which was restricted to females at the
target dose levels of 4, 40 and 80 mg/kg bw/day showed no adverse effects of treatment.

Assessment of estrous cycles, pre-coital interval, mating performance and fertility assessment did not show any adverse effects of treatment. At 40 mg/kg/day there was a shift towards a slightly longer gestation length (p<0.05) but the gestation index was 100%. Assessment of the gestation length at 160 mg/kg bw/day and 320 mg/kg bw/day was limited to females that littered before the groups were terminated prematurely and showed a shift towards a slightly longer gestation length.

At scheduled termination males at target dose levels of 160 or 320 mg/kg bw/day showed statistically significantly low hematocrit, hemoglobin level, mean cell haemoglobin, mean cell haemoglobin concentration and mean cell volume (p<0.01); these was no evidence for a dose response. Males at 40 mg/kg bw/day also showed a low mean cell haemoglobin (p<0.05). Platelet counts for males that received 40, 160 or 320 mg/kg bw/day were slightly high (p<0.05; p<0.01). Alkaline phosphatase activity and cholesterol levels for males and females at 40 mg/kg bw/day and for males at 160 and 320 mg/kg bw/day were high (p<0.05; p<0.01, except female cholesterol); at 4 mg/kg bw/day the mean cholesterol levels for males was also slightly high (p<0.05). Other minor differences included: high alanine amino transferase activity and calcium for males at 320 mg/kg bw/day (p<0.01); slightly low chloride levels for males at 320 mg/kg bw/day (p<0.01); high phosphorous levels for females at 40 mg/kg bw/day (p<0.05); high albumin/globulin ratio for females at 40 mg/kg bw/day (p<0.05).

At scheduled termination the body weight relative liver weight for males that received target dose levels of 160 or 320 mg/kg bw/day were high when compared with Controls (p<0.01); the body weight relative liver weight at 80 mg/kg bw/day also looked slightly high for male and female animals but in the absence of a concurrent control for this dose group a true assessment could not be made.

At scheduled termination, Silver Acetate-related dark coloration of the liver was observed in males administered 40, 160 and 320 mg/kg bw/day and correlated with pigment deposition observed at microscopic examination. The same macroscopic lesion was observed in females administered 40 and 80 mg/kg/day. Silver Acetate-related dark content of cecum was observed in males administered 160 and 320 mg/kg bw/day and in females administered 80 mg/kg bw/day; dark content of rectum was observed in males administered 320 mg/kg/day and in females administered 80 mg/kg bw/day. Silver Acetate-related abnormal color of the pancreas and mesenteric lymph nodes was observed in males administered 40, 160 and 320 mg/kg bw/day and in females administered 40 and 80 mg/kg bw/day.
Silver Acetate-related dark coloration of the uterus, kidney, thymus, urinary, bladder and salivary gland was observed in females administered 80 mg/kg bw/day; one female at 40 mg/kg bw/day exhibited dark mandibular salivary glands and uterus. Pigment was observed in Kupffer cells and to a lesser extent in hepatocytes and in the vascular walls of the liver of males administered 160 and 320 mg/kg bw/day and correlated with macroscopic dark liver color. Centrilobular hepatocellular hypertrophy was observed in males administered 160 and 320 mg/kg bw/day and correlated with increased liver weight.

F1 Litter Responses
Group 4 (160 mg/kg/day) and Group 5 (320 mg/kg/day) were terminated prematurely late gestation/late lactation after four total litter losses at 320 mg/kg/day and two at 160 mg/kg/day. Assessment of litter responses was therefore restricted to target dose levels of 4, 40 or 80 mg/kg/day.

There was no clear effect of treatment on the clinical condition of offspring at 4, 40 or 80 mg/kg/day; one litter at 80 mg/kg/day no. 174 was observed with whole body pallor from Day 7 to Day 14 of age and the coat was recorded as patchy on Days 20/21 of age.

Litter size, offspring survival, sex ratio and ano-genital distance showed no adverse effects of treatment at target dose levels up to and including 80 mg/kg bw/day. Offspring body weight on Day 1 of age and subsequent body weight gain up to Day 7 of age was unaffected by parental treatment at dose levels up to and including 80 mg/kg bw/day. At 4 and 40 mg/kg bw/day offspring bodyweight gain from Days 7-11 of age was slightly low (p<0.05) when compared with Controls.

Offspring body weight at 80 mg/kg bw/day did not show any signs of overt toxicity but in the absence of concurrent Control assessment was limited Offspring Brain Weight on Day 21 of age was unaffected by administration of Silver Acetate at dose levels up to and including 80 mg/kg bw/day.

Macroscopic examination of offspring that died prior to scheduled termination did not reveal any findings at dose levels up to and including 80 mg/kg bw/day that could be attributed to administration of Silver Acetate.

At scheduled termination of unselected offspring on Day 21 of age macroscopic findings were restricted to one litter at 80 mg/kg/day (litter no. 174) where all offspring exhibited a pale liver and generalized hairloss, one offspring had a diaphragmatic hernia and one offspring had a liver mass on the median liver lobe.

Detailed brain histopathology of F1 offspring at Day 21 of age did not reveal any pathological changes or developmental abnormalities.

F1 Generation
Group 4 (160 mg/kg bw/day) and Group 5 (320 mg/kg bw/day) were terminated prematurely late gestation/late lactation after four total litter losses at 320 mg/kg bw/day and two at 160 mg/kg bw/day. Assessment of F1 responses was therefore restricted to phase 1 animals receiving 4 or 40 mg/kg bw/day; litters at 80 mg/kg bw/day (Group 7) were terminated on Day 21 of age.

The target dose levels on this phase of the study were 4 and 40 mg/kg bw/day and the achieved dose levels were as follows:

• F1 males: 4.5 and 45.0 mg/kg bw/day (113% of intended)
• F1 females: 4.3 and 46.2 mg/kg bw/day (108-116% of intended)

There were no clinical signs at routine physical examination that could be attributed to administration of Silver Acetate at 4 or 40 mg/kg bw/day At weaning on Day 21 of age mean body weight was similar across the groups for both males and females.

The mean body weight for selected males on Day 1 of the F1 generation was similar across the groups, however the overall gain for males from Day 1 to Day 29 was low at 4 and 40 mg/kg bw/day. The resultant mean body weight at these target dose levels were low when compared with Controls.
On Day 1 of the F1 generation the mean bodyweight for females at the target dose of 40 mg/kg bw/day was slightly low and this difference in mean body weight persisted up to Day 29 of the F1 generation. Overall the body weight gain for females at 40 mg/kg bw/day was slightly low at 90% of Controls but this difference did not attain statistical significance.

Food consumption for selected F1 animals from Days 1 to 29 of the F1 generation was unaffected by administration of Silver Acetate at 4 or 40 mg/kg bw/day. The age at completion of sexual maturation for both males and females was unaffected by administration of Silver Acetate at target dose levels of 4 and 40 mg/kg bw/day. The mean body weight on the day of completion of preputial separation was slightly but statistically significantly low for males receiving 4 or 40 mg/kg bw/day; females at 40 mg/kg bw/day also showed slightly low mean body weight at vaginal opening but this difference did not attain statistical significance.

The absolute mean brain weight for selected F1 females that received 40 mg/kg bw/day was slightly but statistically significantly low when compared with Controls (p<0.05); this difference was not evident for males at this dose level. Macroscopic examination of F1 animals at scheduled termination following attainment of sexual maturation did not reveal any abnormality.

Conclusion
Based on the findings in this study, dose levels of 160 and 320 mg/kg bw/day were not tolerated and were considered unsuitable for the subsequent OECD 443 study. As no significant adverse effects were observed in parnetal animals or in F1 pups (to weaning) at 80 mg/kg bw/day, the optimal high dose level for the OECD 443 study is considered to be between these levels, at 120 mg/kg bw/day.

Reference 3

Endpoint:

reproductive toxicity, other

Remarks:

Investigation of reproductive organs of animals exposed to test item during 13 weeks

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Weight of evidence approach described in 'Silver metal (massive and powder): Weight of Evidence' (document attached in IUCLID section 13 - "CSR Annex 11 - Weight of Evidence Justification for Silver metal - human health endpoints).

Qualifier:

no guideline available

Principles of method if other than guideline:

Seven-week-old rats (10 rats per sex per group) were exposed nanosilver 110 nm (AgNP-110nm) at 9, 18 and 36 mg/kg bw; and controls (2mM sodium citrate (CIT)at termination the productive system were analysis

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

In order to determine the amount of silver in the AgNP stock suspensions that was in ionic form, filtrates of the AgNP suspensions were prepared by centrifugation through a microcentrifuge cellulose filter that had a molecular weight cut-off of 3 kDa (AmiconVR Ultra-4 centrifugal filter units; Millipore Corporation, Billerica, Massachusetts). Subsamples (1 ml) of the pre-mixed AgNP stock suspensions were applied to the Amicon filter units and centrifuged at 4000g for 30 min in an Eppendorf 5810 R centrifuge that was equipped with an Eppendorf A-4-81 swinging bucket.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- 3-week-old male and female Sprague Dawley/CD-23 rats with specific pathogen-free health status were obtained from the NCTR breeding colony.
- At 6 week of age, the rats were weight-ranked and randomly assigned to treatment groups. Male and female rat were housed conventionally in separate animal rooms with 2 animals per cage.
- The environment of the animal rooms was set to maintain a 12h light cycle, temperature of 22+/-4°C, relative humidity of 40%-70%, and air changes of 10-15 per hour.
- The animals were provided NIH-41 gamma-irradiated pellets and Millipore-filtered drinking water ad libitum.
- Rats were dosed initially at 7 weeks of age.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

The high-dose formulation (0.9 mg/ml) for each size of AgNP was prepared in the CIT/CMC vehicle (2mM sodium citrate/ 0.1% CMC; final concentration; wt/wt), CIT= trisodium citrate dihydrate

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared weekly and were based on the total silver mass concentrations obtained by ICP-MS for the batch stock AgNP suspensions. The high-dose formulation (0.9 mg/ml) for each size of AgNP was prepared in the CIT/CMC vehicle (2mM sodium citrate/ 0.1% CMC; final concentration; wt/wt),

- The total silver concentrations of the high-dose formulations were confirmed by ICP-MS and, subsequently, the AgNP high-dose formulations were serially diluted in the CIT/CMC vehicle to achieve the 0.45 and 0.225 mg/ml concentrations for the mid- and low-dose formulations, respectively.

DOSE CHARACTERIZATION: The total silver concentration by mass of each of the prepared dose formulations (high-, mid-, and lowdose) and the controls (CIT/CMC and water/MC) was determined by ICP-MS in acid- and microwave-digested samples. The hydrodynamic sizes of particles in the dosed solutions were determined by DLS, and the core diameters and aspect ratios of the AgNP were evaluated by TEM. The median number of particles
evaluated by TEM per sample was 214 (range 44–498). Characterization analyses were conducted on triplicate samples. Dose concentration acceptability was set at 610% targeted
values.

VEHICLE
Carboxymethyl cellulose (CMC) and trisodium citrate dihydrate (CIT) were purchased from Sigma Aldrich (St. Louis, Missouri), and methyl cellulose (MC) was purchased from Fisher Scientific (Fair Lawn, New Jersey). The water used in dose formulations was 18 megaohm and autoclave-sterilized.

Route Exposure: Ingestion is the primary route of exposure in humans for silver, silver compounds, and colloidal silver (Silver, 2003). Daily intakes for silver in humans are estimated in the range 0.4–27 mg/day (Clemente et al., 1977; Gibson and Scythes, 1984; Hamilton and Minski, 1972/1973). The Environmental Protection Agency lists the reference dose for silver as 0.5 mg/kg/day or 35 mg/day for an average 70 kg adult (EPA, 2014).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In order to determine the amount of silver in the AgNP stock suspensions that was in ionic form, filtrates of the AgNP suspensions were prepared by centrifugation through a microcentrifuge cellulose filter that had a molecular weight cut-off of 3 kDa (AmiconVR Ultra-4 centrifugal filter units; Millipore Corporation, Billerica, Massachusetts). Subsamples (1 ml) of the pre-mixed AgNP stock suspensions were applied to the Amicon filter units and centrifuged at 4000g for 30 min in an Eppendorf 5810 R centrifuge that was equipped with an Eppendorf A-4-81 swinging buck

Duration of treatment / exposure:

Groups of rats (10 males and 10 females) were exposed daily by oral gavage to dose formulations of AgNP-110 nm at 9, 18, and 36 mg/kg bw; or to the respective control formulations (CIT/CMC) for a period of 13 weeks.

Frequency of treatment:

Gavage dosing was conducted using computer-controlled MicroLabVR 500 series dispensers (Hamilton Co., Reno, Nevada) equipped with gastight syringes and capable of dispensing 1 ml to 50 ml. The syringes were fitted with flexible plastic gavage needles, and the rats were provided equal volume doses based on the daily body weight of the individual rats. The MicroLab dispensers were programmed to administer the total daily dose in 2 daily gavage administrations per day, with half of the dose administered at the start of the light cycle and half of the dose administered just prior to start of the dark cycle. The dose volumes did not exceed 20 ml/kg bw (OECD, 1998). Animals were dosed 7 days each week and the study period was 13 weeks.

Dose / conc.:

9 mg/kg bw/day (actual dose received)

Dose / conc.:

18 mg/kg bw/day (actual dose received)

Dose / conc.:

36 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The aim of this study was to investigate the potential toxicity of AgNP at doses selected to provide the maximal concentrations that could be achieved with the citrate stabilize particles
and that would also ensure stability of the nanoparticles. The nominal concentrations (1 mg/ml) of the AgNP stock suspensions and the maximum gavage dose volume of 20 ml/kg bw per dosing episode (OECD, 1998) set the high dose level for AgNP at 36 mg/kg bw (0.9mg/ml40 ml). Oral gavage, rather than drinking water or dosed feed, was the selected method of dose administration to ensure that the suspensions of AgNP were dispersed at the time of introduction to the animals and to ensure the precision of the dosed amount.

- Fasting period before blood sampling for clinical biochemistry: terminal sacrifices were conducted on over-night fasted rats.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly clinical observations on individual animals were recorded in the animal records database (NCTR Multi-generation Computer Support System; MGSS).

BODY WEIGHT: Yes
- Time schedule for examinations: body weights recorded daily in MGSS for dose administration.

FOOD CONSUMPTION AND COMPOUND INTAKE: Feed and water consumptions were measured and recorded weekly in MGSS.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at terminal and moribund sacrifices
- Anaesthetic used for blood collection: Yes: Animals were euthanized humanely by carbon dioxide asphyxiation after the 72h blood sample collection.
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at terminal and moribund sacrifices
- Animals fasted: Yes

IMMUNOLOGY: A section of the ileum and scrapping of the ileal mucosa were collected for intestinal microbiota and immune response evaluations (Williams et al., 2015).

Oestrous cyclicity (parental animals):

Estrous cycling check in female rats was conducted by vaginal lavage each of 16 consecutive days prior to terminal sacrifice. A sterile glass medecine dropper was used to gently flush the vagina (2 or 3 times) with physiological saline, and the vaginal lavage were placed on glass slides, air dried, and fixed by immersion in methanol. the slides were stained with 0.5% toluidine blue and the cells were exaluated by light microscopy for stage of estrous cycle (diestrus, proestrus, or estrous). Estrous cycle length was assessed in female rats for 16 days prior to sacrifice, and the proportion of days spend in each estrous phase (diestrus, proestrus, and estrus) was analyzed for treatment differences using the arcsine-square root transformed proportion with a 1-way ANOVA.

Sperm parameters (parental animals):

The left testis and the caudal epididymis of male rats were used to determine sperm counts, motility, and morphology. Sperm motility, testes count, cauda count, and sperm morphology (number of normal/abnormal of 200 sperm) were analyzed for treatment differences using a 1-way ANOVA, and pairwise comparisons were performed with Bonferroni adjustments.

The left testis and the caudal epididymis of male rats were used to determine sperm counts, motility, and morphology. For testis sperm count, the tunica albuginea was removed, and the parenchyma was weighed and homogenized in distilled water. A sample of the testis suspension (100 ml) was added to a vial, and the total sperm count evaluated with a sperm analyzer (12.3 Tox Integrated Visual Optical System, Hamilton Thorne Biosciences, Beverly, Massachusetts). Sperm motility and morphology were determined on the caudal epididymis. The caudal portion of the epididymis was placed in buffer (1% bovine serum albumin in phosphate-buffered saline), and the distal portion of
the cauda was pierced 3 times to allow sperm to migrate. The onset and completion times were recorded for the migration period. Aliquots of the caudal suspension were loaded onto a chamber of the sperm analyzer (Hamilton Thorne Biosciences), and sperm motility was determined by the instrument automatically in 5 scanned image fields. For caudal sperm count, a sample of the caudal suspension (100 ml) was added to a vial, and the total sperm count evaluated with the sperm analyzer (Hamilton Thorne Biosciences). Morphological features of sperm were evaluated on the same suspensions used for sperm motility. Aliquots of the suspensions were placed on glass slides, dried, and stained with a 5% eosin aqueous solution. Morphology was examined manually by light microscopy in a total count of 200 normal sperm.

