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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:
weight of evidence
Study period:
From 2018-12-17 to 2018-12-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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:
2016
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,3-dihydro-1,4-benzodioxine-2-carboxylic acid
EC Number:
609-276-2
Cas Number:
3663-80-7
Molecular formula:
C9H8O4
IUPAC Name:
2,3-dihydro-1,4-benzodioxine-2-carboxylic acid
Test material form:
solid
Specific details on test material used for the study:
Purity: 99.7%
Batch No.: 80031745a

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 29674 Kit K and L

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature
- Temperature of post-treatment incubation (if applicable): 36.8 - 37.6 °C (actual range)

REMOVAL OF TEST MATERIAL AND CONTROLS: washed with phosphate buffered saline to remove residual test item, rinsed tissues were kept in 24 well plates on 300 μL DMEM until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL diluted (1:5)
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® 1200 Pro Plate Reader.
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 0.8 - 2.8

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 20 - 100%

NUMBER OF REPLICATE TISSUES: two tissues for 3-minute exposure and two for a 1-hour exposure, 2 tissues for the negative and positive controls for both the 3-minute and 1-hour time point

DECISION CRITERIA
- The test substance is considered to be corrosive to skin if [complete, e.g. 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 [complete, e.g. 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%.]
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 27.0 - 32.3 mg

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 minutes and 1 hour
Number of replicates:
two tissues for 3-minute exposure and two for a 1-hour exposure, 2 tissues for the negative and positive controls for both the 3-minute and 1-hour time point

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Value:
93
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean tissue viability at 3-minute application
Irritation / corrosion parameter:
% tissue viability
Value:
53
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean tissue viability at 1-hour application
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: the test item did not interfere with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean relative tissue viability of 6.6% after the 1-hour exposure.
- Acceptance criteria met for variability between replicate measurements: In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤13%

Applicant's summary and conclusion

Interpretation of results:
other: unclassified
Conclusions:
In conclusion, the test substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the test substance for its ability to induce skin corrosion in accordance with OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method).

The possible corrosive potential of the test substance was tested through topical application for 3 minutes and 1 hour. Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of the test substance was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 6.6% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 13%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 93% and 53%, respectively. The mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment.

In conclusion, the test substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.