Silver cyanide

Endpoint summary

Description of key information

In vitro skin irritation and corrosion assessment in a reconstructed human epidermis model (EpiSkin) (OECD439).
In vitro eye irritation/corrosion test (BCOP) (OECD437).
In vivo eye irritation test in the rabbit (OECD405).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 October 2013 to 04 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant, guideline study, available as an unpublished report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

yes

Remarks:

The control groups were shared and the raw data are filed with another study. This deviation was considered not to affect the purpose or integrity of the study.

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

yes

Remarks:

The control groups were shared and the raw data are filed with another study. This deviation was considered not to affect the purpose or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Species:

other: EPISKIN (TM) reconstructed human epidermis model

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

Not applicable.

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: Not applicable

Amount / concentration applied:

- Treatment group: Test carried out in triplicate. Approximately 10 mg of the test item was applied topically, ensuring an even covering, to the epidermis surface which had previously been moistened with 5 µL sterile, distilled water to improve contact between the solid test item and the epidermis.
- Negative control: 10 µL Dulbecco's Phosphate Buffered Saline (DPBS)
- Positive control: 10 µL Sodium Dodecyl Sulphate (SDS) at 5% w/v aqueous solution spread over entire surface of the epidermis using a pipette tip with the process being repeated after 7 minutes.

Duration of treatment / exposure:

- Treatment period: 15 minutes
- Post-exposure incubation: At the end of the exposure period, tissues were rinsed with DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to a second column of three wells, each containing 2 mL of maintenance medium, and incubated for 42 hours, at 37°C and 5% CO2 in air.

Observation period:

Not applicable.

Number of animals:

Not applicable.

Details on study design:

Triplicate samples of epidermis tissue were uniformly covered with approximately 10 mg of test item and moistened with 5µL sterile distilled water to improve contact with the solid test item. At the end of the 15 minute exposure period, each tissue was removed and rinsed with DPBS with Ca++ and Mg++. The tissues were then incubated for 42 hours at 37°C and 5% CO2 in air, in wells containing 2 mL maintenance media.

The measurement of tissue viability (cytotoxicity) was measured by means of the colourimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (by the mitochondrial succinate dehdroganase in viable cells) in the test item treated tissue relative to the negative control using the following procedure. After incubation, the triplicate tissue samples were transferred into three wells each containing 2ml of a 0.3 mg/L MTT solution, care being taken to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37ºC, 5% CO2 in air and then the epidermis was carefully separated from the collagen matrix using forceps and both parts placed into 1.5 mL microtubes containing 500 µL of acidified isopropanol. Each tube was plugged and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10ºC for 3 days to allow the extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was thoroughly mixed on a vortex mixer to produce a homogeneous coloured solution. The optical density of the extracted solution was measured at 540 nm against an acidified isopropanol blank.

Irritation / corrosion parameter:

other: other: Percent Relative Viability

Value:

8.8

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minute exposureand 42-hour post-exposure incubation. Max. score: 10.4. Reversibility: no data. Remarks: Negative control item (set at 100). Standard deviation of relative mean viability 1.4 %. (migrated information)

Irritant / corrosive response data:

- Viability: The relative mean (n = 3) viability of the treated tissues was 8.8 after a 15-minute exposure period, with standard deviation of 1.4 %
- Optical density: The mean optical density of the treated tissues at 562 nm was 0.084
- Conclusion: Test item is considered to be irritant using the EPISKIN (TM) human epidermis model (viability ≤ 50 %)

Other effects:

- Positive control: The relative mean tissue viability was 10.4 % relative to the negative control and the standard deviation was 4.5 %
- Negative control: The mean optical density was 0.950 and the standard deviation was 0.037.

Direct MTT reduction: The MTT solution containing the test item did not turn blue, indicating that test item did not directly reduce MTT.

The acceptance criteria were satisfied according to the protocol criteria for both the positive control (relative mean viability ≤ 40% and standard deviation ≤ 18%) and negative control (OD562 ≥ 0.6 and standard deviation of individual tissue viability ≤18%). The standard deviation of the triplicate treated tissues was 1.4 % and hence the test item acceptance criterion (≤ 18%) was also satisfied.

Table 1. Mean OD₅₄₀Values and Percentage Viability for Negative Control Item, Positive Control Item and Test Item

Item	OD540of tissue	Mean OD540of triplicate tissues	±SD of OD540	Relative Individual tissue viability (%)	Relative mean viability (%)	±SD of Relative mean viability (%)
Negative Control Item^1	0.931	0.950	0.037	98.0	100*	3.9
	0.927			97.6
	0.993			104.5
Positive Control Item^1	0.102	0.099	0.043	10.7	10.4	4.5
	0.054			5.7
	0.140			14.7
Test Item	0.099	0.084	0.013	10.4	8.8	1.4
	0.078			8.2
	0.074			7.8

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

¹Control group shared with test laboratory project number 41202604

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Silver cyanide is considered to be irritant to the skin using the EPISKIN (TM) human epidermis model.

