Registration Dossier

Diss Factsheets

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20th April 2010 to 7th July 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary guideline study, compliant with GLP.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 439: In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guideline B46: In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects.
Deviations:
no
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Thioanisole
IUPAC Name:
Thioanisole
Constituent 2
Chemical structure
Reference substance name:
Methyl phenyl sulphide
EC Number:
202-878-2
EC Name:
Methyl phenyl sulphide
Cas Number:
100-68-5
Molecular formula:
C7H8S
IUPAC Name:
(methylsulfanyl)benzene
Details on test material:
- Name of test material (as cited in study report): Thioanisole
- Molecular formula (if other than submission substance): Not documented
- Molecular weight (if other than submission substance): 124.19
- Smiles notation (if other than submission substance): Not documented
- InChl (if other than submission substance): Not documented
- Structural formula attached as image file (if other than submission substance): see Fig. Not documented
- Substance type: Not documented
- Physical state: Colourless to slightly yellowish liquid
- Analytical purity: 99% minimum
- Impurities (identity and concentrations): Not documented
- Composition of test material, percentage of components: Not documented
- Isomers composition: Not documented
- Purity test date: Not documented
- Lot/batch No.: 100310
- Expiration date of the lot/batch: March 23rd, 2011
- Radiochemical purity (if radiolabelling): Not documented
- Specific activity (if radiolabelling): Not documented
- Locations of the label (if radiolabelling): Not documented
- Expiration date of radiochemical substance (if radiolabelling): Not documented
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature protected from light under nitrogen
- Other: Use amber-coloured glassware or wrap container in tin-foil.

Test animals

Species:
human
Strain:
other: EPISKIN Standard Model.
Details on test animals or test system and environmental conditions:
Not relevant. EPISKIN Standard Model, a three-dimensional human epidermis model consisting 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 was used. The keratinocytes were cultured for 13 days, resulting in a highly differentiated and stratified epidermis model comprising the main absal, supra basal, spinous and granular layers and a functional stratum corneum.

Test system

Type of coverage:
not specified
Preparation of test site:
other: Standard EPISKIN Model prepared
Vehicle:
unchanged (no vehicle)
Controls:
other: negative control tissues treated with PBS; positive control tissues treated with 5% SDS
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µl undiluted test substance.


Duration of treatment / exposure:
15 minutes followed by washing and incubation for 42 hours at 37°C
Observation period:
Following incubation the cell culture inserts were dried anda total biopsy obtained by biopsy punch. The epidermis was separated from the collagen matrix and both parts separately extracted and the extracted formazan determined spectrophotometrically.
Number of animals:
Not relevant
Details on study design:
On the day the tissues were received, they were transferred into 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37°C. All incubations were carried out in a humid atmosphere of 80-100% (actual range 76-92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37 ±1°C.

The test was performed on a total of 3 tissues per test substance together with negative and positive controls. 10ul of the undiluted test substance was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 10ul PBS (negative control) and 3 tissues with 10ul 5% SDS (positive control) respectively. The positive control was respread 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 2ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

After incubation the cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-well plate prefilled with 2ml MTT-medium (0.3mg/ml). The tissues were incubated for 3 hours at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were palced in prelabelled microtubes and extracted with 500ul isopropanol. Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570nm in duplicate with the Multiskan Spectrum.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: mean tissue viability
Value:
11
Remarks on result:
other:
Remarks:
Basis: mean. Reversibility: no data. Remarks: The relative mean tissue viability obtained after 15 minutes treatment with Thioanisole compared to the negative controls was 11%. . (migrated information)

In vivo

Irritant / corrosive response data:
Thioanisole was checked for possible direct MTT reduction by adding the test susbtance to MTT medium. As no colour change was observed, it was concluded that Thioanisole did not interact with MTT. Skin irritation was expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Thioanisole compared to the negative control tissues was 11%. Since the mean relative tissue viability for Thioanisole was below 50%, it was considered to be irritant. The positive control had a mean cell viability after 15 minutes exposure of 7%.
Other effects:
No information provided

Any other information on results incl. tables

No additional information provided.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this in vitro test, the test substance, Thioanisole, should be considered to be an irritant as the cell viability was less than 50%. Based on this result, the test substance should be classified as a Category 2 irritant in accordance with Regulation EC No. 1272/2008 and should have the signal word Warning and the Hazard statement H315: Causes skin irritation associated with it. According to Directive 67/548/EEC, the test substance should be classified as an Irritant (Xi) and have the risk phrase R38: Irritating to skin associated with it.
Executive summary:

In a study conducted by Verbaan (2010), the test substance, Thioanisole, was investigated for its potential to cause irritation when tested using an in vitro human skin model, a three dimensional epidermal model (EPISKIN Standard Model). 10ul of Thioanisole was applied undiluted directly on top of the skin tissue for an exposure period of 15 minutes. After a 42 hour incubation period, determination of sytotoxic effect was determined. Skin irritancy was expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Thioanisole compared to the negative control tissues was 11%. As the mean relative tissue viability for Thioanisole was below 50% after 15 minutes treatment, it was considered to be an irritant under the experimental conditions of this test. Based on this result, the test substance should be classified as a Category 2 irritant in accordance with Regulation EC No. 1272/2008 and should have the signal word Warning and the Hazard statement H315: Causes skin irritation associated with it. According to Directive 67/548/EEC, the test substance should be classified as an Irritant (Xi) and have the risk phrase R38: Irritating to skin associated with it.