Silver nitrate

Toxicity to reproduction

Administrative data

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:

key study

Study period:

12-02-2021 to 02-12-2022

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:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

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.

Test material

Test material information

Constituent 1

Reference substance name:

Silver acetate

EC Number:

209-254-9

EC Name:

Silver acetate

Cas Number:

563-63-3

Molecular formula:

C2H4O2.Ag

IUPAC Name:

silver(1+) acetate

Test material form:

solid

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

80 mg/kg bw/day (nominal)

Doses / concentrations 3

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.

Examinations

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"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 be 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.
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.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

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.

Effect levels (P0)

1

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.

2

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

Results: F1 generation

General toxicity (F1)

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.

.

Developmental neurotoxicity (F1)

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 (F1)

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).

Effect levels (F1)

1

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

2

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

3

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

Target system / organ toxicity (F1)

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

Overall reproductive toxicity

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.