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Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-04-18 to 2014-05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted 13 April 2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Succinic anhydride
EC Number:
203-570-0
EC Name:
Succinic anhydride
Cas Number:
108-30-5
Molecular formula:
C4H4O3
IUPAC Name:
succinic anhydride
Specific details on test material used for the study:
- Name of the test material used in the report: Succinic anhydride
- Batch no.: LEBA3A7021
- Purity: 99.7%
- Storage temperature: In refrigerator (2 to 8 °C)
- Expiry date: 31 December 2014

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No correction was made for the purity/composition of the test substance. The test substance was crushed and ground in a mortar with pestle. Twenty-five mg of Succinic anhydride was applied directly on top of the skin tissue. Succinic anhydride was spread to match the size of the tissue.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The test is based on the experience that corrosive chemicals show cytotoxic effects following short-term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin corrosion potential of a test substance by assessment of its effect on a three-dimensional human epidermis model.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200). MatTek Corporation, Ashland MA, USA
- Tissue batch number(s): Lot no.: 19989 kit D

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 63 - 83%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.3 - 36.8 °C). Temperature and humidity were continuously monitored throughout the experiment.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test substance. Rinsed tissues were kept in 24 well plates on 300 μL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL, MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM)
- Incubation time: The DMEM medium was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37 °C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: 4 tissues per test substance (two tissues were used for 3 min and two for a 1-hour exposure)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test substance is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test substance considered non-corrosive (viability  50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test substance is decreased below 15%.
A test substance is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: Succinic anhydride was crushed and ground in a mortar with pestle. Twenty-five mg of Succinic anhydride was applied directly on top of the skin tissue which was moistened with 25 μl of Milli-Q water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl Milli-Q water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl 8N KOH
Duration of treatment / exposure:
3 min or 1 hour
Number of replicates:
The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to Succinic anhydride and two for a 1-hour exposure

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure, mean of replicates
Value:
96
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: in combination with the 1-hour exposure: corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min exposure, mean of replicates
Value:
12
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: in combination with the 3-min exposure: corrosive
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The maximum inter-tissue variability in viability between two tissues treated identically was less than 23% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 13%, indicating that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes , The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range
- Acceptance criteria met for positive control: The mean relative tissue viability following
3-minute exposure to the positive control was 9%.
- Acceptance criteria met for variability between replicate measurements: The maximum inter-tissue variability in viability between two tissues treated identically was less than 23% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 13%.

Any other information on results incl. tables

Table 1: Mean absorption in the in vitro skin corrosion test with Succinic anhydride

3-minute application 1-hour application
A (OD540) B (OD540) Mean (OD540) SD A (OD540) B (OD540) Mean (OD540) SD
Negative control 1.493 1.347 1.42 0.103 1.408 1.361 1.385 0.033
Succinic anhydride 1.279 1.454 1.366 0.124 0.189 0.147 0.168 0.03
Positive control 0.124 0.118 0.121 0.004 0.097 0.085 0.091 0.008

Table 2: Mean tissue viability in the in vitro skin corrosion test with Succinic anhydride

3-minute application
viability (percentage of control)
1-hour application
viability (percentage of control)
Negative control 100 100
Succinic anhydride 96 12
Positive control 9 7

Applicant's summary and conclusion

Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
In this study under the given conditions the test item showed corrosive effects. The mean relative tissue viability (% negative control) was below 15% after 60 min treatment and above 50% after 3 min treatment. Therefore, the test item is classified as corrosive.
Executive summary:

In a primary skin corrosion study conducted according to OECD testing guideline 431, two EpiDerm tissues per dose group were exposed to 25 mg of Succinic anhydride (99.7% purity) for 60 min and 3 min. Cytotoxicity was measured in comparison to the concurrent negative controls via the MTT reduction assay and irritation was scored by the method of mean relative tissue viability. The test item showed corrosive effects. The mean relative tissue viability (% negative control) was below 15% (12%) after 60 min treatment and greater than 50% (96%) after a 3 min treatment. The controls confirmed the validity of the study and all acceptance criteria were fulfilled. Based on the results from this study, the test item can be classified as corrosive.

The study is acceptable and satisfies the guideline requirements for an in vitro skin corrosion study (OECD 431).