Postmortem examinations (parental animals):

At necropsy, organs and tissues were examined for grossly visible lesions, and protocol designated tissues and organs were weighed and preserved in 10% neutral buffered formalin (NBF), with the exception that modified Davidson’s fixative was used for the right testes and eyes. Femur bone marrow was collected for histopathology (left) and ICP-MS (right). The testes and epididymides were collected for sperm analysis (left) and histopathology (right).

Statistics:

A Cox proportional hazard model was used to analyze survival data and to test the effect of treatment relative to control. All tests were conducted as 2-sided with significance at the .05 probability level.
Body weights, feed consumption, water intake, and blood were analyzed using a 1-way repeated measures, mixed model ANOVA for each sex, with terms for dose, week, and all interactions.

The statistical analysis of hematology and clinical chemistry data was performed using a non-parametric method with midranks (Brunner et al., 2002).
Pairwise comparisons were performed with Bonferroni adjustments. All tests were conducted as 2-sided with significance at the .05 probability level.

The organ weights were analyzed using a 1-way analysis of covariance with necropsy body weights as a covariate. Pairwise comparisons were performed with Bonferroni adjustments.

Sperm motility, testes count, cauda count, and sperm morphology (number of normal/abnormal of 200 sperm) were analyzed for treatment differences using a 1-way ANOVA, and pairwise comparisons were performed with Bonferroni adjustments.

Estrous cycle length was assessed in female rats for 16 days prior to sacrifice, and the proportion of days spend in each estrous phase (diestrus, proestrus, and estrus) was analyzed for treatment differences using the arcsine-square root transformed proportion with a 1-way ANOVA.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no significant differences in survival between the vehicle and water controls or between any AgNP group and the vehicle control group in either the female or male rats.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Five animals in this study were either moribund or died with severe acute nephropathy. These types of renal changes have been noted in Sprague Dawley rats in previous studies conducted at NCTR and are considered spontaneous findings and not treatment related.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant differences in feed and water intakes between the vehicle and water controls. When compared with respective control animals, sporadic differences in feed and water intakes were observed in female and male rats administered AgNP

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

Hematological and plasma clinical chemistry parameters recorded for rats exposed to AgNP show no significant differences in the values for these parameters between the vehicle and
water controls, and values recorded for the AgNP-treated groups did not differ significantly from the vehicle controls.

Clinical biochemistry findings:

effects observed, non-treatment-related

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A notable observation by light microscopy was the occurrence of diffuse brown pigmentation in numerous organs and tissues of rats exposed to AgNP.

In many respects, the detection of pigmentation was more a measure of silver mobility rather than toxicity, and pigmentation was graded for severity as (1) minimal, (2) mild, (3) moderate, or (4) marked.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No statistically significant differneces between control groups or between any treatment and control groups in proportion of days in each phase of the estrous cycle or in the frequency of transition between normal and abnormal states.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The evaluation of rat testes revealed that there were no statistically significant differences in sperm motility, testis sperm count, caudal sperm count, or sperm morphology.

Reproductive performance:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

36 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

Key result

Remarks on result:

not determinable because of methodological limitations

Remarks:

F1 generation was not performed

Key result

Reproductive effects observed:

no

Characterization of AgNP stock suspension and dose formulation:

The proportion of silver in the AgNP stock solutions that was present in
dissolved ionic form was determined by means of ultrafiltration over a 3-kDa membrane. TEM examination of filtrates confirmed that AgNP did not cross the membrane, and ICP-MS determinations of the pre-filtered samples, filtrates, and resuspensions of the concentrated solutes and filtrates indicated an average recovery>98%, irrespective of AgNP size (data not shown). The mean concentration of ionic silver in the AgNP suspensions was similar across nanoparticle sizes and averaged 0.01%–0.02% of the AgNP stock suspensions. The size, morphology, and distribution parameters of the AgNP stock suspensions were determined by TEM and DLS. AgNP size derived from TEM determinations indicated that mean core sizes for the 110 nm particles were 101.56 +/-15.8 nm. These results closely corroborate the data provided by the manufacturer with each shipment of the AgNP suspensions. The morphological characteristics of the AgNP were also evaluated by TEM, and the mean aspect ratios confirmed that the AgNP particles were mostly spherical in shape.

The hydrodynamic size was determined for the AgNP by DLS (z-average). In the examples, the largest peak in the graph represents 100% intensity, with z-average values of 103.7nm for the 110 nm AgNP. The size determinations by DLS were larger than those measured by TEM for the same particle suspension. The AgNP used in this study were stabilized with the citrate capping agent, likely accounting for the larger hydrodynamic size when measured by DLS, suggesting that the actual size of the particles reside somewhere within the upper TEM and lower DLS range of values.

The concentrations of total silver for the AgNP dose and control formulations
were determined by ICP-MS. The levels of total silver for each of the control formulations were below the limit of quantitation (40 ng/mL) by ICP-MS thourghout the study. Dose formulations were prepared on a weekly basis at 3 concentrations level for each size of AgNP (0.225, 0.45 and 0.9 mg/mL) and the toal silver concentration of each formulation was determined by ICP-MS prior issuance of the dose formulation to animals on the study. The mean percentage of targeted dose for all doses over the study period was 104+/-2% for the 110nm AgNP formulations.

Homogeneity and Stability of the AgNP Suspensions
The results of homogeneity and stability tests indicate that the mixing procedure provided evenly dispersed particles throughout the vessel. Stability testing by
DLS and TEM was conducted on the same combined lot for each particle size suspension and by ICP-MS on filtrates for ionic silver determinations for 90 days. The hydrodynamic size of the particles determined by DLS and the core size of the particles determined by TEM remained stable over the testing period. ICP-MS analysis of filtrate samples showed only a very slight increase in soluble silver formed over the 90-day period.

Dose formulations for the AgNP were prepared in their respective control formulations that contained either 0.1% CMC. CMC was used in this study as
bulking agents to inhibit somewhat the gastrointestinal passage of the mostly aqueous dose formulations. This compounds is not toxic and do not promote allergic reactions in humans or rodents (Frawley et al., 1964; Shelanski and Clark, 1948). The hydrodynamic size of particles were higher in the dose formulations,
when compared with the stock solutions, and the CMC was likely responsible for the increase in particle size, as determined by DLS. The mean core diameters
and aspect ratios of the particles were similar to those of the stock solutions, suggesting that little change occurred in the particle size and shape during the preparation of dose formulations.

Histopathology further details:

The incidences of pigmentation observed by light microscopy among rats that were administered the AgNP treatments conveyed a similar story as told by the results of silver distribution obtained by ICP-MS. The severity of pigmentation in AgNP treatment groups was graded as minimal, with only a few instances of mild pigmentation. Pigmentation was more prevalent in female rats than male rats, especially in the mesenteric lymph nodes, the large intestine (except the rectum), the stomach, kidneys, and spleen. However, the high incidences
(~80%–100%) of pigmentation in most every tissue of female rats tended to mask any differences due to AgNP size. Male rats administered AgNP showed a similar pattern of pigmentation as female rats; however, incidences of pigmentation were much lower and demonstrated a size-dependent prominence, with the AgNP-10 nm having greater prominence than either the AgNP-75nm or 110 nm treatments. Evaluations by light microscopy were able to discern the presence or absence of silver pigmentation in tissue and organ specimens; however, this method could not quantify differences in the intensity of the pigmentation at the levels of accumulation observed for AgNP-exposed rats.

Disposition and accumulation of silver:

The analysis of silver concentrations by ICP-MS in tissue and organ samples collected from the control groups showed no significant differences in silver concentrations between the water and vehicle controls for any reported parameter; however, all tissues and organs analyzed from rats exposed to AgNP for 13 weeks, with the exception of bone marrow, revealed statistically significant
(P<.05) dose-dependent increases in silver concentrations. 

The silver content in blood and bone marrow averaged 3–4 times lower than the silver content of the heart, which had the lowest content of silver among the major organs analyzed and indicated that the contribution of silver accumulation in the blood and bone marrow was minimal. Furthermore, a repeated measures statistical analysis of these data found no significant differences for silver concentrations in rat blood collected at weeks 1 and week 12. As blood levels of silver were not significantly higher after 12 weeks of dosing than levels found after only 1 week of dosing, these results suggest rapid clearance of silver from the blood for all groups irrespective of silver form. In preliminary studies, the half-time for elimination of silver from the blood
was ~24 h regardless of sex of form of silver.

Results of pairwise comparison tests indicated that silver concentrations were significantly higher in the blood and bone marrow of rats exposed to AgNP-10 nm, especially at the 2 higher doses, when compared with those of rats exposed to
AgNP- 75 or 110nm and suggested that the concentration of silver in these samples was inversely related to particle size. 

In female and male rats exposed to AgNP the kidney and spleen represented sites of significant silver accumulation. As observed in the intestinal tract, sex differences in silver concentrations were observed. The concentrations of silver in all organs were significantly lower in male rats exposed to AgNP. In addition, the pattern of distribution in the kidneys and spleens of male rats differed from that
of female rats.

The concentrations of silver in all organs were several folds higher in rats exposed to AgAc, and, as observed with rats exposed to AgNP, the concentrations of silver in all organs appeared to be higher in females than males.

Conclusions:

In conclusion, rats vaginal cytology, or sperm count, motility and morphology is not affected by the exposure to AgNP-110nm up to 36 mg/kg bw/day

Executive summary:

Seven-week-old rats (10 rats per sex per groups) were randomy assigned to treatments: AgNP-110nm at 9, 18 and 36 mg/kg bw/d, and control (CMC/CIT). At termination, compete necropsies were conducted, histopathology, hematology, serum chemistry and reproductive system were performed. Estrous cycle length was assessed in female rats for 16 days prior to sacrifice.

Rats vaginal cytology, or sperm count, motility and morphology is not affected by the exposure to silver acetate (up to 36mg/kg bw/dy (highest dose tested).

In conclusion, rats reproductive system is not affected by exposure to nanosilver (75nm)

 

 

 

 

 

Reference 4

Endpoint:

developmental immunotoxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

lack of GLP compliance, lack of individual animal data, immunotoxicology testing parameters not optimised in line with latest testing approaches (including those integrated into the OECD TG 443 for the EOGRTS), monitors of certain other confounding influences relevant to silver were not included

Justification for type of information:

Weight of evidence approach described in 'Silver metal (massive and powder): Weight of Evidence' (document attached in IUCLID section 13 - "CSR Annex 11 - Weight of Evidence Justification for Silver metal - human health endpoints).

Qualifier:

according to guideline

Guideline:

other: US protocols for food additives (FDA CFSAN Redbook, 2000)

GLP compliance:

no

Remarks:

publication lack of GLP compliance

Limit test:

no

Specific details on test material used for the study:

The test article was obtained from Sigma-Aldrich and stored according to the conditions (dry, at room temperature) detailed in the MSDS Sheets supplied by the manufacturer.
Stability of the test material was determined to be greater than 21 days at room temperature.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague-Dawley rats [Crl:CD@ (SD) IGS BR] were obtained from Charles River Laboratories. Upon receipt, all animals were identified by ear tag.
- Females nulliparous: yes
- Age at study initiation: 4 weeks
- Weight at study initiation: (P) Males: 119.5 ± 2.2 g; Females: 104.8 ± 2.3 g
- Fasting period before study: not specified
- Housing: all rats were individually housed in polycarbonate tubs with shredded cardboard bedding throughout the study except during mating. During mating males and females were cohabitated overnight (approximately 17 h) in polycarbonate tubs containing a metal grate insert flooring.
- Diet: ad libitum; Rodent diet (Tekland Certified Global 18% Protein Rodent Diet (meal) 2018CM; Lot Number 2018CM-020812MA) obtained from Harlan laboratory (P.O. Box 44220, Madison, WI 53744-4220); Feed was provided in Holtge feeders; A certification analysis was performed on the rodent diet by the manufacturer; Eurofins analysis of the diet indicated that it contained no contaminants know to interfere with the study.
- Water: ad libitum; Hydropure water
- Acclimation period: approximately 1 week during which time they were observed daily for clinical signs of disease.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-78
- Air changes (per hr): not specified
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: drinking water

Vehicle:

water

Remarks:

Deionized double distilled HydroPure water (Hydro-Pico Systems, Inc., Research Triangle Park, NC, USA); analysis indicated that the concentration of silver in the Hydropure water was not detectable.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The target concentrations of the dosing solutions utilized in this study were 0.0 mg/kg bw, 0.4 mg/kg bw, 4.0 mg/kg bw and 40.0 mg/kg bw (equivalent to approximately 0, 0.26, 2.6 or 26 mg ionic Ag/kg bw).
- All dosing solutions were formulated in ultra-pure water from the same source as the vehicle controls.
- All doses were administered through water bottles. Animals received either control or dosing solutions in 8 ounce water bottles fitted with straight sipper tubes during the 10 week pre-mating treatment and gestation periods and 16 ounce water bottles fitted with straight sipper tubes during lactation. Water bottles were changed every 3 days.
- Dosing solutions were prepared fresh every 3-6 days and analyzed before they were administered to the study animals.
- The concentration of the dosing solutions for a specific group was formulated so that experimental animals received the appropriate exposure dose of the test solution. The concentration of the test article in the dosing solutions for each experimental dose group were predicted three to six days in advance based on average fluid consumption and body weight gain for animals in a given group.

Details on mating procedure:

- M/F ratio per cage: not specified
- Length of cohabitation: overnight (approximately 17 h); until evidence of mating was obtained or for a maximum of 14 days
- Proof of pregnancy: copulation plug in the vagina or bottom of the mating cage and/or presence of sperm in a vaginal smear
- Females that did not mate at the end of 1 week were paired the following week with a different randomly selected male from the same treatment group.
- Males were subsequently euthanized and necropsied after exposure day 90.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing solution concentrations were verified using an Orion Star pH/ISE meter (Thermo) and Silver/Sulfide electrode with corresponding double-junction reference electrode (Thermo) using direct calibration method following the manufacturer’s recommendation in Electrode User Guide.
Maintenance of the target dose was achieved throughout the pre mating, gestation and lactation periods. The actual dose received for all phases of the study was within 10-15% of the target dose. Only P generation males in the 0.4 mg/kg dose group received a slightly higher dose of silver acetate (0.51 ± 0.06 mg/kg) than the specified target dose. However, potential adsorption losses of silver (e.g. on to container surfaces) were not specifically investigated.

Duration of treatment / exposure:

P generation males and females were exposed to the test material for 10 weeks prior to mating. Treatment continued through the mating period until study termination and included the gestation and lactation periods for females. Selected F animals were not exposed to the test material after Lactation Day 21.
At lactation day 4, litters were culled by random selection to 5 males and 5 females when possible. Randomly selected culled animals (PD4) were utilized for immunological evaluations (n = 10 pups/gender/dose group). At lactation day 21, randomly selected F generation male and female pups were individually housed but were not exposed to silver acetate. At postnatal day 26 randomly selected pups were utilized for immunological evaluations (n = 10 pups/gender/dose group).

Frequency of treatment:

Continuously (through drinking water)

Dose / conc.:

0.4 mg/kg bw/day (nominal)

Dose / conc.:

4 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 for P generation exposure; 10 for F generation immunological observations

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: P generation males and females were randomized into 4 treatment groups through a standard (by weight) block randomization procedure. Sperm/plug positive P generation females remained within their dose groups and were continuously exposed to silver acetate through gestation and lactation up through lactation day 21. At lactation day 4, litters were culled by random selection to 5 males and 5 females when possible.
Randomly selected culled animals (PD4) were utilized for immunological evaluations (n = 10 pups/gender/dose group). At lactation day 21, randomly selected F generation male and female pups were individually housed but were not exposed to silver acetate. At postnatal day 26 randomly selected pups were utilized for immunological observations (n = 10 pups/gender/dose group).

Positive control:

No positive control was included in the study.

Parental animals: Observations and examinations:

Reported in separate publication (see Sprando et al. 2017)

Oestrous cyclicity (parental animals):

Not investigated.

Sperm parameters (parental animals):

Not investigated.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- 10 pups/litter (5/sex/litter when possible)
- randomly selected culled animals (PD 4) were utilized for immunological investigations (n = 10 pups/gender/dose group). At lactation day 21, randomly selected F generation male and female pups were individually housed but were not exposed to silver acetate. At postnatal day 26 randomly selected pups were utilized for immunological observations (n = 10 pups/gender/dose group).

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Yes

(Other examinations are reported in separate publication - see Sprando et al. 2017)

Postmortem examinations (parental animals):

Reported in separate publication (see Sprando et al. 2017)

Postmortem examinations (offspring):

SACRIFICE
PD 4 pups were euthanized by decapitation and PD 26 pups were euthanized with carbon dioxide. The thymus and spleens from PD 4 and PD 26 pups were collected from 10 pups/gender/dose group.

