Potassium dicyanoargentate

Endpoint summary

Description of key information

In vitro human epidermis model (he-EpiDerm) conducted in accordance with OECD431 test guideline and EU method B.40.

In vitro Bovine Corneal Opacity and Permeability (BCOP) eye irritation test conducted in accordance with OECD437 test guideline and EU method B.47.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliable without restriction - GLP-compliant and conducted according to guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

GLP compliance:

yes

Species:

human

Strain:

other: EpiDerm EPI-200 Skin Model

Details on test animals or test system and environmental conditions:

- Source: MatTek Corporation, Ashland MA, USA
- Lot no.: 21902 kit P
- Culture substrate: Polycarbonate membranes of 10 mm cell culture inserts

Type of coverage:

open

Preparation of test site:

other: Skin tissue

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30.0 to 38.1 mg

Duration of treatment / exposure:

3 minutes and 1 hour exposures

Observation period:

3 hours

Number of animals:

2 replicates per exposure duration

Details on study design:

The test substance was checked for colour interference in aqueous conditions and possible direct MTT (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction by adding the test substance to MTT medium. The tissues were treated with test substance for 3 minutes and 1-hour and incubated in Dulbecco's Modified Eagle's Medium (DMEM). The level of the DMEM medium was just beneath the tissue. Skin was moistened with 25 μl Milli-Q water and 30.0 to 38.1 mg of Potassium dicyanoargentate 54.2% was applied directly on top of the skin tissue, spread to match the size of the tissue. 50 μl Milli-Q water was used as a negative contol and 50 μl 8.0 N potassium hydroxide was used as the positive control. All exposures were carried out in a humid atmosphere of 80 - 100% (actual range 63 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.6 – 37.2°C), apart from the 3 minute incubation which was maintained at room temperature. After the exposure period, the tissues were washed with phosphate buffered saline (PBS) to remove residual test substance and kept in DMEM until 6 tissues (= one application time) were dosed and rinsed. The DMEM medium was replaced by 300 μl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with isopropanol over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm. Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

Irritation / corrosion parameter:

other: other: Relative tissue viability

Value:

12

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 minutes. Max. score: 100.0. Reversibility: no data. (migrated information)

Irritation / corrosion parameter:

other: other: Relative tissue viability

Value:

3

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1 hour. Max. score: 100.0. Reversibility: no data. (migrated information)

Other effects / acceptance of results:

The maximum inter-tissue variability in viability between two tissues treated identically was up to 16.5%. Both viabilities at the 3 minute exposure were below 50% (15.9 and 8.7%) and therefore the outcome of both tissues was clearly positive. In addition at the 1 hour exposure both viabilities were below 15% (2.5 and 2.9%) and clearly positive.

The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 3-minute exposure to the positive control should be ≤ 30%.
c) The maximum inter-tissue variability (in viability) is ≤ 30% between two tissues treated identically.
d) The maximum difference in percentage between the mean viability of two tissues and one of the two tissues is ≤ 15%.

All validity criteria were met. Although, the maximum inter-tissue variability in viability between two tissues treated identically and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues treated with Potassium dicyanoargentate 54.2% were above the acceptability criteria at the 3 minute exposure period (up to 45.5 and 29.4% respectively), both viabilities were below 50% (15.9 and 8.7%) and therefore the outcome of both tissues was clearly positive. In addition at the 1 hour exposure both viabilities were below 15% (2.5 and 2.9%) and clearly positive. Therefore this deviation does not affect the study integrity.

Interpretation of results:

Category 1A (corrosive) based on GHS criteria

Remarks:

Criteria used for interpretation of results: OECD GHS

Conclusions:

Potassium dicyanoargentate 54.2% is corrosive in the in vitro skin corrosion test at 3 minute and 1 hour exposures.

Executive summary:

The potential of potassium dicyanoargentate 54.2% to induce skin corrosion was tested on a human three dimensional epidermal model. Potassium dicyanoargentate 54.2% was applied directly on top of the skin tissue, spread to match the size of the tissue. Milli-Q water was used as the negative control and 8.0N potassium hydroxide served as the positive control. Tissues were incubated for 3 minutes at room temperature and for 1 hour at biological temperature (37.0 ± 1.0°C). The amount of extracted formazan was determined spectrophotometrically and results were used to calculate cell viability as percentage of the mean of the negative control tissues. Potassium dicyanoargentate 54.2% was determined to be corrosive at both exposure times. This study is reliable without restriction as it was GLP-compliant and was performed according to guideline.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliable without restriction - GLP-compliant and conducted according to guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Qualifier:

according to guideline

Guideline:

other: INVITTOX protocol 127 (Bovine Opacity and Permeability (BCOP) Assay)

