Sodium thiocyanate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 June 2010 to 07 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Draft Proposal for a New Guideline: In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Sodium thiocyanate
- Substance type: White crystalline powder
- Physical state: Solid
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark under nitrogen

Test animals

Details on test animals or test system and environmental conditions:

Test system - EPISKIN Standard Model (EPISKIN-SM(TM), 0.38 cm2, Lot no.: 10-EKIN-020), SkinEthic Laboratories, Nice, France.
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37°C. The level of Maintenance Medium was just beneath the tissue. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Nice, France.

Environmental conditions
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 76 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.7 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 35.7- 36.0°C) and humidity (with a maximum of 4%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg, moistened with 5 µl water. Sodium thiocyanate was spread to match the size of the tissue.

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 10 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 10 µl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate

Duration of treatment / exposure:

Exposure:5 minutes
Post incubation period: 42 hours

Details on study design:

STUDY DESIGN
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. At least 10 mg solid (with a small glass weight boat) with 5 μl Milli-Q water was added into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μl PBS (negative control) and 3 tissues with 10 μl 5% aq. SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully. The skin tissues were kept in new 12-well plates on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

TEST SITE
- Area of exposure: human epidermis model
- % coverage: 0.38 cm2

REMOVAL OF TEST SUBSTANCE
- Washing (if done): phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: percentage viability

Value:

106

Remarks on result:

other:

Remarks:

Basis: other: percentage of control. Time point: 15 minutes. (migrated information)

Any other information on results incl. tables

Preliminary test for reduction of MTT by the test substance

Sodium thiocyanate was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that Sodium thiocyanate did not interact with MTT.

Acceptability of assay:

The positive control had a mean cell viability after 15 minutes exposure of 7%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Results:

The mean absorption at 570 nm measured after treatment with Sodium thiocyanate and controls are presented in Table 1.

Table 2 shows the mean tissue viability obtained after 15 minutes treatment with Sodium thiocyanate compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Sodium thiocyanate compared to the negative control tissues was 106%.

Table 1 Mean absorption in the in vitro skin irritation test with Sodium thiocyanate

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	0.889	0.879	0.883	0.884	±	0.005
Sodium thiocyanate	0.942	0.939	0.927	0.936	±	0.008
Positive control	0.053	0.062	0.068	0.061	±	0.008

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption. Isopropanol was used to measure the background absorption.

Table 2 Mean tissue viability in the in vitro skin irritation test with Sodium thiocyanate

	Mean tissue viability (percentage of control)
Negative control	100
Sodium thiocyanate	106
Positive control	7

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Sodium thiocyanate is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

In vitro skin irritation test with Sodium thiocyanate using a human skin model.

This report describes the ability of Sodium thiocyanate to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SM^TM)). The possible skin irritation potential of Sodium thiocyanate was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Sodium thiocyanate was a white crystalline powder with a purity of 99.9% (dried material). Skin tissue was moistened with 5 μl of Milli-Q water and at least 10 mg of Sodium thiocyanate was applied directly on top of the skin tissue (0.38 cm2). After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Sodium thiocyanate compared to the negative control tissues was 106%. Since the mean relative tissue viability for Sodium thiocyanate was above 50% after 15 minutes treatment Sodium thiocyanate is considered to be non-irritant.

Sodium thiocyanate did not cause direct MTT reduction. The positive control had a mean cell viability after 15 minutes exposure of 7%. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that Sodium thiocyanate is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.