Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study with acceptable restrictions Deviation from the guideline: - according to the guideline, animals of the satellite groups will not be mated and, consequently, are not used for the assessment of reproduction/developmental toxicity. In this tudy the animals of the recovery groups were mated. - it was not clear if a detailed clinical observation was carried out - female weights on day of delivery and day 4 post-partum were missing - no full gross pathology and histopathology were carried out. Histopathology of testes and epidydimis was not conducted - behaviour of pups was not recorded
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. deficiencies" above
GLP compliance:
yes
Remarks:
in accordance with GLP principles
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Orient Bio (Gyeonggi-do, Korea)
- Age at study initiation: 8 weeks old
- Housing: two rats per cage were housed in stainless-steel cages during the administration period, and one female was housed with one male during the mating period. During the gestation and lactation periods, mated females and foetuses were housed individually in polycarbonate cages.
- Diet (ad libitum)
- Water (ad libitum)
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 20.5–23.5 °C
- Relative humidity: 47.7–62.0%
- Air changes: 12 times each hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The suspension of silver nanoparticles for oral administration was prepared by diluting the stock silver nanoparticles (20% w/v) with distilled water and adjusting for the volume of 10 mL/kg bw of the rats. Formulations were freshly prepared daily prior to use. Suspensions were routinely stable in solution during the experiments. The size distribution of the silver nanoparticles in suspension was measured using a submicron particle size analyser (NICOMPTM, Santa Barbara, CA, USA) and energy-filtering (EF) TEM using a LIBRA120 apparatus (Carl Zeiss, Jena, Germany). To obtain EF-TEM images, AgNPs dispersed in ethanol were spread on the TEM grid.
The average size distribution was 8.8 ± 5.2 nm when measured immediately after preparation and 7.7 ± 4.8 nm 1 day after preparation. TEM images
also supported the size distribution.

A daily application volume (10 mL/kg) was adjusted according to the most recent body weight and the vehicle control rats were treated with an equivalent volume of distilled water.
Details on mating procedure:
- M/F ratio per cage: one female was housed with one male during the mating period
- Length of cohabitation: generally, the maximal mating period was 2 weeks
- Proof of pregnancy: each morning, the females were examined for the presence of sperm and vaginal plug. Day 0 of pregnancy was defined as the day when a vaginal plug or sperm was found.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
Male rats: 14 days before mating, 14 days during the mating period and 14 days of post-mating until necropsy (daily).
Female rats (maximum of 52 days): 2 weeks before mating, during the mating and gestation period, and during 4 days of lactation.
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
62.5, 125 and 250 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Main groups
Vehicle control group: 10 males/10 females
62.5 mg/kg: 10 males/10 females
125 mg/kg: 10 males/10 females
250 mg/kg: 10 males/10 females

Recovery groups:
Vehicle control group: 5 males/5 females)
250 mg/kg: 5 males/5 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the high dose of 250 mg/kg for 52-day treatment was selected based on the preliminary dosage test of this study, in which salivation was shown in a few of pregnant rats during the treatment period of 7 days. No deaths occurred during the 7-day administration period.
- Post-exposure recovery period in satellite groups: after completion of the treatment period, animals were maintained untreated during recovery for 14 days in the case of males and 16 days in the case of females.
Positive control:
no data
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day
- Cage side observations checked: clinical signs including mortality, motility, general appearance and autonomic activity
During the lactation period, nursing behaviours of dams was observed.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights of both sexes were measured once a week during pre-mating, mating and recovery periods. During the pregnancy and lactation periods, body weights were measured at day 0 (day of pregnancy), gestation day 3, 6, 9, 12, 15, 18 and 20 for pregnant rats.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption was measured during the pre-mating, pregnancy, lactation and recovery periods. During the mating period, food consumption was not measured.

WATER CONSUMPTION AND COMPOUND INTAKE: No data

Ag ANALYSIS
Tissues including liver, kidney and lung were obtained from four female rats sacrificed after 4 days lactation. The concentration of Ag in these tissues was analysed using ICP-MS (Elan6100/Perkin Elmer, Manhattan, NY, USA).