Executive summary:

The skin irritation potential of silver cyanide was evaluated using EPISKIN (TM) reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure period of 42 hours using a colourimetric MTT reduction assay following OECD guideline 439. Silver cyanide considered to be irritant to the skin using the EPISKIN (TM) human epidermis model. The quality criteria required for the acceptance of results in the test were satisfied and the study is considered reliable and relevant for use.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as an unpublished report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: 12- 14 weeks old
- Weight at study initiation: 2.54 kg
- Housing: in a suspended cage
- Diet: 2930C Tekland Global Rabbit Diet (Harlan Laboratories UK Ltd., Oxon, UK) ad libitum
- Water: drinking water avaialble ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23
- Humidity (%): 30 to 70
- Air changes (per hr): at least 15 air changes per hour
- Photoperiod: 16 h darkness : 8 h light

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 100 mg

Duration of treatment / exposure:

The test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released.

Observation period (in vivo):

Assessment of ocular damage or irritation was made approximately 1 and 2 hours after treatment.

Number of animals or in vitro replicates:

The test item was applied to the right eye of one rabbit.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: none

SCORING SYSTEM: Assessment of ocular damage/irritation was made according to the numerical evaluation (Draize 1977).

TOOL USED TO ASSESS SCORE: Standard ophthalmoscope.

Irritation parameter:

overall irritation score

Basis:

animal #1

Time point:

other: 1 hour

Score:

21

Max. score:

110

Irritation parameter:

overall irritation score

Basis:

animal #1

Time point:

other: 2 hours

Score:

41

Max. score:

110

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: 1 hour

Score:

0

Max. score:

80

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: 2 hours

Score:

20

Max. score:

80

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: 1 hour

Score:

5

Max. score:

10

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: 2 hours

Score:

5

Max. score:

10

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: 1 hour

Score:

16

Max. score:

20

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: 2 hours

Score:

16

Max. score:

20

Irritant / corrosive response data:

Iridial inflammation and severe conjunctival irritation were noted 1 hour after treatment. Diffuse corneal opacity, iridial inflammation, severe conjunctival irritation were noted 2 hours after treatment.

Other effects:

Blood stained discharge was noted and the animal appeared lethargic 2 hours after treatment.

Interpretation of results:

highly irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Silver cyanide was considered to be at least a severe irritant to the rabbit eye and would be classified in category 1 (serious eye damage/irreversible effects on the eye) according to GHS and EU CLP 1272/2008.

Executive summary:

The study was performed to assess the irritancy potential of silver cyanide to the eye of the New Zealand White rabbit. The test was conducted according to OECD 405 and EC B.5 guidelines. A single application of silver cyanide to the non-irrigated eye of one rabbit produced diffuse corneal opacity, iridial inflammation, severe conjunctival irritation and blood stained discharge. The animal was also lethargic two hours after dosing. The test item, silver cyanide, was considered to be at least severely irritant to the rabbit eye (based on one rabbit only) and, therefore, would be classified in category 1 (serious eye damage/irreversible effects on the eye) according to GHS and EU CLP 1272/2008.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 06 November 2013 to 26 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline test, available as an unpublished report. Reliable without restriction.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The control group and other common data incl. eye receipt and preparation, technical documentation, reagent record and preparation, initial opacity readings, group allocation and optical density print out were shared with study 41302740.

Principles of method if other than guideline:

The 'open-chamber' method was adopted. The window-locking ring and glass were removed from the chamber prior to treatment. The 'open-chamber' method was also followed for the control groups although solutions were applied. Following application the ring and glass were replaced during the exposure period until rinsing.

GLP compliance:

yes (incl. QA statement)

Species:

other: not applicable

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: Approximately 0.213 g of the solid test item was applied to the cornea. It covered adequately the corneal surface.

Duration of treatment / exposure:

240 minutes at 32±1°C

Observation period (in vivo):

Not applicable.

Number of animals or in vitro replicates:

Three corneas were allocated to the test item, three to the negative control and three to the positive control.

Details on study design:

Treatment of corneas
Approximately 0.213 g of the solid test item was found to adequately cover the corneal surface. 750 uL of each control item was applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32± 1°C for 240 minutes. At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete minimum essential medium (MEM) containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post treatment opacity reading was taken and each cornea was visually observed.

Application of sodium fluorescein
Following the opacity measurement the permability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32± 1°C for 90 minutes.

Permability determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 uL of medium representing each cornea was applied to a designed well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Opacity measurement
The change in opacity for each cornea as calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. the mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permability measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

SCORING SYSTEM:
Results from the two test method endpoints, opacity and permability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
In Vitro Irritancy Score = mean opacity value + (15 x mean OD 492 value)

Additionally, the opacity and permability values were evaluated independently to determine whether the test item induces a response through only one of the two endpoints.
A test item that induces an In Vitro Irritancy Score ≥55.1 is defined as an ocular corrosive or severe irritant and will be labelled EU DSD (67/548/EEC) R41 and EU CLP/UN GHS Category 1.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

5.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritant / corrosive response data:

The test item was considered not to be an ocular corrosive or severe irritant.