In this study, the effects of silver acetate exposure during pre-mating and pre and post-gestational periods on post natal (PD4 and PD26) splenic and thymic cell populations and functional adaptive and innate immunocompetence were determined using ex vivo models.
(Other examinations are reported in separate publication - see Sprando et al. 2017)

IMMUNOLOGICAL EVALUATION
- Isolation of mononuclear cells from spleen and thymus: Spleens and thymuses were minced in Hanks' Balanced Salt Solution, filtered and the single cell suspensions were over-layered on Histopaque (density 1.083 g/ml). Mononuclear cells (MNC) were obtained from the interface after centrifugation at 400g for 30 min at room temperature. Cells were washed and the final pellet was adjusted to 4 x 10^6 cells/ml for proliferation assay (in vitro mitogen reactivity) or to 1 x 10^7 cells/ml for natural killer (NK) activity measurement and for flowcytometric analyses of phenotypic cell surface markers.

- Mitogenic reactivity in vitro: Mitogenic reactivity to concanavalin A (Con A, Sigma, St Louis, MO) and E. coli lipopolysaccharide (LPS, Sigma) was assessed. Aliquots of splenic MNC (2 x 10^5 cells) in RPMI 1640 medium with 10% fetal bovine serum were incubated with Con A (0.4, 0.8 or 1.6 mg/well) or LPS (1.25, 2.5 or 5.0 mg/well) for 72 h at 37 °C and in 5% CO2 atmosphere. At the end of 48 h, cells were incubated with 0.5 mCi 3H-thymidine (PerkinElmer, Waltham, MA), for an additional 24 h. Cells were harvested using a 96 well automatic harvester and counted in a microbeta counter (PerkinElmer). Data are presented as stimulation indices, which is the ratio of the counts from stimulated cultures and those from non-stimulated cultures.

- Immunophenotyping by flow cytometry: Immunophenotyping was carried out using freshly isolated MNC from spleen and thymus, and quantified using FACS Aria II flow cytometer (BD Biosciences, CA). Briefly, splenic and thymic MNCs were incubated with the monoclonal antibodies specific to rat cell surface antigens including CD3 labeled with Phycoerythrin (PE)-conjugated anti-rat CD3 (clone G4.18, total T), CD4 labeled with fluorescein isothiocyanate (FITC)-conjugated anti-rat CD4 (clone OX-35, T helper/ inducer), CD8 labeled with FITC- conjugated anti-rat CD8a (clone OX-8), anti-rat CD8b (clone 341,T suppressor/cytotoxic), CD45 labeled with FITC-conjugated-anti rat CD45RA (clone OX-33, B cell marker) and ab TCR labeled with FITC-conjugated-anti rat TCR (clone R73), for at least 45 min at 4 C. Samples were then washed and fixed until further analysis. Antibody pairing was done for two-color analysis. Before sample analysis, flow cytometer was optimized using isotypic IgG controls and five thousand cells were analyzed for each antibody combination. All the antibodies that were conjugated with FITC were paired with CD3 PE.

- Interleukin-2 measurement in Con A induced spleen cell supernatants: Splenic MNC were cultured in the presence or absence of Con A and the supernatants were collected 48 h later and stored at 80 °C until analyzed for interleukin-2 (IL-2) using commercially available kit from Bio-rad laboratories (Hercules, California), according to the manufacturer's recommendations.

- Natural killer cell activity: YAC-1 target cells were incubated with 300 µCi [51Cr] NaCrO4 overnight. After washing thoroughly with PBS, the target cells were resuspended in RPMI containing 10% fetal bovine serum at 10^5 cells/ml. Target cells (100 µl) were incubated with a proportionate volume of splenic MNC (effector cells) using the ratio of
1 target: 25-100 effector cells for about 6 h at 37 °C in 5% CO2 atmosphere. Maximum release of radioactivity was measured by adding 100 µl 0.1% triton-X100 to target cells. To measure spontaneous release, cells were incubated with RPMI medium without effector cells. Radioactivity released in the supernatants was measured using the microbeta counter. Percent NK activity was calculated using the standard formula, [(experimental release-spontaneous release)/(maximum release-spontaneous release)] x 100.

Statistics:

Student-t tests were used to compare the splenic and thymic phenotypic changes and mitogen-induced proliferative response between PD4 and PD26 pups for each treatment. Furthermore, one-way analysis of variance (ANOVA) was used followed by pair-wise multiple comparisons (Holm-Sidak method using Sigmastat
version 3.5 software) to evaluate differences among treatment groups for various thymic and splenic phenotypic and functional changes, including mitogen-induced proliferation and NK activity.
For all the statistical analyses, differences were considered significant at p < 0.05.

Reproductive indices:

Reported in separate publication (see Sprando et al. 2017)

Offspring viability indices:

Reported in separate publication (see Sprando et al. 2017)

Clinical signs:

not examined

Description (incidence and severity):

Not examined in this publication but reported in separate publication (see Sprando et al. 2017)

Mortality:

not examined

Description (incidence):

Not examined in this publication but reported in separate publication (see Sprando et al. 2017)

Body weight and weight changes:

not examined

Description (incidence and severity):

Not examined in this publication but reported in separate publication (see Sprando et al. 2017)

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

Not examined in this publication but reported in separate publication (see Sprando et al. 2017)

Food efficiency:

not examined

Description (incidence and severity):

Not examined in this publication but reported in separate publication (see Sprando et al. 2017)

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

not examined

Description (incidence and severity):

Not examined in this publication but reported in separate publication (see Sprando et al. 2017)

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not examined in this publication but reported in separate publication (see Sprando et al. 2017)

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No histopathological changes in either the spleen or thymus

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Description (incidence and severity):

Not examined in this publication but reported in separate publication (see Sprando et al. 2017)

Dose descriptor:

other: Study focusses on developmental toxicity. Details reported in Sprando et al 2017

Based on:

other: not assessed

Remarks on result:

other: study focusses on developmental toxicity. Details reported in Sprando et al 2017

Clinical signs:

not specified

Mortality / viability:

not examined

Description (incidence and severity):

Not examined in this publication but reported in separate publication (see Sprando et al. 2017)

Body weight and weight changes:

not examined

Description (incidence and severity):

Not examined in this publication but reported in separate publication (see Sprando et al. 2017)

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:

not examined

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not examined in this publication but reported in separate publication (see Sprando et al. 2017)

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

Not examined in this publication but reported in separate publication (see Sprando et al. 2017)

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathological changes in either the spleen or thymus

Histopathological findings:

no effects observed

Description (incidence and severity):

No histopathological changes in either the spleen or thymus

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

effects observed, treatment-related

Description (incidence and severity):

- Effect of silver exposure on postnatal splenic and thymic immuno phenotypes:
Lymphocyte subpopulations were evaluated by flow cytometric immunophenotyping. PND 4 pups had lower percentages of splenic CD8+ (cytotoxic) T cells in the 4 and 40 mg AgAc/kg bw/day groups. Changes in phenotypic markers in splenocytes from PND 26 pups in the same groups included significantly lower TcR+ cells, and an elevated B lymphocyte population (as represented by CD45+ cells) at 40 mg AgAc/kg bw. Although statistically significant, the magnitude of some of the splenocyte population shifts was not major and a monotonic dose-response was not always evident (e.g. at PND 4). No effects on the thymus, including on thymocyte phenotypic markers, were detected (although tissue exposure to silver was considered as having been demonstrated).

- Effect of silver acetate exposure on immune function indices – mitogenic stimulation response to Concanavalin A (ConA) or E.coli-derived lipopolysaccharide (LPS):
In respect of PD4 pup findings, group mean splenic T-lymphocytic stimulation indices (Con A) were significantly lower for all silver acetate treated groups. Although pup PD26 mean values were also lower for treated groups, only the value at the high-dose (40 mg/kg) achieved statistical significance. There were no differences in IL-2 release by Con A stimulated lymphocytes across the treated groups.
Thymic cell proliferation to Con A was not affected by silver acetate exposure for PD4 or PD26 pups. B-cell LPS stimulation indices were unaffected by treatment among PD4 pups. The high-dose group LPS stimulation index mean value showed a statistically significant decrease versus control at PD26.

- Effect of silver acetate exposure on splenic natural killer activity - an index for innate immune response
Based on measures of splenic NK activity, no adverse treatment-related effects were observed.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

0.4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental immunotoxicity

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental immunotoxicity

Reproductive effects observed:

no

Conclusions:

Rats were orally exposed via drinking water to silver acetate (AgAc) at treatment levels corresponding to 0, 0.4, 4 and 40 mg/kg bw/day (0, 0.26, 2.6 or 26 mg ionic Ag/kg bw/day). Splenic and thymic lymphocyte subsets from postnatal day (PND) 4 and 26 pups were assessed by flow cytometry for changes in phenotypic markers. Natural killer cell (NK) activity and mitogen-induced lymphocyte proliferation were selected as immune system function markers. PND 4 pups had lower percentages of splenic CD8+ (cytotoxic) T cells in the 4 and 40 mg AgAc/kg bw/day groups (2.6 or 26 mg ionic Ag/kg bw/day). Changes in phenotypic markers in splenocytes from PND 26 pups in the same groups included significantly lower TcR+ cells, and also an elevated B lymphocyte population at 40 mg AgAc/kg bw/day (26 mg ionic Ag/kg bw/day). Evidence of immune function disturbance was also presented in the paper, viz. mitogen-induced lymphocyte proliferation was apparently depressed in all three AgAc-treated groups at PND 4. However, no effects on the thymus, including on thymocyte phenotypic markers, were detected even though tissue exposure to Ag+ was evident. Innate immune response (splenic NK activity) was unaffected by treatment. Thymic development was unaffected. An important outcome was the inability to define a no observed (adverse) effect level for the immune function marker of the Concanavalin A response (mitogen-stimulated lymphocyte proliferation) in the F1 generation. It is argued that: (a) the apparent effect on splenic T-cell mitogenic stimulation response (Con A) is the only response affecting all treated groups at any timepoint; (b) clear adversity is not evident; and (c) the impacted parameter is not an optimal functional immune system marker (in comparison to TDAR). The balance of evidence (considering both splenic lymphocyte cellularity and mitogenic response patterns) supports a conclusion of indicative developmental immunotoxicity being demonstrated at 4 and 40 mg/kg bw.
It is important to note that the functional immune system assessment was not based on use of the T-cell dependent antibody response (TDAR) assay. In summary, it is considered that this study provides indicative, but not firm, evidence that exposure to ionic silver might give rise to developmental immunotoxicity.

Reference 5

Endpoint:

reproductive toxicity, other

Remarks:

Investigation of reproductive organs of animals exposed to test item during 13 weeks

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Weight of evidence approach described in 'Silver metal (massive and powder): Weight of Evidence' (document attached in IUCLID section 13 - "CSR Annex 11 - Weight of Evidence Justification for Silver metal - human health endpoints).

Qualifier:

no guideline available

Principles of method if other than guideline:

Seven-week-old rats (10 rats per sex per group) were exposed silver acetate (AgAc) at 100, 200, and 400 mg/kg bw; and controls (2mM sodium citrate (CIT) or water)at termination the productive system were analysis

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Test item was purchased 99% pure as a single lot from Gelest, Inc. (Morrison, Pennsylvania), and batches of stock aqueous solutions (nominal concentration 10.4 mg/mL) were prepared in 18 megaohm and autoclave-sterilized water on an "as needed" or bi-weekly basis and stored at room temperature. Samples were submitted to inductively coupled mass spectrometry (ICP-MS) analysis to determine the actual silver mass concentrations. The test item solutions remained clear and colorless throughout the useful life of the solution of 2 weeks, indicating that the silver did not precipitate or become reduced.

Characterization of test item solutions:
Test material characterisation was conducted weekly at the National Center for Toxicology Research (NCTR) on freshly prepared test item solutions.

The samples were diluted to 50 mL with and acid mixture of 1 N each of nitric and hydrochloric acids.
For the stability study, total silver concentrations were determined by ICP-MS, initially for the test item stock suspensions and filtrates, and subsequently for filtrates collected from the same stock suspensions after storage in the dark at 4C–8C for 1 and 90 days. Homogeneity testing was conducted on test item stock suspensions from the same shipment as the stability study; however, triplicate subsamples were collected from the top, middle, and bottom of each vessel and analyzed in triplicate.

The average total silver concentrations by mass of the test item samples were determined with a Thermo Scientific WSERIES 2 Quadrupole ICP-MS (Franklin, Massachusetts), using 107Ag, 109Ag, and 103Rhodium (Rh) at 50 ng/ml and 115Indium (In) at 100 ng/ml as internal standards.

The concentration of Ag was quantified against an external calibration curve (NIST traceable silver samples). The limit of quantification (LOQ) for the test item suspensions and filtrates was estimated to be 40 ng/ml.

The identity of the test item was confirmed by 1H nuclear magnetic resonance spectroscopy on an AVANCE III spectrometer equipped with a BBFO Plus Smart Probe (Bruker Instruments, Billerica, Massachusetts) operating at 500 MHz. The samples were dissolved in D2O and the acquisition was conducted at room temperature using a standard 1H acquisition sequence.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- 3-week-old male and female Sprague Dawley/CD-23 rats with specific pathogen-free health status were obtained from the NCTR breeding colony.
- At 6 week of age, the rats were weight-ranked and randomly assigned to treatment groups. Male and female rat were housed conventionally in separate animal rooms with 2 animals per cage.
- The environment of the animal rooms was set to maintain a 12h light cycle, temperature of 22+/-4°C, relative humidity of 40%-70%, and air changes of 10-15 per hour.
- The animals were provided NIH-41 gamma-irradiated pellets and Millipore-filtered drinking water ad libitum.
- Rats were dosed initially at 7 weeks of age.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

water/0.1%methyl cellulose. Methylcellulose was used in this study as bulking agents to inhibit somewhat the gastrointestinal passage of the mostly aqueous dose formulation. The compound is not toxic and do not promote allergic reactions in rodents.

Details on exposure:

Gavage dosing was conducted using computer-controlled MicroLab 500 series dispensers (Hamilton Co., Reno, Nevada) equipped with gastight syringues and capable of dispensing 1µL to 50mL.
Syringue were fitted with flexible plastic gavage needles, and the rats were provided equal volume doses based on the daily body weight of the individual rats.
The MicroLab dispersion were programmed to administer the total daily dose in 2 daily gavage administrations per day, with half of the dose administered just prior to start of the dark cycle.

Route Exposure: Ingestion is the primary route of exposure in humans for silver, silver compounds, and colloidal silver (Silver, 2003). Daily intakes for silver in humans are estimated in the range 0.4–27 mg/day (Clemente et al., 1977; Gibson and Scythes, 1984; Hamilton and Minski, 1972/1973). The Environmental Protection Agency lists the reference dose for silver as 0.5 mg/kg/day or 35 mg/day for an average 70 kg adult (EPA, 2014).

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared weekly and were based on the total silver mass concentrations obtained by ICP-MS for silver acetate solution. The high-dose formulation of test item (10 mg AgAc/mL or approximately 6.46 mg Ag/mL) was prepared in water/0.1% methylcellulose (final concentration wt/wt).

- The silver concentration of the high-dose formulation were confirmed by ICP-MS and, then serially diluted in water/0.1% methylcellulose to achieve the 3.23 and 1.62 mg Ag/mL concentrations for the mid- and low-dose AgAc formulation respectivelly.

DOSE CHARACTERIZATION: The total silver concentration by mass of each of the prepared dose formulations (high-, mid-, and lowdose) and the controls ( water/MC) was determined by ICP-MS in acid- and microwave-digested samples. Characterization analyses were conducted on triplicate samples. Dose concentration acceptability was set at 610% targeted values.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The total silver concentration by mass of each of the prepared dose formulations (high-, mid-, and lowdose) and the controls (water/MC) was determined by ICP-MS in acid- and microwave-digested samples.

The AgAc solutions remained clear and colorless throughout the useful life of the solution of about 2 weeks, indicating that the silver did not precipitate or become reduced.

Duration of treatment / exposure:

Groups of rats (10 males and 10 females) were exposed daily by oral gavage to dose formulation of AgAc at 100, 200 and 400 mg/kg bw or to the respective control formulations (Water/MC) for a period of 13 weeks.

Frequency of treatment:

Gavage dosing was conducted using computer-controlled MicroLabVR 500 series dispensers (Hamilton Co., Reno, Nevada) equipped with gastight syringes and capable of dispensing 1 ml to 50 ml. The syringes were fitted with flexible plastic gavage needles, and the rats were provided equal volume doses based on the daily body weight of the individual rats. The MicroLab dispensers were programmed to administer the total daily dose in 2 daily gavage administrations per day, with half of the dose administered at the start of the light cycle and half of the dose administered just prior to start of the dark cycle. The dose volumes did not exceed 20 ml/kg bw. Animals were dosed 7 days each week and the study period was 13 weeks.

