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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13-Feb-2012 to 23-Apr-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Also complies with OECD GLP regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
Deviations:
no
Qualifier:
according to
Guideline:
other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Name of test material (as cited in study report): S-10793
- IUPAC nomenclature - Sodium diisobutyldithiophosphinate
- Lot S-20227-170B
- Appearance - White powder with lumps
- CAS No. 13360-78-6
- Molecular Formula - C8H18PS2.Na
- Molecular Weight - 232 g/mole
- Purity 93-94%
- Expiration date of the lot/batch: 16 December 2013
- Storage condition of test material: At room temperature in the dark

In vitro test system

Test system:
human skin model
Remarks:
EPISKIN-SM, 0.38 cm²
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin small model (EPISKIN-SM, 0.38 cm³) from SkinEthic Laboratories, Lyon, France
- Tissue batch number(s):12-EKIN-016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ca. 37 °C
- Temperature of post-treatment incubation (if applicable): ca. 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After 15 minutes, the tissues were washed with PBS to remove residual test substance.
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3h
- Spectrophotometer: Tecan Infinite M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): Living epidermis was transferred to 12 well plates and incubated with 2 ml Mili-Q for 48 +/-1 hours. After incubation, killed epidermis was stored at <= - 15 °C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium. Further use of killed tissues was similar to living tissues.
- N. of replicates : 3
- Method of calculation used: The non-specific reduction of MTT by the test material was 21% of the negative control tisues. The net OD of the treated killed tissues was substracted from the ODs of the test substance treated viable tissues.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to the skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 44 hours of post incubation is =< 50 % of the mean viability of the negative controls.
- The test substance is considered to be non-irritating to the skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 44 hours of post incubation is > 50 % of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 17.7 to 19.2 mg, moistened with 5 µl water

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 25 µl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
83
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test material was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because a colour change was observed it was concluded that S-10793 did interact with MTT.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that the test material is non-irritant in the in vitro skin irritation test.

Executive summary:

In vitro skin irritation of the test material was investigated using a human skin model according to OECD TG 439 and under GLP conditions. EPISKIN-SM was used and the topical application lasted 15 minutes.

Skin tissue was moistened with 5 µl of Milli-Q water and 17.7 to 19.2 mg of the test material was applied directly on top of the skin tissue for 15 minutes. After a post-incubation period of approximately 44 hours, 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. The test material did interact with 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test material was 21% of the negative control tissues. The net optical density (OD) of the treated killed tissues was subtracted from the ODs of the test substance treated viable 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 S-10793 compared to the negative control tissues was 83%. Since the mean relative tissue viability for the test material was above 50% after 15 minutes treatment it is considered to be non-irritant.

The positive control had a mean cell viability of 4% after 15 minutes exposure. 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 18%, indicating that the test system functioned properly.

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