Silver nitrate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-06-10 to 2009-06-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals 431: In vitro Skin Corrosion: Human Skin Model Test (2004)

Deviations:

no

Principles of method if other than guideline:

The In vitro Skin Corrosion (Human Skin Model Test) consists of topical application of the test material to the tissue for 3 minutes and 1 hour, followed by immediate determination of the cytotoxic effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the exposure period.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Silver nitrate

EC Number:

231-853-9

EC Name:

Silver nitrate

Cas Number:

7761-88-8

Molecular formula:

AgNO3

IUPAC Name:

Silver nitrate

Details on test material:

- Name of test material (as cited in study report): Silver nitrate
- Physical state: crystalline
- Storage condition of test material: at room temperature in a thightly closed container, protected from light effect and humidity, the test item has a corrosive effect on aluminium or steel

Test animals

Species:

other: in vitro: human skin model

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

not applicable, in vitro testing

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

other: not applicable

Controls:

other: not applicable, in vitro testing

Amount / concentration applied:

About 25 mg of the test material were applied to the tissues and wetted with 25 µL deionised water. The test item was spread to cover the surface of the tissue.
Prior to the exposure to the test item the pH of a test item solution in deionised water was determined as 6.49, while the weight volume ratio corresponded to the ratio applied to the skin equivalents

Duration of treatment / exposure:

3 minutes and 1 hour, respectively

Number of animals:

not applicable, in vitro testing

Details on study design:

The In vitro Skin Corrosion (Human Skin Model Test) consists of topical application of the test material to the tissue for 3 minutes and 1 hour, followed by immediate determination of the cytotoxic effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the exposure period.

Cell culture:
EST-1000 kits were purchased from CellSystems Biotechnologievertrieb GmbH. The EST-1000 tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EST-1000 tissues (surface 0.6cm²) were cultured on specially prepared cell culture inserts.

Controls:
- Deionised water was used a the negative control. Volumes of 50 µL (>30 µL/cm²) were applied to each set of duplicate tissues for the exposure periods.
- Potassium Hydroxide as an 8.0 N ready-made solution was used as a positive control material. Volumes of 50 µL (> 30 µL/cm²) were applied to each set of duplicate tissues for the exposure periods.

Experimental performance:
At least 2 hours before dosing the EST-1000 tissues were removed from the refrigerator where they were stored. Under sterile conditions, the inserts were transferred into 6-well plates containing the pre-warmed assay medium. Two 24-well plates were prepared as holding plates, each well containing 300 µL assay medium per well. The holding plates were pre-warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until use.
Duplicate EST-1000 tissues were exposed to the test item, positive control or negative control for each of two different exposure periods.
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a wash bottle containing PBS to remove any residual test material.

MTT assay:
After the exposure procedure was completed for all tissues of each time point, the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period the MTT solution was aspirated from the wells. The inserts were transferred into new 24 -well plates. The inserts were immersed in extractant solution. The 24 -well plate was sealed to minimise isopropanol evaporation. The formazan salt was extracted for 2 hours while shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for approx. 15 minutes until the solution was homogeneous in colour.
The optical density (OD) was read in a microplate reader at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue insert.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: Relative absorbance value (%) at 570 nm

Run / experiment:

After 3 min exposure

Value:

8.4

Results 2

Irritation / corrosion parameter:

other: Relative absorbance value (%) at 570 nm

Run / experiment:

After 1 hour

Value:

25.1

Any other information on results incl. tables

After exposure to the test item Silver nitrate the relative absorbance values were decreased to 8.4% after 3 minutes. This value is well below the threshold for corrosivity of 50% for the 3 minutes treatment. After the 1 hour exposure relative absorbance values were reduced to 25.1%. Although this value was not below the threshold of 15% for the 1 hour exposure, the test item was nevertheless considered to be corrosive, firstly, since the value for the 3 minutes exposure was very convincing, and secondly, since the test item precipitated and the precipitate could not be washed of the tissues completely after the 1 hour exposure. This fact distorted the measurement of the absorbance values. Probably, the poorly soluble precipitate was silver chloride, which was formed during the washing step with PBS (which contains 0.8% / 8 g/L NaCl). In addition, the cells of the 1 hour treatment tissues were not able to reduce the MTT at all (no blue coloration at all) indicating complete death of the cells due to exposure to the test item.

Applicant's summary and conclusion

Interpretation of results:

Category 1A (corrosive) based on GHS criteria

Conclusions:

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item Silver nitrate was corrosive to skin.