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In studies with mice, Horner et al. (1983) determined that the oral lethal dose, 90% (LD90) of AgAc was 2505 mg/kg bw. In studies with rats to evaluate AgAc for developmental toxicity, the lowest observed adverse effect level by the oral
route was 30 mg/kg bw/day AgAc, and the no observed adverse effect level for development toxicity was 100 mg/kg bw/day (NTP, 2002). Based on the wide range of doses in toxicity tests a low dose of 100mg AgAc/kg bw/day (64.6mg Ag/kg bw/day) was selected.

- Fasting period before blood sampling for clinical biochemistry: terminal sacrifices were conducted on over-night fasted rats.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly clinical observations on individual animals were recorded in the animal records database (NCTR Multi-generation Computer Support System; MGSS).

BODY WEIGHT: Yes
- Time schedule for examinations: body weights recorded daily in MGSS for dose administration.

FOOD CONSUMPTION AND COMPOUND INTAKE: Feed and water consumptions were measured and recorded weekly in MGSS.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at terminal and moribund sacrifices
- Anaesthetic used for blood collection: Yes: Animals were euthanized humanely by carbon dioxide asphyxiation after the 72h blood sample collection.
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at terminal and moribund sacrifices
- Animals fasted: Yes

IMMUNOLOGY: A section of the ileum and scrapping of the ileal mucosa were collected for intestinal microbiota and immune response evaluations (Williams et al., 2015).

Oestrous cyclicity (parental animals):

Estrous cycling check in female rats was conducted by vaginal lavage each of 16 consecutive days prior to terminal sacrifice. A sterile glass medecine dropper was used to gently flush the vagina (2 or 3 times) with physiological saline, and the vaginal lavage were placed on glass slides, air dried, and fixed by immersion in methanol. the slides were stained with 0.5% toluidine blue and the cells were exaluated by light microscopy for stage of estrous cycle (diestrus, proestrus, or estrous). Estrous cycle length was assessed in female rats for 16 days prior to sacrifice, and the proportion of days spend in each estrous phase (diestrus, proestrus, and estrus) was analyzed for treatment differences using the arcsine-square root transformed proportion with a 1-way ANOVA.

Sperm parameters (parental animals):

The left testis and the caudal epididymis of male rats were used to determine sperm counts, motility, and morphology. Sperm motility, testes count, cauda count, and sperm morphology (number of normal/abnormal of 200 sperm) were analyzed for treatment differences using a 1-way ANOVA, and pairwise comparisons were performed with Bonferroni adjustments.

The left testis and the caudal epididymis of male rats were used to determine sperm counts, motility, and morphology. For testis sperm count, the tunica albuginea was removed, and the parenchyma was weighed and homogenized in distilled water. A sample of the testis suspension (100 ml) was added to a vial, and the total sperm count evaluated with a sperm analyzer (12.3 Tox Integrated Visual Optical System, Hamilton Thorne Biosciences, Beverly, Massachusetts). Sperm motility and morphology were determined on the caudal epididymis. The caudal portion of the epididymis was placed in buffer (1% bovine serum albumin in phosphate-buffered saline), and the distal portion of
the cauda was pierced 3 times to allow sperm to migrate. The onset and completion times were recorded for the migration period. Aliquots of the caudal suspension were loaded onto a chamber of the sperm analyzer (Hamilton Thorne Biosciences), and sperm motility was determined by the instrument automatically in 5 scanned image fields. For caudal sperm count, a sample of the caudal suspension (100 ml) was added to a vial, and the total sperm count evaluated with the sperm analyzer (Hamilton Thorne Biosciences). Morphological features of sperm were evaluated on the same suspensions used for sperm motility. Aliquots of the suspensions were placed on glass slides, dried, and stained with a 5% eosin aqueous solution. Morphology was examined manually by light microscopy in a total count of 200 normal sperm.

Postmortem examinations (parental animals):

At necropsy, organs and tissues were examined for grossly visible lesions, and protocol designated tissues and organs were weighed and preserved in 10% neutral buffered formalin (NBF), with the exception that modified Davidson’s fixative was used for the right testes and eyes. Femur bone marrow was collected for histopathology (left) and ICP-MS (right). The testes and epididymides were collected for sperm analysis (left) and histopathology (right).

Statistics:

A Cox proportional hazard model was used to analyze survival data and to test the effect of treatment relative to control. All tests were conducted as 2-sided with significance at the .05 probability level.
Body weights, feed consumption, water intake, and blood were analyzed using a 1-way repeated measures, mixed model ANOVA for each sex, with terms for dose, week, and all interactions.

The statistical analysis of hematology and clinical chemistry data was performed using a non-parametric method with midranks (Brunner et al., 2002).
Pairwise comparisons were performed with Bonferroni adjustments. All tests were conducted as 2-sided with significance at the .05 probability level.

The organ weights were analyzed using a 1-way analysis of covariance with necropsy body weights as a covariate. Pairwise comparisons were performed with Bonferroni adjustments.

Sperm motility, testes count, cauda count, and sperm morphology (number of normal/abnormal of 200 sperm) were analyzed for treatment differences using a 1-way ANOVA, and pairwise comparisons were performed with Bonferroni adjustments.

Estrous cycle length was assessed in female rats for 16 days prior to sacrifice, and the proportion of days spend in each estrous phase (diestrus, proestrus, and estrus) was analyzed for treatment differences using the arcsine-square root transformed proportion with a 1-way ANOVA.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings suggested severe gastrointestinal symptoms, loss of body weight and unthrifty appearance among these animals, likely due to the bactericidal activity of the silver ion on the intestinal microbiota.

Mortality:

mortality observed, treatment-related

Description (incidence):

Morbidity was pronounced in groups of rats exposed to the high (400 mg/kg bw/d) dose with a majority (70%) of female (100%) male rats being removed prior to the scheduled terminal sacrifice.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant lower mean body weights were observed in female rats administered test item at dose of 100 and 400 mg/kg bw/d; the orverall mean body weights were 88.5% and 74.4% of the control group, respectively. Male rats administered 400 and 200 mg/kg bw/d of test item demonstrated significantly lower mean body weights than the controls, beginning at week 1 and at week 3, respectively. Male rats in low dose (100 mg/kg bw/d; body weights in this group were not significantly affected.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant differences in feed and water intakes between the vehicle and water controls when compared with espective control animals, sporadic differences in feed and water intakes were observed in female and male rats administered test item.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology and plasma clinical chemistry parameters recorded for rats exposed to test item are provided in the table section "Any other information on results incl. table".
Sporadic differences in the haematological and biochemistry parameters were found for silver acetate groups when compared with their controls. MCV (mean cells volume) were significantly lower for mid and high dose treated female rats and for test item low and mid dose treated male rats.
Red Blood Cell (RBC) counts were significantly higher (p>0.05 vs control) for the test item low and mid dose treated female rats and for low dose male rats. The significantly lower MCV value and high RBC counts by tes titem treated rats are suggestive of low iron status, however, minerals other than sivler were not evaluated in blood samples.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology and plasma clinical chemistry parameters recorded for rats exposed to test item are provided in the table under section "Any other information on results incl. tables".

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Non-neoplastic lesions were confined primarly to rats administered test item. with the exception of an increased dose-response in the incidence of histiocutic infiltrates in the lungs of female and male groups, there were no meaningful treatment-related differences from controls in the 100 and 200 mg/kg bw/d groups.

Increase incidences and severities of lesions were detected in rats exposed to highest dose of test item, and included mucosal hyperplasia in the small and large intestine, as well as thymic atrophy or necrosis - a stress-response to the gastrointestinal distrurbances experienced by these animals.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No statistically significant differneces between control groups or between any treatment and control groups in proportion of days in each phase of the estrous cycle or in the frequency of transition between normal and abnormal states.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The evaluation of rat testes revealed that there were no statistically significant differences in sperm motility, testis sperm count, caudal sperm count, or sperm morphology.

Reproductive performance:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Key result

Dose descriptor:

other:

Generation:

F1

Based on:

not specified

Sex:

not specified

Remarks on result:

not measured/tested

Remarks:

F1 generation was not performed

Key result

Reproductive effects observed:

no

Characterization of test item solutions and dose formulation:

The results of the characterisation for each of the individual lots of test item solutions. Stock of aqueous solution of test item (nominal concentration 10.4 mg/mL) were prepared in 18 megaohm sterilized water on a bi-weekly basis. The total silver and ioniic silver concentrations of the test materials were determined by ICP-MS, value presented the mean +/- SD of 22 shipments of 16 batch preparations of test item. 

The average total silver concentration of test item solution was 6.73 +/- 0.048 mg/mL and approximated the solubility (10.4 mg/mL) of test item in water; silver acetate is composed of 64.6% silver by weight.

Dose formulations were prepared on a weekly basis at 3 concetrations levels for silver acetate (1.62, 3.23 and 6.46 mg Ag/mL) and the total silver concetration of each formulation was determined by ICP-MS prior to insuance of the dose formulation to animals on the study.

 

Ratio of determined total silver concentration and targeted silver concentration:

	Target dose concetration (mg/mL)	Total silver concentration (mg/mL) +/- SD	Percentage of targeted dose +/- SD	Particle hydrodynamic size (nm) +/- SD
Silver acetate	1,62	1,684 (±0,05)	104 (±3,02)	225,14 (±14,45)
	3,23	3,327 (± 0,07)	103,20 (± 2,12)	225,77 (± 14,94)
	6,46	6,657 (±0,15)	103,10 (± 2,30)	226,03 (± 7,15)

 

Hematology and Clinical Chemistry of Sprague Dawley rats in the 13-weeks study of silver acetate:

	Female rats				Male rats
	H2O/MC	100 mg/kg bw/d	200 mg/kg bw/d	400 mg/kg bw/d	H2O/MC	100 mg/kg bw/d	200 mg/kg bw/d	400 mg/kg bw/d
Basophil (%)	0.2 ±0.0	0.4 ±0.1	0.2 ±0.0	0.2 ±0.1	0.2 ±0.0	0.2 ±0.0	0.2 ±0.0	n.a.
Eosinophil (%)	1.2 ±0.1	0.9 ±0.1	1.6 ±0.2	1.2 ±0.1	1.5 ±0.2	1.5 ±0.2	2.1 ±0.3	n.a.
Lymphocyte (%)	79.0 ± 1.0	80.3 ± 2.2	79.5 ± 1.5	75.7 ± 2.1	75.7 ± 0.9	76.9 ± 1.6	77.1 ± 2.5	n.a.
Monocyte (%)	5.1 ± 0.7	4.6 ± 0.6	5.2 ± 0.8	4.8 ± 1.3	6.8 ± 0.6	8.3 ± 0.5	6.8 ± 1.0	n.a.
Neutrophil (%)	14.5 ± 0.7	13.8 ± 1.8	13.6 ± 1.4	18.1 ± 1.9	15.8 ± 0.9	13.2 ± 1.2	13.9 ± 1.5	n.a.
Basophils (10^3/mL)	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	n.a.
Eosinophils (10^3/mL)	0.1 ± 0.0	0.1 ± 0.0	0.2 ± 0.0	0.1 ± 0.0	0.2 ± 0.0	0.2 ± 0.0	0.2 ± 0.0	n.a.
Lymphocytes (10^3/mL)	6.1 ± 0.5	7.3 ± 0.5	7.1 ± 0.7	7.3 ± 1.4	8.2 ± 0.7	8.4 ± 0.7	6.7 ± 0.6	n.a.
Monocytes (10^3/mL)	0.4 ± 0.1	0.4 ± 0.0	0.5 ± 0.1	0.5 ± 0.2	0.7 ± 0.1	0.9 ± 0.1	0.6 ± 0.1	n.a.
Neutrophils (10^3/mL)	1.1 ± 0.2	1.3 ± 0.2	1.2 ± 0.1	1.7 ± 0.4	1.7 ± 0.1	1.5 ± 0.2	1.2 ± 0.2	n.a.
Hematocrit (%)	45.5 ± 0.5	46.8 ± 0.5	44.9 ± 0.5	39.6 ± 1.5	47.3 ± 0.4	46.2 ± 0.5	44.9 ± 0.8	n.a.
MCHC (g/dL)	35.1 ± 0.2	35.1 ± 0.1	35.0 ± 0.2	33.4 ± 0.3	34.5 ± 0.2	34.0 ± 0.1	33.8 ± 0.2	n.a.
MCV (mm3)	55.2 ± 0.4	53.9 ± 0.6	51.6 ± 0.5	46.3 ± 0.9	51.3 ± 0.6	47.0 ± 0.3	46.6 ± 0.5	n.a.
PCV (mm3)	45.6 ± 0.5	46.8 ± 0.5	45.0 ± 0.5	39.7 ± 1.6	46.9 ± 0.4	46.1 ± 0.5	44.8 ± 0.8	n.a.
PLT (10^3/mL)	737 ± 34	764 ± 28	839 ± 37	775 ± 184	715 ± 20	805 ± 28	824 ± 31	n.a.
RBC (10^3/mL)	8.2 ± 0.1	8.7 ± 0.1	8.7 ± 0.1	8.6 ± 0.5	9.2 ± 0.1	9.8 ± 0.1	9.6 ± 0.2	n.a.
WBC (10^3/mL)	7.8 ± 0.7	9.1 ± 0.6	9.0 ± 0.9	9.7 ± 2.0	10.8 ± 0.8	11.0 ± 1.0	8.7 ± 0.8	n.a.
Albumin (g/dL)	4.2 ± 0.1	4.0 ± 0.1	4.1 ± 0.1	3.5 ± 0.0	3.6 ± 0.1	3.6 ± 0.1	3.6 ± 0.1	n.a.
Alkalin phosphatase (U/L)	57.4 ± 5.5	85.6 ± 6.1	106 ± 8	120 ± 28	100 ± 4	154 ±10	157 ± 13	n.a.
Alanine transferase (U/L)	33.1 ± 2.5	35.0 ± 2.3	41.8 ± 2.7	55.0 ± 8.3	35.8 ± 1.1	40.8 ± 2.0	45.7 ± 3.1	n.a.
Blood Urea nitrogen (mg/dL)	17.1 ± 0.6	19.9 ± 0.8	19.2 ± 0.3	20.7 ± 1.8	16.0 ± 0.6	16.9 ± 0.6	18.2 ± 1.0	n.a.
Cholesterol (mg/dL)	100 ± 6	122 ± 7	136 ± 5	118 ± 4	73.7 ± 5.1	110 ± 6.5	102 ± 5	n.a.
Creatine kinase (U/L)	190 ± 36	200 ± 28	212 ± 25	338 ± 146	201 ± 48	240 ± 46	232 ± 34	n.a.
Creatinine (mg/dL)	0.5 ± 0.0	0.4 ± 0.0	0.5 ± 0.0	0.3 ± 0.0	0.4 ± 0.0	0.3 ± 0.0	0.4 ± 0.0	n.a.
Glucose (mg/dL)	130 ± 6.2	115 ± 3.1	102 ± 3.6	136 ± 27	138 ± 4.6	127 ± 7.4	112 ± 4.1	n.a.
Sorbitol dehydrogenase (U/L)	8.1 ± 3.0	6.7 ± 2.4	8.8 ± 2.7	7.6 ± 6.9	7.5 ± 1.7	9.9 ± 1.7	9.4 ± 2.5	n.a.
Total bile acids (mmol/L)	42.4 ± 5.6	68.3 ± 13.1	76.2 ± 7.6	123 ± 37	39.1 ± 4.5	54.7 ± 9.0	92.6 ± 8.6	n.a.
Total protein (g/dL)	7.6 ± 0.2	7.0 ± 0.1	7.0 ± 0.1	6.0 ± 0.2	6.5 ± 0.1	6.3 ± 0.1	6.2 ± 0.1	n.a.
Triglycerides (mg/dL)	88.4 ± 10.6	70.3 ± 7.0	109 ± 14	54.0 ± 9.3	98 ± 8.7	103 ± 14	91.3 ± 9.6	n.a.