The corneas treated with the test item had cloudy areas post incubation. The corneas treated with the negative control item were clear post incubation. The corneas treated with positive control item were cloudy post incubation.

The positive control In Vitro Irritancy Score was 82.1 and the negative control In Vitro Irritancy Score was 3.7.

Criterion for an acceptable test.

The positive control In Vitro Irritancy Score was within the range of 65.9 to 140.8. The positive control acceptance criterion was therefore satisfied.

The In Vitro irritancy scores are summarized as follows:

 

Treatment	In Vitro Irritancy Score
Test Item	5.6
Negative Control	3.7
Positive Control	82.1

 

Table 1. Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD)		In VitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	1	1	4	3		0.035
	2	1	4	3		0.067
	3	2	5	3		0.028
				3.0*		0.043¨		3.7
Positive Control	4	2	69	67	64.0	1.590	1.547
	5	2	58	56	53.0	0.759	0.716
	6	1	78	77	74.0	1.477	1.434
					63.7·		1.232·	82.1
Test Item	7	2	9	7	4.0	0.141	0.098
	8	2	10	8	5.0	0.098	0.055
	9	1	8	7	4.0	0.139	0.096
					4.3·		0.083·	5.6

OD= Optical density; * = Mean of the post-treatment -pre‑treatment values; ¨= Mean permeability; ·= Mean corrected value

Table 2. Corneal Epithelium Condition Post Treatment

 

Treatment	Cornea Number	Observation
		Post Treatment
Negative Control	1	clear
	2	clear
	3	clear
Positive Control	4	cloudy
	5	cloudy
	6	cloudy
Test Item	7	cloudy areas
	8	cloudy areas
	9	cloudy areas

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Silver cyanide was considered not to be an ocular corrosive or severe irritant.

Executive summary:

Silver cyanide was tested in vitro for its ocular irritancy potential to the isolated bovine cornea according to the OECD 437 guideline. The test item was applied neat for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Silver cyanide induces an In Vitro Irritancy Score of 5.6 and therefore was considered not to be an ocular corrosive or severe irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The EPISKIN reconstructed human epidermis model was used to test for skin irritation and corrosion. Triplicate tissue samples were exposed to 10 mg silver cyanide applied topically for 15 minutes followed by a 42-hour post-exposure incubation period, according to OECD guideline 439 and EU method B.46 (Warren 2014a). Silver cyanide is considered to be irritating based on EU criteria. In the skin corrosion test, duplicate tissue samples were exposed for 3, 60 and 240 minutes to 20 mg silver cyanide applied topically followed by a 3-hour post-exposure incubation period, according to OECD guideline 431 and EU method B.40 (Warren 2014b). Silver cyanide is considered to be non-corrosive based on EU criteria. Both studies were GLP-compliant and are suitable for use as key studies.

Eye irritation was tested in vitro and in vivo. In the Bovine Corneal Opacity and Permeability (BCOP) assay following OECD guideline 437, eyes from adult cattle were treated with approximately 0.213 g of silver cyanide for 240 minutes (Warren 2014c). Results of the opacity and permeability endpoints were combined to give an In Vitro Irritancy Score of 5.6. Silver cyanide is not considered to be an ocular corrosive or severe irritant based on EU criteria. In the in vivo test performed according to OECD guideline 405 and EU method B.5, one New Zealand white rabbit was treated with a single application of 100 mg silver cyanide in the right eye. Diffuse corneal opacity, iridial inflammation, severe conjunctival irritation, blood-stained discharge and lethargy were observed within 2 hours of application. Silver cyanide is considered to be at least a severe irritant to the rabbit eye based on EU criteria, resulting in classification in category 1 (serious eye damage/irreversible effects on the eye) according to GHS and EU CLP 1272/2008. Both studies were GLP-compliant and are suitable for use as key studies.

Overall, silver cyanide is not corrosive to eye or skin, but is however a skin irritant and a severe eye irritant.

Justification for selection of skin irritation / corrosion endpoint:
Silver cyanide was determined to be an irritant but non-corrosive to the skin based on EU criteria in a skin irritation and corrosion study on a human epidermis model.

Justification for selection of eye irritation endpoint:
Silver cyanide was determined to be a severe eye irritant but non-corrosive based on EU criteria in a BCOP assay and in vivo eye irritation study.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: highly irritating

Justification for classification or non-classification

According to GHS and EU CLP 1272/2008, silver cyanide is classified as category 2 for skin irritation, based on the results of in vitro human epidermis model studies, and category 1 (serious eye damage/irreversible effects on the eye) for effects on the eye, as serious eye damage was observed in an in vivo eye irritation study.