OTHER:
- gestation length was observed and recorded.
Oestrous cyclicity (parental animals):
For the examination of oestrus cycle, a vaginal smear was taken daily from each female during the pre-mating period. Regularity and length of the cycle was examined.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations: testis weight and epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
- on the parturition date, live and dead pups, sex, and external anomalies of live pups were observed and recorded.
- sex rate and survival rate at day 4 postpartum and body weight of live pups on day 0 (day of delivery) and day 4 postpartum were recorded.
- during the lactation period, viability of pups was observed.

Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
At scheduled termination, all live males and females were necropsied after repeated dosing for at least 28 or more days, and at day 5 of lactation, respectively. In recovery groups, males and females were terminated at 14 days after the first scheduled sacrifice of animals of the main groups. For all animals, gross necropsy consisted of a complete external and internal examination and identification of all abnormal findings with external surface and all orifices, cranial cavity, external surface of the brain and spinal cord, nasal cavity and paranasal sinus, thoracic-, abdominal- and pelvic cavities, and their viscera, cervical tissues and organs. Organs with gross lesions were preserved in 10% neutral buffered formalin.

Absolute organ weights were measured and their relative organ weights (organ-to-body weight ratios) were calculated from the terminal body weight for the following organs of parent and recovery animals when they were sacrificed: liver, spleen, heart, lung, brain, thymus, kidneys (left and right), adrenal glands (left and right), thyroid, pituitary gland, salivary gland, ovaries (left and right), testis (left and right), epididymis (left and right), seminal vesicles and uterus.
On completion of the gross pathology examination, histopathological examination of tissues was carried out for liver, kidney, adrenal gland, heart, lung, spleen, prostate gland, vagina, thymus, thyroid gland, stomach, urinary bladder and pancreas. Histopathological examination was performed on the aforementioned tissues from animals in the vehicle control and 250 mg/kg dose groups.
At necropsy, the numbers of corpora lutea and implantation sites were counted in all females.
Postmortem examinations (offspring):
For the necropsy of day 4 postpartum pups, five pups of five females in the vehicle control and 250 mg/kg/day groups were selected randomly at schedule termination. Liver, kidney, lung and brain were collected to obtain samples for electron microscopy.
Statistics:
Statistical analyses were performed by comparing the treatment groups with the vehicle control group using SPSS 10.1 Base Statistical Analysis System (SPSS Korea Data Solution, Seoul, Korea). The data were presented as mean ± SD. Variance in the numerical data was checked using Levene’s test. If the variance was homogeneous, the one-way analysis-of-variance test was conducted to determine which pairs of group comparison were significantly different. If this test showed significance between the groups, the data were analysed by the multiple-comparison procedure of the Dunnet’s post hoc test.
Reproductive indices:
Mating index, fertility index and pregnancy rate were recorded.
Delivery rate was recorded.
Pre- and post-implantation losses were calculated.
Offspring viability indices:
no data
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- deaths were not observed in any treated or control group.
- alopecia was observed in one male in the 125 mg/kg-treated group at 37 days after oral administration and two males in the 250 mg/kg-treated
group 38 days after treatment; the rats did not recover. Alopecia was observed in two pregnant rats in the vehicle-treated group, one in the 62.5 mg/kgtreated group, two in the 125 mg/kg-treated group and six in the 250 mg/kg-treated group. Recovery did not occur, except for two rats in the 250 mg/kg-treated group.
- transient salivation was observed immediately after administration of silver nanoparticles in a female in the high-dose group (250 mg/kg/day) on day 1 after gestation. Salivation was not observed in any other female rat during the entire experimental period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- no statistically significant changes in weight gain in any of the treatment groups during the pre-mating and mating periods in male rats, or in females during the pre-mating, gestation and lactation periods.
- compared to the entire experimental period, the weight gain during the first week of treatment was markedly more in male rats, while no difference
between control and treated groups was observed. This tendency was not observed in female rats.
- in both male and female rats, there was no significant difference in food consumption during the entire experimental period including pre-mating, gestation and post-parturition periods
- early during the study, food consumption was much more in male rats than in female rats, perhaps reflecting the difference in body sizes.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- no statistically significant difference in oestrus cycle between the groups in female rats.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- no statistically significant differences in mating, fertility and pregnancy rate among the groups, except that one non-pregnant rat and one premature birth were observed in the vehicle group
- no statistically significant differences were observed in the gestation period, number of corpora lutea and implantation, delivery rate, percentage of live and dead pups to implantations, preimplantation loss and post-implantation loss