Absolute and relative organ weights of rats treated orally by daily gavage for 13-weeks with silver acetate

female	H2O/CM	100 mg/kg bw/day	200 mg/ kg bw/day	400 mg/kg bw/day
	Mean± SE	Mean± SE	Mean± SE	Mean± SE
Necropsy BW	312.9.6 ± 9.6	270.3 ±  4.3	269 ±  4.9	238 ±  5.9
Heart absolute	1.091 ±  0	1.034 ±  0	0.99 ±  0	0.877 ±  0
Heart relative	0.349 ±  0	0.383 ± 0	0.37 ±  0	0.367 ±  0
Kidney, left absolute	0.967 ±  0	0.979 ±  0	0.85 ±  0	0.9 ± 0
Kidney, left relative (%)	0.31 ± 0	0.325 ± 0	0.32 ± 0	0.377 ± 0
Kidney, right absolute	0.964 ± 0	0.884 ± 0	0.88 ± 0	0.887 ± 0
Kidsney, right relative	0.309 ± 0	0.327 ± 0	0.33 ± 0	0.371 ± 0
Liver absolute	7.855 ± 0.3	6.892 ± 0.2	7.15 ± 0.1	8.126 ± 0.9
Liver relative (%)	2.515 ± 0	2.548 ± 0	2.66 ± 0	3.387 ± 0.3
Lung absolute	1.309 ± 0	1.256 ± 0	1.25 ± 0	1.311 ± 0
Lung relative (%)	0.421 ± 0	0.466 ± 0	0.47 ± 0	0.55 ± 0
Ovary, left absolute	0.05 ± 0	0.046 ± 0	0.05 ± 0	0.045 ± 0
Ovary, left relative (%)	0.016 ± 0	0.017 ± 0	0.02 ± 0	0.019 ± 0
Ovary, right absolute	0.047 ± 0	0.039 ± 0	0.05 ± 0	0.041 ± 0
Ovary, right relative (%)	0.015 ± 0	0.015 ± 0	0.02 ± 0	0.017± 0
Thymus absolute	0.319 ± 0	0.259 ± 0	0.22 ± 0	0.131 ± 0
Thymus relative (%)	0.101 ± 0	0.096 ± 0	0.08 ± 0	0.055 ± 0
male	H2O/CM	100 mg/kg bw/day	200 mg/ kg bw/day	400 mg/kg bw/day
	Mean± SE	Mean± SE	Mean± SE	Mean± SE
Necropsy BW	530± 17.3	514.5 ±  18.8	466.6 ±  8.8	n.a.
Epididymis left absolute	0.59 ± 0	0.672 ± 0	0.060 ± 0	n.a.
Epididymis left relative	0.11 ± 0	0.144 ± 0	0.145 ± 0	n.a.
Epididymis right, absolute	0.6 ± 0	0.609 ± 0	0.607 ± 0	n.a.
Epididymis right, relative	0.12 ± 0	0.131 ± 0	0.146 ± 0	n.a.
Heart absolute	1.59 ± 0	1.663 ± 0.1	1.148 ± 0	n.a.
Heart relative	0.3± 0	0.356 ± 0	0.355± 0	n.a.
Kidney, left absolute	1.52 ± 0.1	1.337 ± 0.1	1.224 ± 0	n.a.
Kidney, left relative (%)	0.29 ± 0	0.287 ± 0	0.295 ± 0	n.a.
Kidney, right absolute	1.53 ± 0	1.381 ± 0.1	1.212± 0	n.a.
Kidsney, right relative	0.29 ± 0	0.296 ± 0	0.291± 0	n.a.
Liver absolute	13.3 ± 0.5	13.08 ± 0.5	12.24 ± 0.4	n.a.
Liver relative (%)	2.51 ± 0	2.799± 0.1	2.939± 0.1	n.a.
Lung absolute	1.74± 0	1.771± 0.1	1.529± 0.1	n.a.
Lung relative (%)	0.33± 0	0.381± 0	0.368 ± 0	n.a.
Testes, left absolute	1.75± 0.1	1.881 ± 0.1	1.749 ± 0.1	n.a.
Testes, left relative	0.33 ± 0	0.404 ± 0	0.421 ± 0	n.a.
Testes right absolute	1.68 ± 0.1	1.788 ± 0.1	1.76 ± 0	n.a.
Tested right relative	0.32 ± 0	0.385 ± 0	0.424 ± 0	n.a.
Thymus absolute	0.27 ± 0	0.277 ± 0	0.198 ± 0	n.a.
Thymus relative (%)	0.05 ± 0	0.059 ± 0	0.048 ± 0	n.a.

 

		Water/CM	100 mg/kg bw/d	200 mg/kg bw/d	400 mg/kg bw/d
Female	Blood Wk-1 (ng/mL)	53 ± 28*	1171 ± 91**	1631 ±188**	9047 ± 1883**
	blood Wk-2 (ng/mL)	18 ± 4*	792 ± 95**	1753 ± 212**	6960 ±
	Bone Marrow, femur (ng/mL)	3 ± 0*	800 ± 168**	3973 ± 1314**	13433 ± 2385**
	Heart (ng/g)	76 ± 69*	375244 ± 310684**	116150 ± 18153**	189000 ± 5686**
	Ileum (ng/g)	33 ± 21*	259000 ± 53550**	262000 ± 34697**	588667 ± 61167**
	Jejunum (ng/g)	12 ± 5*	336333 ± 52145**	574900 ± 49119**	1416667 ± 133708**
	Kidney (ng/g)	14 ± 9*	1481444 ± 205799**	1895000 ± 202640**	25256667 ± 188886**
	Liver lateral (ng/g)	4 ± 1*	759556 ± 69202***	1377600 ± 159674**	843667 ± 132640**
	Lymph node, mes. (ng/g)	10 ± 3*	830444 ± 107536**	2119000 ± 237821**	5556667 ± 788973**
	Colon (ng/g)	9 ± 2*	793778 ± 61053**	1175800 ± 87121**	117800 ± 232975**
	Spleen (ng/g)	24 ± 19*	560111 ± 85092**	2373000 ± 247557**	2843333 ± 101708**
	Uterus (ng/g)	67 ± 37*	56844 ± 6010**	76350 ± 5098**	126100 ± 40771**
Male		Water/MC	100mg/kg bw/d	200 mg/kg bw/d	400 mg/kg bw/d**
	Blood Wk-1 (ng/mL)	27  ± 8*	1121  ± 151**	2482  ± 546**	12270  ± 1846**
	blood Wk-2 (ng/mL)	34  ± 15*	595  ± 45**	1618  ± 227**	n.a.
	Bone Marrow, femur (ng/mL)	3  ± 0*	1020  ± 430**	6302  ± 1189**	n.a.
	Heart (ng/g)	2   ± 0*	58500  ± 5696**	77330  ± 8890**	n.a.
	Ileum (ng/g)	19  ± 10*	228920  ± 28619**	357100  ± 46308**	n.a.
	Jejunum (ng/g)	12  ± 5*	423800  ± 43898**	929100  ± 189193**	n.a.
	Kidney (ng/g)	8  ± 3*	440000  ± 60950**	768000  ± 96142**	n.a.
	Liver lateral (ng/g)	3  ± 0*	472620  ± 66058**	813300  ± 115528**	n.a.
	Lymph node, mes. (ng/g)	7  ± 0*	549200  ± 45321**	1661400  ± 215854**	n.a.
	Colon (ng/g)	18  ± 7*	310100  ± 44888**	658100  ± 52337**	n.a.
	Spleen (ng/g)	6  ± 1*	601000  ± 138471**	1207700  ± 214244**	n.a.

The limit of quantification for endpoint of interest is as follows:

blood: 20 ng/mL

bone marrow: 5ng/mL

heart: 4 ng/g

ileum and jejunum: 13 ng/g

kidney: 10 g/g

liver: 5ng/g

mesenteric lymph nodes and colon: 13g/g

spleen: 10 ng/g

uterus: 22ng/g

* In the control groups, signifies significant linear dose trend effects (P< 0.05) based on Bonferoni adjustments.

**Signifies values thar are significantly different (P<0.05) from the control group by Bonferoni adjustment

Disposition and accumulation of silver:

Tissue and organs from animals exposed to AgAc for 13 weeksn with exception of bone marrow, revealed statistically significant (p<0.05) dose-dependent increases in silver concentrations.

The silver content in blood and bone marriw averages 3-4 times lower than silver content of the heart, which had the lowest content of silver among the major organs analysed and indicated that the contribution of silver accumulation in the blood and bone marrow was minimal.

Blood levels of silver were not significantly higher after 12 weeks of dosing than levels found after only 1 week of dosing, these results suggest rapid clearance of silver from blood for all groups irrespective of silver form.
 
In preliminary studie, the half-time for elimination of silver from the blood was 24h regardless of sex of form of silver.

Silver concentrations were significantly higher in the blood and bone marrow of rats exposed to AgOAc when compared to AgNP. Results indicate that a much higher uptake of silver occurred following the administration of AgOAc, than could be accounted for by the differences in silver content of the dose.

In female and male rats exposed to AgAc, the kidney and spleen represented sites of significant silver accumulation. As observed in the intestinal tract, sex differences in silver concentrations were observed. In addition, the pattern of distribution in the kidneys and spleens of male rats differed from that of female rats.

The concentrations of silver in all organs appeared to be higher in females than males. The accumulation and pattern of silver distribution was similar in the kidney and spleen of female and male rats administered AgAc.

Localisation of silver in rat tissue

In rats exposed to AgAc (100 mg/kg bw), the accumulation of silver granules was detected primarily along the epithelial basement membrane of the jejunum ileum  and colon. The surface morphology of the accumulated electron dense silver granules in the intestine of rats exposed to AgAc was irregular and segmented. Mean diameters were larger for granules accumulated in the intestinal tract of rats exposed to AgAc.

The accumulation of silver was observed along the basement membranes of the glomeruli of kidneys from rats exposed to AgAc similarly found the accumulation of silver granules primarily along the basement membranes of the glomeruli in kidneys from rats exposed to AgAc.Those deposited in the kidneys of AgAc-exposed rats were large and irregular in shape, often with surface bulges.

AgAc appeared to have an affinity for extracellular membranes.

EDS was used to determine the compositional analysis of the granules within rat tissues. Strong energy signals were observed for silver, and the presence of selenium, sulfur, and chloride was noted in association with the silver
granules for AgAc.
Signals corresponding to copper and lead were also identified. These originated from the grids that were used in the visualization procedures; an osmium signal was observed and originated from the tissue fixation procedures. Whereas, only signals for lead, copper and osmonium were detected by EDS in tissues of the vehicle control and water control animals.

Conclusions:

In conclusion, rats vaginal cytology, or sperm count, motility and morphology is not affected by the exposure to silver acetate up to 400 mg/kg bw/day

Executive summary:

Seven-week-old rats (10 rats oer sex per groups) were randomy assigned to treatments: silver acetate at 100, 200 and 400 mg/kg bw/d, and control (water). At termination, compete necropsies were conducted, histopathology, hematology, serum chemistry and reproductive system were performed. Estrous cycle length was assessed in female rats for 16 days prior to sacrifice.

Rats vaginal cytology, or sperm count, motility and morphology is not affected by the exposure to silver acetate (up to 400 mg/kg bw/dy (highest dose tested).

In conclusion, rats reproductive system is not affected by exposure to silver acetate.

 

 

 

 

 

Reference 6

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

lack of GLP compliance, lack of individual animal data, lack of certain key investigations (oestrus cycle, sperm parameters and incomplete histopathological analyses of reproductive tissues)

Justification for type of information:

Weight of evidence approach described in 'Silver metal (massive and powder): Weight of Evidence' (document attached in IUCLID section 13 - "CSR Annex 11 - Weight of Evidence Justification for Silver metal - human health endpoints).

Qualifier:

according to guideline

Guideline:

other: US protocols for food additives (FDA CFSAN Redbook, 2000)

GLP compliance:

no

Remarks:

Publication: lack of GLP compliance

Limit test:

no

Specific details on test material used for the study:

The test article was obtained from Sigma-Aldrich and stored according to the conditions (dry, at room temperature) detailed in the MSDS Sheets supplied by the manufacturer.
Stability of the test material was determined to be greater than 21 days at room temperature.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague-Dawley rats [Crl:CD@ (SD) IGS BR] were obtained from Charles River Laboratories. Upon receipt, all animals were identified by ear tag.
- Females nulliparous: yes
- Age at study initiation: 4 weeks
- Weight at study initiation: (P) Males: 119.5 ± 2.2 g; Females: 104.8 ± 2.3 g
- Fasting period before study: not specified
- Housing: all rats were individually housed in polycarbonate tubs with shredded cardboard bedding throughout the study except during mating. During mating males and females were cohabitated overnight (approximately 17 h) in polycarbonate tubs containing a metal grate insert flooring.
- Diet: ad libitum; Rodent diet (Tekland Certified Global 18% Protein Rodent Diet (meal) 2018CM; Lot Number 2018CM-020812MA) obtained from Harlan laboratory (P.O. Box 44220, Madison, WI 53744-4220); Feed was provided in Holtge feeders; A certification analysis was performed on the rodent diet by the manufacturer; Eurofins analysis of the diet indicated that it contained no contaminants known to interfere with the study.
- Water: ad libitum; Hydropure water
- Acclimation period: approximately 1 week during which time they were observed daily for clinical signs of disease.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-78
- Air changes (per hr): not specified
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: drinking water

Vehicle:

water

Remarks:

Deionized double distilled HydroPure water (Hydro-Pico Systems, Inc., Research Triangle Park, NC, USA); analysis indicated that the concentration of silver in the Hydropure water was not detectable.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The target concentrations of the dosing solutions utilized in this study were 0.0 mg/kg bw, 0.4 mg/kg bw, 4.0 mg/kg bw and 40.0 mg/kg bw (equivalent to approximately 0, 0.26, 2.6 or 26 mg ionic Ag/kg bw).
- All dosing solutions were formulated in ultra-pure water from the same source as the vehicle controls.
- All doses were administered through water bottles. Animals received either control or dosing solutions in 8 ounce water bottles fitted with straight sipper tubes during the 10 week pre-mating treatment and gestation periods and 16 ounce water bottles fitted with straight sipper tubes during lactation. Water bottles were changed every 3 days.
- Dosing solutions were prepared fresh every 3-6 days and analyzed before they were administered to the study animals.
- The concentration of the dosing solutions for a specific group was formulated so that experimental animals received the appropriate exposure dose of the test solution. The concentration of the test article in the dosing solutions for each experimental dose group were predicted three to six days in advance based on average fluid consumption and body weight gain for animals in a given group.

Details on mating procedure:

- M/F ratio per cage: not specified
- Length of cohabitation: overnight (approximately 17 h); until evidence of mating was obtained or for a maximum of 14 days
- Proof of pregnancy: copulation plug in the vagina or bottom of the mating cage and/or presence of sperm in a vaginal smear
- Females that did not mate at the end of 1 week were paired the following week with a different randomly selected male from the same treatment group.
- Males were subsequently euthanized and necropsied after exposure day 90.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing solution concentrations were verified using an Orion Star pH/ISE meter (Thermo) and Silver/Sulfide electrode with corresponding double-junction reference electrode (Thermo) using direct calibration method following the manufacturer’s recommendation in Electrode User Guide.
Maintenance of the target dose was achieved throughout the pre mating, gestation and lactation periods. The actual dose received for all phases of the study was within 10-15% of the target dose. Only P generation males in the 0.4 mg/kg dose group received a slightly higher dose of silver acetate (0.51 ± 0.06 mg/kg) than the specified target dose. However, potential adsorption losses of silver (e.g. on to container surfaces) were not specifically investigated.

Duration of treatment / exposure:

P generation males and females were exposed to the test material for 10 weeks prior to mating. Treatment continued through the mating period until study termination and included the gestation and lactation periods for females. Selected F animals were not exposed to the test material after Lactation Day 21 and were maintained on study until PN day 26 when they were euthanized and tissues were collected.

Frequency of treatment:

Continuously (through drinking water)

Dose / conc.:

0.4 mg/kg bw/day (nominal)

Dose / conc.:

4 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: P generation males and females were randomized into 4 treatment groups through a standard (by weight) block randomization procedure. Offspring from the P animals were designated F, and following weaning one male and one female pup, when possible, were randomly selected per litter and raised to PN day 26 at which time the animals were euthanized and tissues were collected. When less than 20 litters were available per group, additional F generation pups were randomly selected from other litters within the same group to provide the additional pups needed.

Positive control:

No positive control was included in the study.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Mortality, morbidity, injury, and availability of food and water were assessed

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: every 3 days (individual body weight)

FOOD CONSUMPTION:
- Food consumption for each animal determined every 6 days (except during mating)

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: every 3 days (except during mating)

CLINICAL CHEMISTRY
At euthanasia blood samples were collected from all animals on study. Once blood clotted the samples were centrifuged and blood serum was collected. The following parameters were evaluated in the serum: albumin, albumin/globulin ratio, alkaline phosphatase, ALT (SGPT), AST (SGOT), bicarbonate, direct bilirubin, indirect bilirubin, total bilirubin, BUN, BUN/creatinine ratio, calcium, chloride, cholesterol, creatinine kinase, creatinine, globulin, glucose, phosphorus, potassium ratio and total protein.

Oestrous cyclicity (parental animals):

Not investigated.

Sperm parameters (parental animals):

Not investigated.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- 10 pups/litter (5/sex/litter when possible); excess pups were randomly selected for use in another study (designed to assess the effect of ionic silver on selected immunological and toxicogenomic endpoints).

PARAMETERS EXAMINED
The following parameters were examined in F offspring:
- Mortality and clinical observations: Gross abnormalities and clinical signs for pups were recorded as soon as possible after birth. F pups were also individually examined for gross external abnormalities on PND 26. Litters were observed twice daily for behavioral alterations in nesting and nursing. All animals were observed daily for clinical signs of toxicity during lactation.
- Body weights, litter size and pup sex: Measurements of individual pup body weights were made as soon as possible after birth. Data on litter size and pup sex were recorded as soon as possible after birth. On days 0, 4, 7, 14, and 21 of lactation each pup was observed, sexed and weighed. F generation pups were also individually weighed on Days 21 and 26. Litters were observed twice daily for pup survival; the presence of dead/missing pups was recorded.