Qualifier:

according to guideline

Guideline:

other: ICCVAM and NICEATM test method March 2006 (Bovine Corneal Opacity and Permeability (BCOP))

GLP compliance:

yes

Species:

other: Bovine

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Vitelco, -'s Hertogenbosch, The Netherlands
- Age at study initiation: Bovine eyes were used as soon as possible after slaughter.
- Storage: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Preparation: The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium) containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum). The isolated corneas were mounted in a corneal holder.
- Acclimation period: Minimum of 1 hour
- Temperature (°C): 32 ± 1°C

Vehicle:

physiological saline

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μl
- Concentration (if solution): 20 % (w/v) potassium dicyanoargentate 54.2% in physiological saline, actual concentration of 10.84% potassium dicyanoargentate

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 μl
- Concentration (if solution): physiological saline or 20% (w/v) Imidazole solution

Duration of treatment / exposure:

240 ± 10 minutes

Number of animals or in vitro replicates:

3 corneas per treatment

Details on study design:

The treatment solution was introduced onto the epithelium of the cornea and the cornea holder was maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium). The cornea was placed in fresh cMEM and opacity and permeability determinations were performed.

Irritation parameter:

in vitro irritation score

Value:

65

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean irritancy score, time point: 240 min

Other effects / acceptance of results:

The corneas treated with Potassium dicyanoargentate 54.2% showed opacity values ranging from 34.0 to 51.0 and permeability values ranging from 1.131 to 1.801. The corneas were turbid after the 240 minutes of treatment with Potassium dicyanoargentate 54.2%. The in vitro irritancy scores ranged from 54.5 to 78.0, with a mean irritancy score of 65, after 240 minutes of treatment with Potassium dicyanoargentate 54.2%.

The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
All validity criteria were met.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Remarks:

Criteria used for interpretation of results: OECD GHS

Conclusions:

A 20% (w/v) solution of Potassium dicyanoargentate 54.2% which contains 10.84% potassium dicyanoargentate induced serious eye damage through both opacity and permeability, resulting in a mean in vitro irritancy score of 65 after 240 minutes of treatment.

Executive summary:

The eye hazard potential of potassium dicyanoargentate 54.2% was determined by its ability to induce opacity and increase permeability in an isolated bovine cornea. A 20% (w/v) solution of Potassium dicyanoargentate 54.2% was prepared in physiological saline, resulting in an actual concentration of 10.84% potassium dicyanoargentate. Physiological saline was used as the negative control and 20 % (w/v) imidazole served as the positive control. 750 μl of treatment solution was introduced onto the epithelium of the cornea and corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. Cornea opacity and permeability were assessed. Exposure to the test material induced serious eye damage through both opacity and permeability, resulting in a mean in vitro irritancy score of 65 after 240 minutes of treatment. This study is reliable without restriction as it was GLP-compliant and was performed according to guideline.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The EpiDerm human epidermis model was used to test for skin corrosion according to OECD guideline 431 and EU method B.40. Duplicate tissue samples were exposed for 3 and 60 minutes to 30 to 38.1 mg potassium dicyanoargentate followed by a 3-hour post-exposure incubation period. The relative tissue viabilities after 3 and 60 minutes were 12% and 3%, respectively. Potassium dicyanoargentate is considered to be corrosive to skin based on OECD GHS criteria. This study was GLP-compliant and is suitable for use as a key study.

Eye irritation was tested in vitro in the Bovine Corneal Opacity and Permeability (BCOP) assay according to OECD guideline 437 and EU method B.47. Fresh eyes were extracted from cattle and treated with 750µL of 10.84% potassium dicyanoargentate in physiological saline solution for 240 ± 10 minutes (Eurlings 2015). Results of the opacity and permeability endpoints were combined to give an In Vitro Irritancy Score of 65. Potassium dicyanoargentate is considered to be an eye irritant based on OECD GHS criteria. This study was GLP-compliant and is suitable for use as a key study.

Overall, potassium dicyanoargentate is corrosive to skin and is an eye irritant.

Justification for selection of skin irritation / corrosion endpoint:
Potassium dicyanoargentate was determined to be corrosive to skin based on OECD GHS criteria in a skin corrosion study on a human epidermis model.

Justification for selection of eye irritation endpoint:
Potassium dicyanoargentate was determined to be an eye irritant based on OECD GHS criteria in a BCOP assay.

Effects on skin irritation/corrosion: corrosive

Effects on eye irritation: irritating

Justification for classification or non-classification

According to GHS and EU CLP 1272/2008, potassium dicyanoargentate is classified as category 1A for skin corrosion, as an adverse effect was observed within an hour with 3 minutes of exposure to the substance, and category 1 for effects on the eye, as a positive result was determined in a BCOP assay.