ORGAN WEIGHTS (PARENTAL ANIMALS)
- in the recovery groups, a statistically significant increase in absolute and relative weights of liver was observed in males, and an increase in absolute weights of kidneys and adrenal gland was observed in females.
- absolute and relative weights of spleen, testes, brain, pituitary gland, lung, heart, thymus, thyroid gland, salivary gland, seminal vesicles and epididymis in the treated groups showed no significant differences from the control group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- yellow discolouration of lung was observed in one male in the 250 mg/kg/day group and multifocal white discolouration of spleen and splenomegaly was observed in one female in the 125 mg/kg/day group.

HISTOPATHOLOGY (PARENTAL ANIMALS)(control and high-dose groups (250 mg/kg)
- hepatocyte vacuolisation was observed in three treated female rats and in one rat from the control group
- interstitial inflammatory cells were observed in the kidney of three female rats in the treated group and three male rats in the control group.
- adrenal gland: cortical vacuolisation was found in three male rats from both the recovery and treated groups in the 250 mg/kg-treated groups.
- cholesterol granuloma in the lung was observed in two male rats in the treated and recovery groups. Granuloma was observed in two female rats in the treated group and one female rat in the recovery group.
- extramedullary haematopoiesis of the spleen was observed in three male rats from both the treated and recovery groups, and in one rat in the vehicle group.
- in other organs including brain, spinal cord, seminal vesicle, testis and ovary, lesions related with the AgNPs were not observed.
- lung granulomatous lesions were observed in 2 of 10 rats. It could not be confirmed whether the granulomatous lesion was related with silver nanoparticles treatment because it was evident only in two rats. Measurement of Ag in the lung revealed the apparent abundant accumulation compared to organs like the liver and kidney. The data were not sufficient to conclusively associate Ag accumulation with granulomatous lesion in the lung. Further study is necessary.

OTHER FINDINGS (PARENTAL ANIMALS)
- tissue distribution of silver in pregnant rats: the average quantity of silver in the livers of treated rats (1117.55 ± 381.68 ng/g) was increased by 34-fold compared to the level in control livers (33.54 ± 15.25 ng/g). The silver levels in lung (treatment value: 4461.22 ± 2726.42 ng/g; control value: 20.91 ± 7.47 ng/g) and kidney (treatment value: 449.78 ± 151.29 ng/g; control value: 34.81 ± 13.97 ng/g) were also significantly increased. Especially, the level of silver in the lungs of treated rats (4461.22 ± 2726.42 ng/g) was markedly increased compared to the level in liver or kidney.
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified Generation not specified (migrated information)
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
VIABILITY (OFFSPRING)
- no statistically significant differences were observed in the number of live and dead pups, sex ratio and survival rate.

BODY WEIGHT (OFFSPRING)
- no statistically significant differences were observed in the body weights of pups on postnatal days 0 and 4.

GROSS PATHOLOGY (OFFSPRING)
- no statistically significant differences were observed in the number of neonates with external anomalies.

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Reproductive effects observed:
not specified
Conclusions:
According to the authors, there was no statistically significant difference in mating, fertility and pregnancy rate among the groups. No statistically significant differences were observed in the following parameters examined: gestation period, number of corpora lutea and implantation, delivery rate, number of live and dead pups, percentage of live and dead pups to implantations, preimplantation loss, post-implantation loss, sex ratio, survival rate, number of neonates with external anomalies, and body weights of pups on postnatal days 0 and 4.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Guideline-compliant two-generation studies specifically on elemental silver or a simple ionic silver compound are not available. Two-generation studies in the rat are available for silver-containing active substances, but due to the coexistence with silver of other constituents which could exert direct toxic effects or else interfere with essential metals homeostasis, the direct read-across of the outcomes from these studies is considered to be insecure.  A 28d combined RDT study with reproduction/developmental screening (OECD 422) with citrate-capped silver nanomaterial demonstrated no effects on reproduction up to the highest dose of 250 mg/kg bw/d. It should be noted that this investigation did not involve a soluble silver salt, and that accompanying toxicokinetics sufficient to allow the extrapolation of its outcome more generally to ionic silver were not part of its design. Therefore, for simple soluble silver compounds it is considered that a formal data gap exists in relation to reproductive performance in males or females (in respect of REACH Annex IX and X requirements) and a testing proposal for an extended one-generation reproductive toxicity study is therefore submitted.