GROSS EXAMINATION OF DEAD PUPS:
Dead pups recovered at birth or during lactation were necropsied if intact.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Reported in separate publication (see Babu et al. 2016)

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were euthanized and necropsied after exposure day 90.
- Maternal animals: All surviving animals were euthanized after the last litter of each generation was weaned.

GROSS NECROPSY
- Gross necropsy consisted of examinations of brain, thymus, heart, lungs, stomach, small intestines, large intestines, liver, spleen, kidneys, ovaries, uterus, seminal vesicles, prostate, epididymis, testes. All organs except the small, large intestines and lungs were weighed.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Tissues examined in the histopathological assessment included the thymus, heart, lungs, liver, spleen, kidneys, stomach, intestine (ileum and colon) and bone marrow (sternum and femur).
- Histopathology for the ovaries, uterus, seminal vesicles, prostate and epididymis were not reported.
- Perfusion-fixed and resin-embedded testicular tissues from a subset of randomly selected male rats (n = 10 per dose groups) were also examined for the control and high-dose groups only.

Postmortem examinations (offspring):

SACRIFICE
F1 pups were sacrificed on PND 26

GROSS NECROPSY
- Gross necropsy consisted of examinations of brain, thymus, heart, lungs, stomach, small intestines, large intestines, liver, spleen, kidneys, ovaries, uterus, seminal vesicles, prostate, epididymis, testes. All organs except the small, large intestines and lungs were weighed.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Tissues examined in the histopathological assessment included the thymus, heart, lungs, liver, spleen, kidneys, stomach, intestine (ileum and colon) and bone marrow (sternum and femur).
- Histopathology for the ovaries, uterus, seminal vesicles, prostate and epididymis were not reported.
- Perfusion-fixed and resin-embedded testicular tissues from a subset of randomly selected male rats (n = 10 per dose groups) were also examined for the control and high-dose groups only.

Statistics:

The numbers were compared across dose groups using ANOVA followed by Dunnett’s -tests to ascertain the source(s) of any significance difference between dose groups and control.

Reproductive indices:

Mating index = (No. produced plugs or sperm-positive females / no. Exposed to mating) x 100
Fertility index 1 = (No. produced litter/no. produced plugs or sperm-positive females) x 100
Fertility index 2 = (No. produced litter/no. Exposed to mating) x 100

Offspring viability indices:

Pup survival (no. implants, mean no. pups born per litter, mean no. live pups born per litter, no. alive day 4/7/14/21)
Pup weight (male/female pup weight on day 0/4/7/14/21)
Number of runts (total no. of runts on day 0/4/7/14/21, no. of runts by gender on day 0/4/7/14/21)

Clinical signs:

no effects observed

Description (incidence and severity):

No deleterious dose-related clinical effects were observed in either adult male or female experimental and control animals.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control group male was found dead in its cage. The animal was necropsied but the cause of its death could not be determined.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant reductions in feed consumption were observed for all treated groups during the lactation period. Though these were not reported to correlate with bodyweight effects, and not of a magnitude considered to represent a notable deleterious treatment-related effect.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

There was a statistically significant reduction in fluid consumption in the 40.0 mg/kg treatment group in P generation male and female animals during the 10-week pre-mating exposure and gestation periods, but not during the lactation period.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Male Rat:
Blood serum phosphorus and potassium were significantly lower and alkaline phosphatase was significantly higher than their respective control values in the 40 mg/kg dose group. Statistically significant decreases from control values were observed for the parameters glucose, calcium, chloride and potassium and for phosphorus for the 4.0 mg/kg dose group.
Female Rat:
Serum Analysis Excluding Dams who Lost litters: A statistically significant increase in serum glucose from the control group was observed in the 40 mg/kg dose group. A statistically significant decrease in creatinine and a statistically significant increase in phosphorus was observed in the 4.0 mg/kg group in comparison to control values.

The serum effects observed were random and believed not to be related to silver acetate exposure.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Deposition of silver (pigmentation) was observed in a number of tissues of P generation animals but was not associated with any significant gross or histopathologic changes in any of the tissues examined.
Pigment deposition occurred in a dose dependent manner with greatest deposition being observed in the 40 mg/kg dose group and none observed in the control group or at the low dose (0.4 mg/kg dose).
Although pigmented material was observed in other tissues the greatest concentration was observed in the kidney. Pigment deposition was also observed in the liver, intestines and stomach, and to a lesser extent in the spleen, lymph node, thymus, and heart. The brain, bone, and bone marrow did not show pigmentation.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Male rat:
The mating index for P males was >= 90% for control and all dose groups.
The fertility indices were not different compared to the control group for the 0.4 and 4.0 mg/kg dose groups but were reduced for the 40 mg/kg dose group.
Female rat:
The mating index for P females was > 99% for control and all dose groups.
The fertility indices were not different compared to the control group for the 0.4 and 4.0 mg/kg dose groups but were reduced for the 40 mg/kg dose group.
All animals were found to be sperm/plug positive however 4 of the 20 animals from the 40 mg/kg dose group, although exposed to mating, did not produce litters. Of the 4 animals that did not produce litters, two of these animals did not conceive (no implantation sites were observed at necropsy) while the uterus of the remaining 2 animals contained resorptions.

Dose descriptor:

NOAEL

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

water consumption and compound intake

reproductive performance

Dose descriptor:

LOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

water consumption and compound intake

reproductive performance

Dose descriptor:

NOAEL

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The number of implants/number of sperm-plug positive dams was significantly reduced (p < 0.05) in the 40 mg/kg dose group as compared to the control, 0.4 and 4.0 mg/kg dose groups. Compared to the control group the mean number of pups born/litter was significantly reduced (p < 0.05) as was the mean number of surviving pups (p < 0.10) in the 40.0 mg/kg dose group however statistically significant differences were not observed between the control group and the other treated groups. The number of viable litters remaining at LD 21 was significantly reduced in the 40 mg/kg dose group in comparison to the control group, the 0.4 mg/kg and the 4.0 mg/kg dose groups, however the pups in the surviving litters thrived.

Runts and cumulative pup loss:
A statistically significant increase (p < 0.05) in the total number of runts was observed at lactation days 4 and 7 at 4 mg/kg.
Males: At lactation day 4, males in the 4.0 mg/kg dose group had a significantly higher number of runts per litter than control group (p < 0.05 pre and post cull).
Females: At lactation day 4, females in the 4.0 mg/kg dose group had significantly higher numbers of runts per litter (p < 0.10) than the control group. Increased numbers of female runts were also observed at lactation day 7 (p < 0.05) and non-statistically higher numbers of runts were observed at lactation day 21 in the 4.0 mg/
kg dose group.
However, it should be noted that similar statistically significant increases in indices for runting were not evident at the high-dose (40 mg/kg), and appropriate comparator historical control data were not reported for this parameter.
Statistically significant differences were not observed in cumulative pup loss when the control and other dose groups were compared.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male pup weight in the 4.0 mg/kg dose group was significantly lower than control values at lactation day 0 (p < 0.1); 4 (p < 0.5) and 7 (p < 0.1). Male pups in the 40 mg/kg dose group weighed less than male pups from the control group at lactation day 21 (p < 0.1).
Female pup weight in the 4.0 mg/kg dose group was significantly lower than the control group at PND 4 (p < 0.5), 7 (p < 0.5) and 21 (p < 0.1).
Overall, male and female pups in the 4.0 mg/kg dose group weighed slightly less than pups in the control group throughout the 21 day lactation period.
Male pup body weight from animals in the 40 mg/kg dose group was significantly lower than control values at PND 26.
Female pup body weights from animals in the 4.0 mg/kg dose group were significantly lower than control values at PND 26.

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:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Male Pups: Although the concentration of BUN detected in the 0.4, 4.0 and 40.0 mg/kg dose groups was found to be slightly lower than control values it only reached statistical significance in the 40 mg/kg group (p < 0.1). BUN/Creatinine ratios (B/C) for animals in the 0.4, 4.0 and 40 mg/kg dose groups were also found to be lower but did not reach statistical significance. Glucose levels in the 40 mg/kg dose group were significantly elevated over control values (p < 0.05). Serum calcium levels in the 0.4 mg/kg dose group (p < 0.1) and in the 4.0 mg/kg dose group (p< 0.1) was slightly higher than control values. A slight statistically significant decrease in globulin from control values was observed in the 40.0 mg/kg dose group (p < 0.1). All of the differences noted above were only marginally different (p < 0.10) from control values and all appeared random and could not be correlated with any morphological or physiological effects. The decreases or increases have been noted but are not thought to have biological relevance.
Female Pups: Serum glucose levels were slightly elevated over control values in both the 4.0 mg/kg (p < 0.05) and 40 mg/kg (p < 0.05) dose groups. Serum potassium levels were also slightly elevated over control values in the 4.0 mg/kg (p < 0.05) and 40 mg/kg (p < 0.10) dose groups. Bicarbonate levels were found to be slightly lower than control values in the 4.0 mg/kg dose group (p < 0.05). A statistically significant increase in SGPT (p < 0.05) and SGOT (p <0.1) was observed in the 4.0 mg/kg dose group. Finally, a statistically significant decrease (p < 0.05) in sodium was observed when the 4.0 mg/kg dose group was compared to the control group.

The serum effects observed were random and believed not to be related to silver acetate exposure.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Female Pups:
A statistically significant (p < 0.05) decrease in stomach weight was observed in the 40 mg/kg dose groups in comparison to the control group. A slight decrease in stomach weight was also observed in the 4.0 mg/kg dose group (p < 0.10). A statistically significant decrease in right kidney weight (p < 0.05) and heart weight (p < 0.1) was observed in the 4.0 mg/kg in comparison to the control group. Exposure-related effects were not observed in any of the other organs examined.
Male Pups:
Exposure-related effects were observed in stomach weight in the 0.4 mg/kg (p < 0.10) and the 40 mg/kg dose group (p < 0.05) in comparison to the control group. A slight statistically significant increase in right epididymal weight compared to the control was observed in the 0.4 mg/kg dose group (p < 0.10). Treatment related effects were not observed in any of the other organs examined.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Description (incidence and severity):

F generation animals exposed in utero and during lactation to silver acetate had no histopathologic changes in the tissues examined. Pigment accumulation was not observed in any of the organs examined. When select F generation animals were stained using the AMG method, pigment (mostly in the form of small droplets) was observed in the glomeruli and kidney tubules. Further analysis of F generation animals with AMG would be required to evaluate the extent of silver deposition in F generation tissues.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

0.4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

organ weights and organ / body weight ratios

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

4 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

Sprague-Dawley rats (n = 20/sex per group) received silver acetate administered in drinking water at dose levels of 0, 0.4, 4 or 40 mg/kg bw/day (equivalent to 0, 0.26, 2.6 or 26 mg ionic Ag/kg bw/day). Parental animals were exposed 10 weeks prior to mating. The F1-pups were sacrificed on postnatal day (PND) 26.
Evidence of appreciable maternal or paternal toxicity was absent in the study (although other potential secondary effect influences were not examined).
Evidence of developmental toxicity was reported including decreased implant numbers, increased pup mortality, and F1 generation weight retardation/delayed growth. The LOAEL and NOAEL for developmental toxicity are defined as 4 mg AgAc/kg bw/d (equivalent to 2.6 mg Ag/kg bw/d) and 0.4 mg AgAc/kg bw/d (equivalent to 0.26 mg Ag/kg bw/d), respectively.
An adverse effect on fertility was described as affecting the high-dose group only. The main parameters impacted were reduced fertility and the number of litters. The LOAEL and NOAEL for fertility effects are defined as 40 mg AgAc/kg bw/d (equivalent to 26 mg Ag/kg bw/d) and 4 mg AgAc/kg bw/d (equivalent to 2.6 mg Ag/kg bw/d), respectively.
The study was not conducted to GLP, and its design aligned with US FDA CFSAN norms rather than an EOGRTS (OECD TG 443). Whilst it generally appeared to be well conducted, its value is impacted by several important limitations, such as an absence of individual animal data, and the omission of certain key investigations (oestrus cycle, sperm parameters, and full histopathology of reproductive tissues).
Marked secondary non-specific effects were not reported as affecting treated groups, but potential secondary influences were not comprehensively examined, e.g. changes arising from gut microbiota disturbances due to the biocidal activity of ionic silver.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Introduction:

An in-vivo comparative toxicokinetic study, via oral route, was performed using a rodent model (according to OECD TG 417 and GLP compliant; Melvin et al., 2021 and Charlton et al., 2021). The test items included two ionic silver salts (silver nitrate and silver acetate), a well-characterized nanosilver reference material (15 nm AgNP) and a powder-form of silver metal (size ~0.3 μm, representing a conservative silver metal powder). Comparative toxicokinetics data were obtained after both single and 28-days repeated dose administration, including the measurements of Ag levels in blood and in tissues.

The key findings were:

• silver metal was substantially less absorbed than soluble silver salts and nanosilver. Based on matched dose assessments, the extent of systemic exposure was about 10 to 30-fold lower in the case of silver metal versus reference ionic silver salts.
• silver metal was considerably less distributed in tissues and organs than silver salts (ionic silver compounds). This links to predictions that silver metal (massive and powder) represents a correspondingly lower health hazard, i.e., is less likely to cause toxicity effects.

It is generally accepted that systemic toxicity of simple silver salts substances is driven by the silver ion (Ag+) as the primary species relevant for tissue exposure, and hence hazard assessment. Thus, a low bioavailability of silver metal (massive and powder), leading to a low internal concentration of silver ions (as toxicophore) leads to a lack of biological interaction and hence an absence of adverse outcome in comparison with high bioavailable silver salts. Therefore, it is assumed that silver metal represents a lower health hazard than the more bioavailable forms of silver at comparable nominal Ag levels.
Therefore, following the new in-vivo TK study findings, a direct Read-Across of mammalian toxicity datasets from simple silver salts and nanosilver to silver metal (massive and powder) is considered not appropriate.

Alternatively, a Weight of Evidence (WoE) approach considering:

1. the available mammalian toxicity data of simple silver salts and nanosilver and
2. the demonstrated difference in bioavailability of simple silver salts and nanosilver vs. silver metal (massive/powder)
   is justified to complete the REACH data requirements for Ag metal (massive/powder) and to avoid any new animal testing.

 

Weight of evidence

1. Problem formulation

No study is available for the reproductive (including fertility and developmental toxicity) of silver metal (massive and powder). However, there are studies available for other silver substances including nanosilver and soluble silver compounds (like silver acetate). Systemic toxicity of silver is driven by released silver ions (Ag+) and the amount of Ag+ release is proportionate to the specific surface area of the particle. Therefore, the studies performed on nanosilver and silver compounds will be considered in a weight-of-evidence (WoE) approach for the repeated toxicity of silver metal (massive and powder) and the difference in bioavailability and systemic distribution between silver metal (massive and powder) vs nanosilver and soluble silver salts -demonstrated in a comparative in-vivo toxicokinetic assay- will be accounted for. In addition, the hypothesised mode of action via copper deficiency is considered in the reproductive toxicity assessment of silver metal (massive and powder).

2. Collection - documentation and quality assessment of all information

The WoE-approach for reproductive toxicity (including fertility and developmental toxicity) of silver metal (massive and powder) is built based on several piece of evidence provided by studies with nanosilver and soluble silver salts (like silver acetate and silver nitrate).

The toxicity to reproduction was assessed considering:

1. Data from studies assessing the fertility and reproductive performance of parental animals – F0 generation capacity and capability to reproduction
2. Data from studies assessing the development and performance of offspring (including developmental neurotoxicity and developmental immunotoxicity).

Note that, based on the outcome of the comparative in-vivo toxicokinetics data, the data of nanosilver and soluble silver salts (like silver acetate) cannot be directly read-across to silver metal (massive and powder) because of the demonstrated difference in bioavailability. Therefore, and since it is acknowledged that silver ion is the toxicophore for systemic toxicity of silver, the outcome of studies performed with nanosilver and soluble silver salts are a worst-case to assess the potential effects of silver metal powder and can not be used in a direct read-across approach to Ag metal (massive and powder). Therefore, the appropriate modification to address this difference in bioavailability (and its consequence on the Cu depletion as presumed Mode of Action for observed reproductive and developmental effects) needs to be accounted for to assess the effects of Ag metal (massive and powder).

3. Integration of data and weight of evidence

The reproductive toxicity assessment of silver metal (massive and powder) is based on several pieces of evidence assessing the effect of nanosilver and soluble silver salts (like silver acetate) on the reproductive toxicity endpoint.

It is assumed that systemic toxicity of silver substances is driven by Ag+ (silver ions) as the primary toxicophore of systemic circulation and thus relevant to tissues exposure, and hence hazard assessment. Thus, a low bioavailability, leading to a correspondingly low internal concentration of Ag+ (toxicophore) leads to a correspondingly low level of biological interaction and hence correspondingly low adverse outcome in comparison with highly bioavailable silver. Therefore, the bioavailability (i.e. Ag+ release) is considered as key piece of evidence in the weight of evidence for the reproductive toxicity of silver metal (massive and powder).

