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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:
12 September 2016 - 11 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
diethyl 2-methylidenebutanedioate
EC Number:
607-321-0
Cas Number:
2409-52-1
Molecular formula:
C9H14O4
IUPAC Name:
diethyl 2-methylidenebutanedioate

In vitro test system

Test system:
human skin model
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Epi-200 SIT kits and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.
Justification for test system used:
The EpiDerm TM is an in vitro system recommended by the corresponding guideline.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT Kit
- Tissue batch number: 23356
- Date of initiation of testing: 20 September 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C for 35 minutes; room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with DPBS at least 15 times
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax Molecular Devices, Softmax Pro
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin (Category 2) if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control
- The test substance is considered to be non-irritant to skin if the mean relative tissue viability of three individual tissues is reduced > 50% of the negative control
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
35 min at 37 °C + 25 min at room temperature
Duration of post-treatment incubation (if applicable):
25 h + 18 h
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
Tissue 1
Value:
100.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
Tissue 2
Value:
127.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
Tissue 3
Value:
108.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

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: yes

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

The test item passed the pre-tests for colour interference and direct MTT reduction. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

The test met the required acceptability criteria.

After treatment with the test item the mean relative absorbance value did not decrease (112.1%) compared to the relative absorbance value of the negative control. This value does not affect the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.