Short description of key information:
Taking into account the availability and robustness of the existing dataset, at least partial data gaps exist in respect of the reproductive toxicity potential of ionic silver. Based on the existing data gap, a testing proposal for an extended one-generation reproductive toxicity study is submitted.

Justification for selection of Effect on fertility via oral route:
Key study

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

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

Effects on developmental toxicity

Description of key information
Read-across to a developmental toxicity study with silver acetate (Price et al., 2002) conducted for the US National Toxicology Program is performed: at all tested doses (i.e. 10, 30 and 100 mg/kg/day as silver acetate), there was an absence of any biologically or statistically significant developmental toxicity. The NOAEL of 100 mg/kg/bw (as silver acetate) corresponds to a NOAEL of 64.6 mg/kg/bw based on silver.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data available
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well-documented, guideline-conform study conducted under GLP (NTP)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. Raleigh, NC
- Housing: pegnant females and control animals were individually housed in solid-bottom polycarbonate cages with stainless steel wire lids and certified Sani-Chip hardwood cage litter.
- Diet: ad libitum; purina Certified Rodent Chow
- Water:ad libitum; tap water
- Acclimation period: 10 days following arrival

ENVIRONMENTAL CONDITIONS
- Temperature (°C): appriximately 21 to 23
- Humidity (%): 47.2-58.5
- Photoperiod: 12 hours dark/light cycle
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
- Each concentration of silver acetate was formulated independently in the vehicle.
- Dose formulations were stored at approximately 5°C in sealed, amber glass bottles.

VEHICLE
- The vehicle, 1% aqueous methylcellulose, was formulated from methylcellulose (CAS No. 9004-67-5), 400cps, obtained from MRI (Batch No. 01; Vendor Lot No. PE0515, Spectrum Chemical Manufacturing Co.).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Samples of each dose formulation were collected prior and after dosing and were submitted to Midwest Research Institute for verification of silver acetate concentrations by potentiometric titration.
- Pre-dosing samples were within 87.5 and 98.8% and post dosing samples were within 88 and 101.1% of their nominal concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1; individual breeding pairs were cohabited overnight.
- Proof of pregnancy: vaginal sperm referred to as day 0 of pregnancy.
Duration of treatment / exposure:
from gestational day (gd) 6 to 19
Frequency of treatment:
daily
Duration of test:
until gd 20
No. of animals per sex per dose:
- A total of 25 time-mated females per group were assigned to this study.
- 10 additional untreated females (sentinels) were cooused.
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: based on a previous screening study in rats (NTP, 2001); the low dose was selected since it was not likely to cause maternal or foetal toxicity; the high dose given over the period of embryo/foetal development was expected to cause significant maternal toxicity, but was unlikely to cause developmental toxicity; an intermediate dose was selected to assist in characterisation of the dose-response curve for critical endpoints.
- Rationale for animal assignment: confirmed-mated females were assigned to treatment groups by stratified randomisation for body weight on gestation day (gd) 0, so that mean body weight on gd 0 did not differ among treatment groups; maternal body weights for confirmed pregnant females ranged from 213.9 to 271.6 g on gd 0.
- Other: the oral route corresponsed to one of the expected routes of humen exposure.
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