In more detail, the bioavailability of AgAc via oral route is consistent between independent estimates presented in the scientific literature (Barraclough and Cotton 2017, Boudreau et al. 2012, Park et al. 2011 and Van der Zande et al. 2012) and the comparative in-vivo TK study – in the order of 3-5%. In comparative terms, silver metal powder (AgMP) is substantially less absorbed. Based on matched dose assessments performed in the in-vivo TK study (Melvin et al., 2021 and Charlton et al., 2021), the extent of systemic exposure was about 10 to 30-fold lower in the case of AgMP vs AgAc. Unlike of situation of AgAc, the degree of uptake is not linear as the amount of administered of AgMP was increased up to a limit dose, but instead there was evidence of absorption plateauing. As a generic observation, the following trend in bioavailability is being observed: AgAc / AgNO3 >> AgNP >>> AgMP.

Therefore, the toxicokinetic findings strongly suggest that the direct read-across of mammalian toxicity data with soluble silver salts (like silver acetate and silver nitrate) and nanosilver to silver metal (powder and massive) is not justified (based on their respective toxicokinetic profiles).

This new evidence demonstrates that oral intake of AgMP results in markedly lower absorption, distribution and systemic tissue/organ exposure to silver than more bioavailable forms like AgAc.

Therefore, the Toxicokinetic / bioavailability / Ag+ release parameter (Melvin et al., 2021; Charlton et al., 2021) is considered as key piece of evidence in the weight of evidence reasoning for the reproductive toxicity of silver metal (massive and powder) by oral route.

Moreover, the copper-depletion mode of action is considered and assessed by an external expert in the context of the EOGRTS on silver acetate:

1. Evidence exists from rodent models that administration of ionic Ag can cause a copper deficiency (Cu-D) state, through Ag+-induced deformation of the key Cu transporter protein ceruloplasmin (Cp). Treatment with ionic Ag, e.g. for 4 weeks or more, has been shown to cause loss of Cp activity and perturbation of Cu homeostasis.
2. In the EOGRTS with silver acetate, serum Cu measurements performed in F0 and F1 rats clearly showed a treatment-related pattern of Cu level depression, with serum Cu concentrations inversely related to increasing blood Ag concentration. At the dose levels associated with the main treatment-related findings in the EOGRTS, the deficiency state can be categorised as either moderately severe or moderate in its degree.
3. The following treatment-related effects were observed in F0 and F1 rats in the EOGRTS: (i) haematological and biochemical shifts (mainly anaemia, increased plasma alkaline phosphatase activity and hypercholesterolemia); (ii) organ weight changes indicative of cardiac hypertrophy and thymic involution; (iii) changes in littering indices (reductions in live birth rate and pup viability); (iv) pup growth rate depression, and development of poor condition or failure to thrive; (v) brain myelopathy, cell loss, and diminution of the size of the hippocampus; and (vi) deficits in motor function and neurobehavioral parameters. As described in detail in the attached expert opinion, all these effects are related to indirect Cu-D mediated by Ag+ exposure.
4. EPMF investigated the Cu status of rats treated with a sub-micron silver metal powder (AgMP) during a recent comparative OECD TG 417 TK study. Findings were compared to those of animals which received an ionic Ag reference form (silver nitrate, with a proven similar TK profile than silver acetate). Even at a limit dose (1000 mg Ag/kg bw/d), AgMP did not cause Cu-D in rats following 28 days treatment (p.o.), in contrast with silver nitrate where circulating Cu levels were already depressed as from 35 mg Ag/kg bw/d. It should be noted that 1) the highest tested Ag equivalent dose level for AgMP was more than 12 times that for silver nitrate and 2) steady-state conditions for Ag are reached after repeated dosing for a period of about two weeks.

In conclusion, based on comparative TK data coupled to Cu serum determinations, it is unlikely to attain a condition of sufficient Cu depletion in the case of orally administered silver metal (massive and powder) at relevant dose levels for hazard classification (up to limit dose).

The weight of evidence is considered justified and conservative when using robust evidence of reproductive toxicity studies performed with nanosilver and soluble silver salts (like silver acetate), provided that -in accordance with the outcome of the comparative in-vivo toxicokinetic study- an appropriate interpretation and evaluation of key effects for silver metal (massive and powder) is performed.

For the reproductive toxicity/fertility, the study of Renaut et al., 2022 (EOGRTS on silver acetate) is considered conservative and robust as key study to assess the reproductive toxicity of silver metal (massive and powder). The No Adverse Effect Level (NOAEL) defined by Renaut et al., 2022 for adverse effect on reproductive performance parameters is 120 mg/kg bw/day (highest dose tested). According to the in-vivo TK study, the absorption of silver metal was assessed at 10 to 30-fold lower than AgAc. Therefore, it can reasonably and confidently be extrapolated that that the corresponding NOAEL for silver metal (massive and powder) will be well above the limit dose (120*10 (most conservative %) = 1200 mg/kg bw/day for male and female animals).

For the reproductive toxicity/developmental toxicity assessment, the study of Renaut et al, 2022 (EOGRTS including DNT and DIT cohorts on silver acetate) is considered conservative and robust as key study to assess the reproductive toxicity of silver metal (massive and powder). The NOAEL defined by Renaut et al, 2022 are for developmental toxicity NOAEL = 80 mg /kg bw/day, for developmental neurotoxicity NOAEL = 40 mg/kg bw/day and for developmental immunotoxicity NOAEL = 120 mg/kg bw/day. According to the in-vivo TK study, the absorption of silver metal was assessed at 10 to 30-fold lower than AgAc. Therefore, it can reasonably and confidently be extrapolated that that the corresponding NOAEL for silver metal (massive and powder) will be at or above (80*10)= 800 mg/kg bw/day, (40*10)= 400 mg/kg bw/day and (120*10) = 1200 mg/kg bw/day, respectively. Additional piece of evidence to consider is that the comparative in vivo TK study demonstrated no effect of repeated Ag powder exposure up to limit dose levels on Cu serum levels, the presumed MoA behind the observed effects in the above studies. This further supports the conservatism of the above extrapolated threshold levels for Ag meta (massive and powder).

4. Conclusion

In conclusion the evaluation of reproductive toxicity of silver metal (massive and powder) was performed via the weight of evidence approach. The approach was built on:

• confident and reliable reproductive toxicity studies using nanosilver and soluble silver salts (like AgAc
• the comparative in vivo TK study justifying a correction factor to adjust the difference in oral absorption/bioavailability between silver metal (massive and powder)
• the observed Cu depletion as presumed Mode of Action behind the observed reproductive effects, which has been demonstrated in the comparative in vivo TK study to not appear up to limit dose levels of Ag metal powder.

A strong weight of evidence using the available data demonstrates that the adverse effects on fertility and reproduction after repeated exposure to nanosilver and soluble silver salts are not expected for silver metal (massive and powder) based on the bioavailability considerations and its direct interaction with the Cu-depletion mode of action.

Please see in Section 13 the CSR Annex 11 - Weight of Evidence Justification for Silver Metal - human health endpoints, for further details. 

 

Justification for selection of Effect on fertility via oral route:
Weight of evidence

Justification for selection of Effect on fertility via inhalation route:
Oral route considered more relevant.

Justification for selection of Effect on fertility via dermal route:
Oral route considered more relevant.

Effects on developmental toxicity

Description of key information

The evaluation of developmental toxicity of silver metal (massive and powder) was performed via the weight of evidence approach. The approach was built on:

• confident and reliable reproductive toxicity studies using nanosilver and soluble silver salts (like AgAc
• the comparative in vivo TK study justifying a correction factor to adjust the difference in oral absorption/ bioavailability between silver metal (massive and powder)
• the observed Cu depletion as presumed Mode of Action behind the observed reproductive effects, which has been demonstrated in the comparative in vivo TK study to not appear up to limit dose levels of Ag metal powder.

A strong weight of evidence using the available data demonstrates that the adverse effects on fertility and reproduction after repeated exposure to nanosilver and soluble silver salts are not expected for silver metal (massive and powder) based on the bioavailability considerations and its direct interaction with the Cu-depletion mode of action.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure

Species:

other: No test species proposed. The conduct of a developmental toxicity study in a second species cannot be expected to contribute any relevant information to the assessment of (otherwise negative) information on reproduction toxicity.

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD443 Extended One Generation Reproductive Toxicity Study

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

oral: feed

Dose / conc.:

120 mg/kg bw/day (actual dose received)

Dose / conc.:

80 mg/kg bw/day (actual dose received)

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Key result

Dose descriptor:

LOAEL

Effect level:

<= 40 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

gross pathology

Remarks on result:

other: degeneration in stomach mucosa in females at all doses - cfr. Renaut et al 2022 under section 'Reproductive toxicity'

Key result

Dose descriptor:

NOAEL

Effect level:

>= 120 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other:

Remarks on result:

other: cfr. Renaut et al 2022 under section 'Reproductive toxicity'

Key result

Dose descriptor:

NOAEL

Remarks:

F1 (cohort 2A)

Effect level:

ca. 40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: neuropathology

Remarks on result:

other: cfr. Renaut et al 2022 under section 'Reproductive toxicity'

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: cfr. Renaut et al 2022 under section 'Reproductive toxicity'

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

It was therefore concluded that the various no observed adverse effect levels (NOAELs) on this study were:

- F1 offspring survival and growth up to weaning: 80 mg/kg/day

- Developmental neurotoxicity in selected F1 animals: 40 mg/kg/day (due to the following effects of treatment at 80 or 120 mg/kg/day: reduced activity and rearing of males and females in the arena, reduced reactivity, abnormal motor movement/gait, intramyelinic edema and neuronal and/or glial cell necrosis and F1 brain morphometry (low mean hippocampus)

- Developmental immunotoxicity in selected F1 animals – 120 mg/kg/day (highest dose tested)

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data available

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well-documented, guideline-conform study conducted under GLP (NTP)

Justification for type of information:

Weight of evidence approach described in 'Silver metal (massive and powder): Weight of Evidence' (document attached in IUCLID section 13 - "CSR Annex 11 - Weight of Evidence Justification for Silver metal - human health endpoints).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. Raleigh, NC
- Housing: pegnant females and control animals were individually housed in solid-bottom polycarbonate cages with stainless steel wire lids and certified Sani-Chip hardwood cage litter.
- Diet: ad libitum; purina Certified Rodent Chow
- Water:ad libitum; tap water
- Acclimation period: 10 days following arrival

ENVIRONMENTAL CONDITIONS
- Temperature (°C): appriximately 21 to 23
- Humidity (%): 47.2-58.5
- Photoperiod: 12 hours dark/light cycle

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
- Each concentration of silver acetate was formulated independently in the vehicle.
- Dose formulations were stored at approximately 5°C in sealed, amber glass bottles.

VEHICLE
- The vehicle, 1% aqueous methylcellulose, was formulated from methylcellulose (CAS No. 9004-67-5), 400cps, obtained from MRI (Batch No. 01; Vendor Lot No. PE0515, Spectrum Chemical Manufacturing Co.).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Samples of each dose formulation were collected prior and after dosing and were submitted to Midwest Research Institute for verification of silver acetate concentrations by potentiometric titration.
- Pre-dosing samples were within 87.5 and 98.8% and post dosing samples were within 88 and 101.1% of their nominal concentrations.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1; individual breeding pairs were cohabited overnight.
- Proof of pregnancy: vaginal sperm referred to as day 0 of pregnancy.

Duration of treatment / exposure:

from gestational day (gd) 6 to 19

Frequency of treatment:

daily

Duration of test:

until gd 20

No. of animals per sex per dose:

- A total of 25 time-mated females per group were assigned to this study.
- 10 additional untreated females (sentinels) were cooused.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: based on a previous screening study in rats (NTP, 2001); the low dose was selected since it was not likely to cause maternal or foetal toxicity; the high dose given over the period of embryo/foetal development was expected to cause significant maternal toxicity, but was unlikely to cause developmental toxicity; an intermediate dose was selected to assist in characterisation of the dose-response curve for critical endpoints.
- Rationale for animal assignment: confirmed-mated females were assigned to treatment groups by stratified randomisation for body weight on gestation day (gd) 0, so that mean body weight on gd 0 did not differ among treatment groups; maternal body weights for confirmed pregnant females ranged from 213.9 to 271.6 g on gd 0.
- Other: the oral route corresponsed to one of the expected routes of humen exposure.

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: females were observed for clinical signs once/day on gd 0 to 5 (prior to initiation of dosing), and twice per day on gd 6 through 19 (once at dosing and once 1 to 2 hours after dosing).

BODY WEIGHT: Yes
- Time schedule for examinations: body weight was recorded on the mornings of gd 0, 6 through 19 and 20, immediately following sacrifice (gd 20).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: feed consumption was monitored during the study, with measurements on the mornings of gd 0, 6, 9, 12, 15, 18, 19 and 20.

WATER CONSUMPTION: Yes
- Time schedule for examinations: water consumption was monitored during the study, with measurements on the mornings of gd 0, 6, 9, 12, 15, 18, 19 and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 by CO2 asphyxiation.
- Organs examined: body, liver and gravid uterus were weighed; thoracic and abdominal cavities were examined; maternal livers were saved in 10% neutral buffered formalin for optional histopathology.

OTHER:
- At necropsy, blood samples were collected by cardiac puncture from 10 sentinel females under terminal CO2 anaesthesia. Serum was submitted for evaluation of viral antibody titers. All assay in the serological panel were negative, including pneumonia virus (PVM), sendai virus (sendai), rat corona virus/sialodacyoadenitis virus (RCV/SDA) and parvo virus (Parvo).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes, pregnancy status was confirmed by uterine examination.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Number of dead and live foetuses: Yes
- Uteri which presented no visible implantation sites were staiend with ammonium sulfide (10%) in order to visulise any implantation sites which might have undergone very early resorption.

Fetal examinations:

External examinations: Yes
- Dead foetuses were counted, weighed and discarded.
- Live foetuses were dissected from the uterus and immediately placed on a moist paper towel over a tray of ice, a procedure which induces anaesthesia.
- All live foetuses were counted, weighed, sexed and examined for external morphological abnormalities, including cleft palate.

Soft tissue examinations: Yes
- Approximately 50% of the fetal carcasses were sexed and examined for visceral morphological abnormalities.

Skeletal examinations: Yes
- All foetal carcasses were eviscerated, and the skeletons macerated and stained with alcian blue/alizarin red S stain.
- Intact foetal skeletons were examined for skeletal morphological abnormalities.

Head examinations: Yes
- Foetal heads were fixed and decalcified in Bouin's solution and subsequently examined.

Statistics:

- The unit for statistical measurement was the pregnant female or the litter.
- Quantitative continuous data were compared among treatment groups by parametric statistical tests whenever Bartlett's test for homogeneity of variance was not significant. If Bartlett's test indicated a lack of homogeneity (p<0.001), then nonparametric statistical tests were applied.
- All statistical procedures applied to select measures from this study were based on SAS software.
- General Linear Models (GLM) procedures were applied to the Analyses of Variance (ANOVA) and the Test for Linear Trand. For litter-derived percentage data, the ANOVA was weighed according to litter size.
- If a asignificant (p<0.05) main effect for dose occurred, Dunnett's Multiple Comparison Test was used to compare each treatment group to the control group for that measure.
- A one-tailed test was used for all pairwise comparisons to the vehicle control group, expect that a two-tailed test was used for maternal body and organ weight parameters, maternal feed and water consumption, foetal body weight and percent males per litter.
- Nonparametric tests applied to continuous variables included the Kruskal-Wallis one-way analysis of variance by ranks for among-group differences and, when significant (p<0.05), the Mann-Whitney U test for pairwise comparisons to the vehicle control group.
- Jonckheere's test for k independent samples was used to identify significant dose-response trends.
- Nominal scale measures were analysed by Chi-Square Test for Independence for differences among treatment groups and by the Cochran-Armitage Test for Linear Trend on Properties.
- The alpha level for each statistical comparison was 0.05, except as noted above for Bartlett's Test. The significance level for each trend test or pairwise comparison (one- or two-tailed) was reported as p<0.05 or p<0.01.

Indices:

no details

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
MATERNAL EXAMINATIONS
- One animal was removed from the high dose group due to a misdirected dose, and one confirmed pregnant female in the high-dose group was euthanized on gd 12 due to morbidity. All remaining females survived until scheduled sacrifice on gd 20.
- Pregancy rates were 96, 92, 100 and 87.5 % in the control, 10, 30 and 100 mg/kg bw/d groups, respectively.
- Clinical findings of weight loss, rooting after dosing (only high dose) or oiloerection were observed in individual animals (1-5 per day) of the mid (30 mg/kg b/d) and high (100 mg/kg bw/d) dose groups.
- Maternal body weights and body weight gains were comparable among groups throughout the study in cluding the treatment period.
- A decreasing trend was noted for maternal body weight on gd 12, although there were not significant differences between control an treated groups.
- No effects on body weights corrected for uterine weights, gravide uterine weights or liver weights were found.
- No dose-related effects on food and water consumption could be established.

