Silver docosanoate

Developmental toxicity / teratogenicity

Administrative data

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

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

Details on test material:

- Name of test material (as cited in study report): Silver (I) acetate
- Molecular formula (if other than submission substance): AgC2H3O2
- Molecular weight (if other than submission substance): 166.92 g/mol
- Physical state: solid
- Analytical purity: purity determination, based on silver ion (Ag+) content, 99.0% as determined by potentiometric titration.
- Purity test date: April 24, 2001
- Lot/batch No.: Batch No. 01; Vendor Lot No. 9021-992203

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

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

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

Administration / exposure

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.

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

1

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

3

Dose descriptor:

NOAEL

Effect level:

> 100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

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.

Effect levels (fetuses)

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.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

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.