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

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

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

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

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

OTHER:
- At necropsy, blood samples were collected by cardiac puncture from 10 sentinel females under terminal CO2 anaesthesia. Serum was submitted for evaluation of viral antibody titers. All assay in the serological panel were negative, including pneumonia virus (PVM), sendai virus (sendai), rat corona virus/sialodacyoadenitis virus (RCV/SDA) and parvo virus (Parvo).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes, pregnancy status was confirmed by uterine examination.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Number of dead and live foetuses: Yes
- Uteri which presented no visible implantation sites were staiend with ammonium sulfide (10%) in order to visulise any implantation sites which might have undergone very early resorption.
Fetal examinations:
External examinations: Yes
- Dead foetuses were counted, weighed and discarded.
- Live foetuses were dissected from the uterus and immediately placed on a moist paper towel over a tray of ice, a procedure which induces anaesthesia.
- All live foetuses were counted, weighed, sexed and examined for external morphological abnormalities, including cleft palate.

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

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

Head examinations: Yes
- Foetal heads were fixed and decalcified in Bouin's solution and subsequently examined.
Statistics:
- The unit for statistical measurement was the pregnant female or the litter.
- Quantitative continuous data were compared among treatment groups by parametric statistical tests whenever Bartlett's test for homogeneity of variance was not significant. If Bartlett's test indicated a lack of homogeneity (p<0.001), then nonparametric statistical tests were applied.
- All statistical procedures applied to select measures from this study were based on SAS software.
- General Linear Models (GLM) procedures were applied to the Analyses of Variance (ANOVA) and the Test for Linear Trand. For litter-derived percentage data, the ANOVA was weighed according to litter size.
- If a asignificant (p<0.05) main effect for dose occurred, Dunnett's Multiple Comparison Test was used to compare each treatment group to the control group for that measure.
- A one-tailed test was used for all pairwise comparisons to the vehicle control group, expect that a two-tailed test was used for maternal body and organ weight parameters, maternal feed and water consumption, foetal body weight and percent males per litter.
- Nonparametric tests applied to continuous variables included the Kruskal-Wallis one-way analysis of variance by ranks for among-group differences and, when significant (p<0.05), the Mann-Whitney U test for pairwise comparisons to the vehicle control group.
- Jonckheere's test for k independent samples was used to identify significant dose-response trends.
- Nominal scale measures were analysed by Chi-Square Test for Independence for differences among treatment groups and by the Cochran-Armitage Test for Linear Trend on Properties.
- The alpha level for each statistical comparison was 0.05, except as noted above for Bartlett's Test. The significance level for each trend test or pairwise comparison (one- or two-tailed) was reported as p<0.05 or p<0.01.
Indices:
no details
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:yes

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

Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

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

FOETAL EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
- No toxicologically relevant differences were observed in the incidences of foetal malformations and variations.
- The percent female fetuses with malformatins per litter exhibited a significant effect for dose (p<0.01), but no significant trend or pairwise differences beween treated groups and the control group were observed.
Dose descriptor:
other:
Basis for effect level:
other: See 'Remark'
Remarks on result:
other: See 'Remarks'
Remarks:
There were no differences among groups for the number of ovarian corpora lutea/dam, number of implantation sites/litter or percent preimplantation loss/litter. Postimplantation/loss (resorptions, late foetal deaths or nonlive implant/litter), live litter size and percent male foetuses/litter did not differ among groups. An increasing trend was observed for the percent litters with late foetal deaths. Average foetal body weight per litter (sexes combined) and average male foetal body weight per litter exhibited a significant decreasing trend, but no significant pairwise differences between treated groups and the control group. No statistically significant effects were noted for average female body weight. No toxicologically relevant differences were observed in the incidences of foetal malformations or variations.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In conclusion, the maternal LOAEL for this study was considered to be 30 mg/kg/day silver acetate (19.4 mg silver/kg/day) based on clinical signs including weight loss. The maternal NOAEL was 10 mg/kg/day silver acetate (6.5 mg Ag/kd/day). The NOAEL for developmental toxicity for this study was 100 mg/kg/day silver acetate (64.6 mg Ag/kg/day), based on the absence of any biologically or statistically significant developmental toxicity.
Executive summary:

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

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

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

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

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

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

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

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
64.6 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A developmental toxicity study with metallic silver is not available. However, in a developmental toxicity study conducted on silver acetate (with dosing during the period of organogenesis), the maternal LOAEL was considered to be 30 mg/kg/day silver acetate (19.4 mg Ag/kg/day) based on clinical signs including weight loss, and the maternal NOAEL was 10 mg/kg/day silver acetate (6.5 mg Ag/kg/day). The NOAEL for developmental toxicity for this study was 100 mg/kg/day silver acetate (64.6 mg Ag/kg/day), based on the absence of any biologically or statistically significant developmental toxicity. Some minimal effects observed like an increasing trend for the percentage of litters with late foetal deaths, or a significant decreasing trend of average foetal body weight per litter (sexes combined) and average male foetal body weight per litter exhibited with no significant pair wise differences between treated groups and the control group were not regarded as being of biological relevance. No toxicologically relevant differences were observed in the incidences of foetal malformations or variations. Silver acetate is a soluble silver substance, showing mild maternal toxicity at 10 mg/kg/day, with a developmental NOAEL at much higher levels (i.e., highest tested dose). This finding can be read-across to other soluble, readily bioavailable silver substances. In the absence of such data for metallic silver, read-across from this study on silver acetate is highly conservative in consideration of higher bioavailability of soluble silver substances in comparison to metallic silver.

Information is available from a one-generation reproductive toxicity study (OGRTS) relevant to ionic silver (Sprando et al., 2017).  Rats received silver acetate administered in the drinking water at dose levels of 0, 0.4, 4 or 40 mg/kg bw/day (equivalent to 0, 0.26, 2.6 or 26 mg Ag/kg bw/day). Parental animals were exposed 10 weeks prior to mating. F1-pups were sacrificed on postnatal day 26. Evidence of appreciable maternal or paternal toxicity was absent in the study (although other potential secondary effect influences were not examined). Adverse effects on fertility (reduced fertility and number of litters) were observed for high-dose group animals receiving 40 mg silver acetate/kg bw/day, with a NOAEL for this effect of 4 mg silver acetate/kg bw/day. Evidence of developmental toxicity was reported including decreased implant numbers, increased pup mortality, and F1 generation weight retardation/delayed growth with LOAEL and NOAEL being defined as 4 and 0.4 mg silver acetate/kg bw/d, respectively (corresponding to 2.6 and 0.26 mg Ag/kg bw/d).

Babu et al. (2016) conducted as a parallel arm of the Sprando et al. (2017) study a developmental immunotoxicity (DIT) study, based on same treatment regimen (0, 0.4, 4 and 40 mg silver acetate/kg bw/day). Natural killer cell (NK) activity and mitogen-induced lymphocyte proliferation were selected as functional immune system markers. Findings for the F generation pups included lower percentages of splenic CD8+ T‑cells (mid- and high-dose) and a non-dose-related reduction in the splenic mitogenic responses to a selective T-cell mitogen in treated groups. Thymic development was unaffected. It is important to note that the functional immune system assessment was not based on use of the T-cell dependent antibody response (TDAR) assay. In summary, it is considered that this study provides indicative, but not firm, evidence that exposure to ionic silver might give rise to developmental immunotoxicity.

Test data on silver nanomaterials involving intravenous and/or intraperitoneal treatment were disregarded because of (i) the lack of relevance of these routes of administration for human health risk assessment of industrial chemicals under REACH, (ii) the absence of conclusive evidence that exposure to silver nanoparticles via physiological routes of exposure (inhalation and oral) leads to any substantial systemic exposure to primary silver nanoparticles, and (iii) any such results are considered to be superseded by a fully guideline-compliant US NTP developmental toxicity study in rats with the highly water-soluble silver acetate which is considered to maximise the systemic availability of ionic silver.

Testing for developmental toxicity in a second species is waived on grounds of (i) low toxicological activity, (ii) negligible systemic bioavailability via oral and inhalation routes, and (iii) no exposure levels of concern in relation to no-effect levels in a developmental toxicity in the rat and sub-chronic toxicity studies.


Justification for selection of Effect on developmental toxicity: via oral route:
Key study, using the soluble silver acetate as a suitable surrogate for other silver substances (read-across). NOAEL expressed as mg Ag/kg/day.

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

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

Justification for classification or non-classification

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