Disilver(1+) sulphate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

According to OECD guideline for testing chemicals in toxicity studies in rodents (Guidelines for the Testing of Chemicals, Section.Health Effects. Test No. 408: Repeated dose 90-day oral toxicity study in rodents.) and NTP specification for the conduct of studies in laboratory animals NTP, 2011)

Principles of method if other than guideline:

The genotoxic effects of silver acetate exposure in mal and female rats were examined with a micronucleus assay, using flow cytometric analysis of peripheral blood (Witt et al., 2008). Blood obtained from the rat at week 1, 4 and 12 of the study were fixed in ultracold methanol and stored at -80°C until analysis for the frequency of micronucleated cells in 20,000 reticulocytes per samples.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Silver acetate

Cas Number:

563-63-3

Molecular formula:

AgC2H3O2

IUPAC Name:

Silver acetate

Test material form:

solid - liquid: suspension

Specific details on test material used for the study:

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

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

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

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

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

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

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Water/0.1% methyl cellulose, methylcellulose was used in this study as bulking agents to inhibit somewhat the gastroinstestinal passage of the mostly aqueous dose formulation. The compounds is not toxic and do not promote allergic reactions in rodents.

Details on exposure:

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

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

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

VEHICLE
Methylcellulose was purchased from Fischer Scientific (Fair Lawn, New Jersey). The water used in dose formulations was 18 megaohm and autoclave-sterilized.

Duration of treatment / exposure:

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

Frequency of treatment:

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

Post exposure period:

none

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Examinations

Tissues and cell types examined:

List of tissues examined:
- jejunum, ileum, colon, kidney, liver, spleen,

Details of tissue and slide preparation:

At necropsy, organs and tissues were examined for grossly visible lesions, and protocol designated tissues and organs were weighed and preserved in 10% neutral buffered formalin (NBF), with the exception that modified Davidson’s fixative was used for the right testes and eyes. Femur bone marrow was collected for histopathology (left) and ICP-MS (right). The testes and epididymides were collected for sperm analysis (left) and histopathology (right). A section of the ileum and scrapping of the ileal mucosa were collected for intestinal microbiota and immune response evaluations (Williams et al., 2015). Sections
of the proximal ileum, jejunum, proximal colon, and mesenteric lymph nodes, the right kidney, median and left lateral liver lobes, and spleen were preserved for 2 weeks at 4°C in Karnovsky’s fixative and stored in 10% NBF at 4°C for TEM evaluations.
Additional sections of these organs and tissues, along with the heart and uterine horn, were collected, flash frozen in liquid nitrogen, and stored at 80°C for total silver analysis by ICP-MS.

Statistics:

Micronuclei analysis was conducted for each sex using a 2-way fixed effect ANOVA to determine the effect of treatment using the arcsin square root transformation for percent data. Pairwise comparisons of the 2 control groups and each of the treatment groups to the appropriate control group were performed with Bonferroni adjustments. All tests were conducted as 2-sided with significance at the .05 probability level.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

ambiguous

Remarks:

high toxicity level

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks on result:

other: 400 mg/kg bw/d small but significant increase in the frequency of micronucleated reticulocytes at week-4, but not subsequent time point. Those animals had severe gastroinestinal distress and only 1 female and no male rats survived to week 12

Applicant's summary and conclusion

Conclusions:

Male and female rats administered with 400 mg/kg bw/day (highest dose) had a small but significant increase in frequency of micronucleated reticulocytes at week-4 only, but not at subsequent time points. In addition, these animals had severe gastrointestinal distress and only 1 female and no male rats survived to week 12.

In conclusion, under the study conditions, silver acetate do not induce micronuclei in reticulocytes of female and male rats.

Executive summary:

The genotoxic effects of silver acetate exposure in mal and female rats were examined with a micronucleus assay, using flow cytometric analysis of peripheral blood (Witt et al., 2008). Blood obtained from the rat at week 1, 4 and 12 of the study were fixed in ultracold methanol and stored at -80°C until analysis for the frequency of micronucleated cells in 20,000 reticulocytes per samples.

Male and female rats administered with 400 mg/kg bw/day (highest dose) had a small but significant increase in frequency of micronucleated reticulocytes at week-4 only, but not at subsequent time points. In addition, these animals had severe gastrointestinal distress and only 1 female and no male rats survived to week 12.

In conclusion, under the study conditions, silver acetate do not induce micronuclei in reticulocytes of female and male rats.