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

> 100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
OVARIES AND UTERINE CONTENTS
- The number of corpora lutea per dam, number of implantation sites per litter, percent implantation loss per litter were comparable among groups.
- Postimplantation mortality (number/percent resorptions, late foetal death and/or nonlive implants) average litter size and percent male foetuses per litter did not differ among groups.
- Although a significant trend (p<0.05) was observed for the percent litters with late foetal deaths (0, 0, 0, and 10% in the control 10, 30, and 100 mg/kg bw/d groups, respectively), no significant tretament-related effects were observed for the percent late foetal death/litter.
- Average foetal body weight per litter (3.52±0.05, 3.53±0.08, 3.40 ±0.04, 3.35±0.10, respectively) and average male foetal body weight per litter (3.61±0.05, 3.60± 0.08, 3.45±0.05 and 3.42±0.10, respectively) exhibited a significant trend (p<0.05), but no significant differences between control and the 10, 30 or 100 mg/kg bw/d groups.

FOETAL EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
- No toxicologically relevant differences were observed in the incidences of foetal malformations and variations.
- The percent female fetuses with malformatins per litter exhibited a significant effect for dose (p<0.01), but no significant trend or pairwise differences beween treated groups and the control group were observed.

Dose descriptor:

other:

Basis for effect level:

other: See 'Remark'

Remarks on result:

other: See 'Remarks'

Remarks:

There were no differences among groups for the number of ovarian corpora lutea/dam, number of implantation sites/litter or percent preimplantation loss/litter. Postimplantation/loss (resorptions, late foetal deaths or nonlive implant/litter), live litter size and percent male foetuses/litter did not differ among groups. An increasing trend was observed for the percent litters with late foetal deaths. Average foetal body weight per litter (sexes combined) and average male foetal body weight per litter exhibited a significant decreasing trend, but no significant pairwise differences between treated groups and the control group. No statistically significant effects were noted for average female body weight. No toxicologically relevant differences were observed in the incidences of foetal malformations or variations.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

In conclusion, the maternal LOAEL for this study was considered to be 30 mg/kg/day silver acetate (19.4 mg silver/kg/day) based on clinical signs including weight loss. The maternal NOAEL was 10 mg/kg/day silver acetate (6.5 mg Ag/kd/day). The NOAEL for developmental toxicity for this study was 100 mg/kg/day silver acetate (64.6 mg Ag/kg/day), based on the absence of any biologically or statistically significant developmental toxicity.

Executive summary:

The developmental toxicity study was conducted in female Sprague-Dawley (CD) rats dosed by gavage with silver acetate in 1% aqueous methylcellulose (10, 30 or 100 mg/kg/day) or vehicle from gd 6 through 19.

One animal was removed from the high dose group due to a misdirected dose, and one confirmed pregnant female in the high-dose group was euthanized on gd 12 due to morbidity. All remaining females survived until scheduled sacrifice on gd 20. Pregnancy was confirmed in 21-25 females per group (i.e. 87.5-100% per group) after necropsy on gd 20.

Treatment-related clinical signs were noted primarily in the mid and high-dose groups and consisted of weight loss, rooting after dosing and piloerection.

Maternal body weight was comparable among groups, as was maternal body weight change. A significant (p<0.05) decreasing linear trend was noted for maternal body weight on gd 12, but there were no statistically significant differences between the control group and any treated group.

Maternal absolute feed and water consumption did not exhibit dose-related differences between the control group and silver acetate-treated group.

There were no differences among groups for the number of ovarian corpora lutea/dam, number of implantation sites/litter or percent preimplantation loss/litter. Postimplantation/loss (resorptions, late foetal deaths or nonlive implant/litter), live litter size and percent male foetuses/litter did not differ among groups. An increasing trend was observed for the percent litters with late foetal deaths. Average foetal body weight per litter (sexes combined) and average male foetal body weight per litter exhibited a significant decreasing trend, but no significant pairwise differences between treated groups and the control group. No statistically significant effects were noted for average female body weight.

No toxicologically relevant differences were observed in the incidences of foetal malformations or variations.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Introduction:

An in-vivo comparative toxicokinetic study, via oral route, was performed using a rodent model (according to OECD TG 417 and GLP compliant; Melvin et al., 2021 and Charlton et al., 2021). The test items included two ionic silver salts (silver nitrate and silver acetate), a well-characterized nanosilver reference material (15 nm AgNP) and a powder-form of silver metal (size ~0.3 μm, representing a conservative silver metal powder). Comparative toxicokinetics data were obtained after both single and 28-days repeated dose administration, including the measurements of Ag levels in blood and in tissues.

The key findings were:

• silver metal was substantially less absorbed than soluble silver salts and nanosilver. Based on matched dose assessments, the extent of systemic exposure was about 10 to 30-fold lower in the case of silver metal versus reference ionic silver salts.

• silver metal was considerably less distributed in tissues and organs than silver salts (ionic silver compounds). This links to predictions that silver metal (massive and powder) represents a correspondingly lower health hazard, i.e., is less likely to cause toxicity effects.

It is generally accepted that systemic toxicity of simple silver salts substances is driven by the silver ion (Ag+) as the primary species relevant for tissue exposure, and hence hazard assessment. Thus, a low bioavailability of silver metal (massive and powder), leading to a low internal concentration of silver ions (as toxicophore) leads to a lack of biological interaction and hence an absence of adverse outcome in comparison with high bioavailable silver salts. Therefore, it is assumed that silver metal represents a lower health hazard than the more bioavailable forms of silver at comparable nominal Ag levels.
Therefore, following the new in-vivo TK study findings, a direct Read-Across of mammalian toxicity datasets from simple silver salts and nanosilver to silver metal (massive and powder) is considered not appropriate.

Alternatively, a Weight of Evidence (WoE) approach considering:

1. the available mammalian toxicity data of simple silver salts and nanosilver and
2. the demonstrated difference in bioavailability of simple silver salts and nanosilver vs. silver metal (massive/powder)
   is justified to complete the REACH data requirements for Ag metal (massive/powder) and to avoid any new animal testing.

 

Weight of evidence

1. Problem formulation

No study is available for the reproductive (including fertility and developmental toxicity) of silver metal (massive and powder). However, there are studies available for other silver substances including nanosilver and soluble silver compounds (like silver acetate). Systemic toxicity of silver is driven by released silver ions (Ag+) and the amount of Ag+ release is proportionate to the specific surface area of the particle. Therefore, the studies performed on nanosilver and silver compounds will be considered in a weight-of-evidence (WoE) approach for the repeated toxicity of silver metal (massive and powder) and the difference in bioavailability and systemic distribution between silver metal (massive and powder) vs nanosilver and soluble silver salts -demonstrated in a comparative in-vivo toxicokinetic assay- will be accounted for. In addition, the hypothesised mode of action via copper deficiency is considered in the reproductive toxicity assessment of silver metal (massive and powder).

2. Collection - documentation and quality assessment of all information

The WoE-approach for reproductive toxicity (including fertility and developmental toxicity) of silver metal (massive and powder) is built based on several piece of evidence provided by studies with nanosilver and soluble silver salts (like silver acetate and silver nitrate).

The toxicity to reproduction was assessed considering:

1. Data from studies assessing the fertility and reproductive performance of parental animals – F0 generation capacity and capability to reproduction
2. Data from studies assessing the development and performance of offspring (including developmental neurotoxicity and developmental immunotoxicity).

Note that, based on the outcome of the comparative in-vivo toxicokinetics data, the data of nanosilver and soluble silver salts (like silver acetate) cannot be directly read-across to silver metal (massive and powder) because of the demonstrated difference in bioavailability. Therefore, and since it is acknowledged that silver ion is the toxicophore for systemic toxicity of silver, the outcome of studies performed with nanosilver and soluble silver salts are a worst-case to assess the potential effects of silver metal powder and can not be used in a direct read-across approach to Ag metal (massive and powder). Therefore, the appropriate modification to address this difference in bioavailability (and its consequence on the Cu depletion as presumed Mode of Action for observed reproductive and developmental effects) needs to be accounted for to assess the effects of Ag metal (massive and powder).

3. Integration of data and weight of evidence

The reproductive toxicity assessment of silver metal (massive and powder) is based on several pieces of evidence assessing the effect of nanosilver and soluble silver salts (like silver acetate) on the reproductive toxicity endpoint.

It is assumed that systemic toxicity of silver substances is driven by Ag+ (silver ions) as the primary toxicophore of systemic circulation and thus relevant to tissues exposure, and hence hazard assessment. Thus, a low bioavailability, leading to a correspondingly low internal concentration of Ag+ (toxicophore) leads to a correspondingly low level of biological interaction and hence correspondingly low adverse outcome in comparison with highly bioavailable silver. Therefore, the bioavailability (i.e. Ag+ release) is considered as key piece of evidence in the weight of evidence for the reproductive toxicity of silver metal (massive and powder).

In more detail, the bioavailability of AgAc via oral route is consistent between independent estimates presented in the scientific literature (Barraclough and Cotton 2017, Boudreau et al. 2012, Park et al. 2011 and Van der Zande et al. 2012) and the comparative in-vivo TK study – in the order of 3-5%. In comparative terms, silver metal powder (AgMP) is substantially less absorbed. Based on matched dose assessments performed in the in-vivo TK study (Melvin et al., 2021 and Charlton et al., 2021), the extent of systemic exposure was about 10 to 30-fold lower in the case of AgMP vs AgAc. Unlike of situation of AgAc, the degree of uptake is not linear as the amount of administered of AgMP was increased up to a limit dose, but instead there was evidence of absorption plateauing. As a generic observation, the following trend in bioavailability is being observed: AgAc / AgNO3 >> AgNP >>> AgMP.

Therefore, the toxicokinetic findings strongly suggest that the direct read-across of mammalian toxicity data with soluble silver salts (like silver acetate and silver nitrate) and nanosilver to silver metal (powder and massive) is not justified (based on their respective toxicokinetic profiles).

This new evidence demonstrates that oral intake of AgMP results in markedly lower absorption, distribution and systemic tissue/organ exposure to silver than more bioavailable forms like AgAc.

Therefore, the Toxicokinetic / bioavailability / Ag+ release parameter (Melvin et al., 2021; Charlton et al., 2021) is considered as key piece of evidence in the weight of evidence reasoning for the reproductive toxicity of silver metal (massive and powder) by oral route.

Moreover, the copper-depletion mode of action is considered and assessed by an external expert in the context of the EOGRTS on silver acetate:

1. Evidence exists from rodent models that administration of ionic Ag can cause a copper deficiency (Cu-D) state, through Ag+-induced deformation of the key Cu transporter protein ceruloplasmin (Cp). Treatment with ionic Ag, e.g. for 4 weeks or more, has been shown to cause loss of Cp activity and perturbation of Cu homeostasis.

2. In the EOGRTS with silver acetate, serum Cu measurements performed in F0 and F1 rats clearly showed a treatment-related pattern of Cu level depression, with serum Cu concentrations inversely related to increasing blood Ag concentration. At the dose levels associated with the main treatment-related findings in the EOGRTS, the deficiency state can be categorised as either moderately severe or moderate in its degree.

3. The following treatment-related effects were observed in F0 and F1 rats in the EOGRTS: (i) haematological and biochemical shifts (mainly anaemia, increased plasma alkaline phosphatase activity and hypercholesterolemia); (ii) organ weight changes indicative of cardiac hypertrophy and thymic involution; (iii) changes in littering indices (reductions in live birth rate and pup viability); (iv) pup growth rate depression, and development of poor condition or failure to thrive; (v) brain myelopathy, cell loss, and diminution of the size of the hippocampus; and (vi) deficits in motor function and neurobehavioral parameters. As described in detail in the attached expert opinion, all these effects are related to indirect Cu-D mediated by Ag+ exposure.

4. EPMF investigated the Cu status of rats treated with a sub-micron silver metal powder (AgMP) during a recent comparative OECD TG 417 TK study. Findings were compared to those of animals which received an ionic Ag reference form (silver nitrate, with a proven similar TK profile than silver acetate). Even at a limit dose (1000 mg Ag/kg bw/d), AgMP did not cause Cu-D in rats following 28 days treatment (p.o.), in contrast with silver nitrate where circulating Cu levels were already depressed as from 35 mg Ag/kg bw/d. It should be noted that 1) the highest tested Ag equivalent dose level for AgMP was more than 12 times that for silver nitrate and 2) steady-state conditions for Ag are reached after repeated dosing for a period of about two weeks.

In conclusion, based on comparative TK data coupled to Cu serum determinations, it is unlikely to attain a condition of sufficient Cu depletion in the case of orally administered silver metal (massive and powder) at relevant dose levels for hazard classification (up to limit dose).

The weight of evidence is considered justified and conservative when using robust evidence of reproductive toxicity studies performed with nanosilver and soluble silver salts (like silver acetate), provided that -in accordance with the outcome of the comparative in-vivo toxicokinetic study- an appropriate interpretation and evaluation of key effects for silver metal (massive and powder) is performed.

For the reproductive toxicity/fertility, the study of Renaut et al., 2022 (EOGRTS on silver acetate) is considered conservative and robust as key study to assess the reproductive toxicity of silver metal (massive and powder). The No Adverse Effect Level (NOAEL) defined by Renaut et al., 2022 for adverse effect on reproductive performance parameters is 120 mg/kg bw/day (highest dose tested). According to the in-vivo TK study, the absorption of silver metal was assessed at 10 to 30-fold lower than AgAc. Therefore, it can reasonably and confidently be extrapolated that that the corresponding NOAEL for silver metal (massive and powder) will be well above the limit dose (120*10 (most conservative %) = 1200 mg/kg bw/day for male and female animals).

For the reproductive toxicity/developmental toxicity assessment, the study of Renaut et al, 2022 (EOGRTS including DNT and DIT cohorts on silver acetate) is considered conservative and robust as key study to assess the reproductive toxicity of silver metal (massive and powder). The NOAEL defined by Renaut et al, 2022 are for developmental toxicity NOAEL = 80 mg /kg bw/day, for developmental neurotoxicity NOAEL = 40 mg/kg bw/day and for developmental immunotoxicity NOAEL = 120 mg/kg bw/day. According to the in-vivo TK study, the absorption of silver metal was assessed at 10 to 30-fold lower than AgAc. Therefore, it can reasonably and confidently be extrapolated that that the corresponding NOAEL for silver metal (massive and powder) will be at or above (80*10)= 800 mg/kg bw/day, (40*10)= 400 mg/kg bw/day and (120*10) = 1200 mg/kg bw/day, respectively. Additional piece of evidence to consider is that the comparative in vivo TK study demonstrated no effect of repeated Ag powder exposure up to limit dose levels on Cu serum levels, the presumed MoA behind the observed effects in the above studies. This further supports the conservatism of the above extrapolated threshold levels for Ag meta (massive and powder).

4. Conclusion

In conclusion the evaluation of reproductive toxicity of silver metal (massive and powder) was performed via the weight of evidence approach. The approach was built on:

• confident and reliable reproductive toxicity studies using nanosilver and soluble silver salts (like AgAc
• the comparative in vivo TK study justifying a correction factor to adjust the difference in oral absorption/bioavailability between silver metal (massive and powder)
• the observed Cu depletion as presumed Mode of Action behind the observed reproductive effects, which has been demonstrated in the comparative in vivo TK study to not appear up to limit dose levels of Ag metal powder.

A strong weight of evidence using the available data demonstrates that the adverse effects on development and reproduction after repeated exposure to nanosilver and soluble silver salts are not expected for silver metal (massive and powder) based on the bioavailability considerations and its direct interaction with the Cu-depletion mode of action.

Please see in Section 13 the CSR Annex 11 - Weight of Evidence Justification for Silver Metal - human health endpoints, for further details. 

 

 

Justification for selection of Effect on developmental toxicity: via oral route:
Weight of evidence

Justification for selection of Effect on developmental toxicity: via inhalation route:
Oral route considered more relevant.

Justification for selection of Effect on developmental toxicity: via dermal route:
Oral route considered more relevant.

Justification for classification or non-classification

No data are currently available indicating that effects on fertility would be of specific concern. Based on the existing data gap, a testing proposal for an extended one-generation reproductive toxicity study is submitted. A developmental toxicity study with oral (gavage) treatment of pregnant rats at different dose levels of silver acetate (Price et al., 2002) conducted within the US National Toxicology Program is available. Silver acetate is considered a soluble (=bioaccessible) substance and a suitable surrogate for other inorganic silver substances in which the silver ion Ag+ is the relevant moiety (worst case read-across for poorly soluble silver compounds). At all tested doses (i. e. 10, 30 and 100 mg/kg/day, based on silver acetate) there was an absence of any biologically or statistically significant developmental toxicity. In lack of data specifically on metallic silver or silver nanomaterials, (conservative) read-across from the study on silver acetate is conducted. In consequence, no classification for fertility or developmental effects is required.