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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 31 August 2015 to 20 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Reddish solid below 25°C / liquid above 25°C
Details on test material:
- State of aggregation: N/A
- Particle size distribution: N/A
- Mass median aerodynamic diameter (MMAD): N/A
- Geometric standard deviation (GSD): N/A
- Shape of particles: N/A
- Surface area of particles: N/A
- Crystal structure: N/A
- Coating: N/A
- Surface properties: N/A
- Density: 1.06 g/cm3
- Moisture content: N/A
- Residual solvent: N/A
- Activation: N/A
- Stabilisation: N/A
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: MR92143651
- Expiration date of the lot/batch: 01 January 2017
- Purity: 86.4% (No correction was made for purity of the test substance)
- Purity test date: 18 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Yes, maximum temperature: 50°C, maximum duration: unknown (until liquefaction).
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was heated to 37°C, to obtain a homogeneous liquid sample.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A

In vitro test system

Test system:
human skin model
Remarks:
Supplier: MatTek Corporation, Ashland MA, U.S.A.
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch numbers: 22660 and 22268
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 31 August 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C
- Temperature of post-treatment incubation: N/A

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline.
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD value = 1.699 and 1.590 in batches 22660 and 22268, respectively (acceptance criteria: 1- Barrier function: ET-50 = 7.77 hrs and 8.71 hrs in batchs 22660 and 22268, respectively (acceptance criteria: 4.77 hrs- Morphology: not specified
- Contamination: No contamination in either batches.
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: 4 tissues per test item together with a negative control and positive control.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: freeze-killed tissues
- Procedure used to prepare the killed tissues (if applicable): Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 ml DMEM medium.
- N. of replicates : 2 tissues treated with test item and 2 negative control treated tissues.
- Method of calculation used: The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues.
- Test for colour interference by the test item: 1-Phenyldecane-1,3-dione was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, 50 μl of 1-Phenyldecane-1,3-dione or 50 μl Milli-Q water as a negative control were added to 0.3 ml Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
- Test for reduction of MTT by the test item: 1-Phenyldecane-1,3-dione was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 μl of 1-Phenyldecane-1,3-dione was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: N/A
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution): as such

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): as such

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): as such
Duration of treatment / exposure:
Two tissues were used for a 3-minute exposure to 1-Phenyldecane-1,3-dione and two for a 1-hour exposure.
Duration of post-treatment incubation (if applicable):
N.A
Number of replicates:
2 per duration of treatment

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Relative mean tissue viability obtained after the 3-minute treatment
Value:
99
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Relative mean tissue viability obtaine after the 1-hour treatment
Value:
78
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No specified
- Direct-MTT reduction: In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each timepoint. The non-specific reduction of MTT by 1-Phenyldecane-1,3-dione was 0.6% and 4% of the negative control tissues after 3 minutes and 1 hour respectively.
- Colour interference with MTT: 1-Phenyldecane-1,3-dione was checked for colour interference in aqueous conditions and for possible direct MTT reduction by adding the test item to MTT medium. Because a colour change was observed by adding MTT-medium it was concluded that 1-Phenyldecane-1,3-dione did interact with the MTT endpoint.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: The maximum inter-tissue variability in viability between two tissues treated identically was less than 26% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 15% for the negative control and test item. For the positive control, the maximum inter-tissue variability in viability between two tissues treated identically was up to 31% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was up to 19%, however since the viabilities were below 20% the acceptability criteria were met. It was therefore concluded that the test system functioned properly.
- Range of historical values if different from the ones specified in the test guideline: N/A

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
It is concluded that this test is valid and that 1-Phenyldecane-1,3-dione is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

This report describes the ability of 1-Phenyldecane-1,3-dione to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of 1-Phenyldecane-1,3-dione was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD guideline no. 431 and EC guideline B.40 Bis.

Batch MR92143651 of 1-Phenyldecane-1,3-dione is a Reddish solid below 25°C / liquid above 25°C with a purity of 86.4%. 1-Phenyldecane-1,3-dione was applied undiluted (50 μl) directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 7% after 3 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 acceptability criteria for 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 were met, indicating that the test system functioned properly.

1-Phenyldecane-1,3-dione did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each timepoint. The non-specific reduction of MTT by 1-Phenyldecane-1,3-dione was 0.6% and 4% of the negative control tissues after 3 minutes and 1 hour respectively. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with 1-Phenyldecane-1,3-dione compared to the negative control tissues was 99% and 78%, respectively. Because the mean relative tissue viability for 1-Phenyldecane-1,3-dione was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, 1-Phenyldecane-1,3-dione is considered to be not corrosive.

Finally, it is concluded that this test is valid and that 1-Phenyldecane-1,3-dione is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.