[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-03-21 to 2019-04-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT)

Version / remarks:

2017-07-11

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012-07-06

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 2015-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of diethyl (E)-2-methylbut-2-enedioate, diethyl (Z)-2-methylbut-2-enedioate and diethyl 2-methylenebutanedioate

Cas Number:

2245967-74-0

Molecular formula:

C9H14O4

IUPAC Name:

reaction mass of diethyl (E)-2-methylbut-2-enedioate, diethyl (Z)-2-methylbut-2-enedioate and diethyl 2-methylenebutanedioate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

other: Not applicable

Justification for test system used:

The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The EpiDerm™ Skin Irritation Test (SIT) was validated in an international prevalidation study performed by ECVAM and have turned out as a sufficiently promising predictor for skin irritancy potential. Laboratory technical proficiency with the test system according to OECD Test Guideline 439 was demonstrated at Envigo CRS GmbH in March 2014.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissue
- Tissue batch number: 28690
- Delivery date: April 02, 2019
- Date of initiation of testing: April 03, 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C and 5 ± 0.5% CO2 (first 35 min) and room temperature (remaining 25 min)
- Temperature of post-treatment incubation: 37 ± 1.5 °C and 5 ± 0.5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: At least 15 times
- Observable damage in the tissue due to washing: Not indicated
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (working solution)
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1
- Wavelength: 570 nm
- Filter: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Concurrent negative controls were used in each run to demonstrate that viability of the tissues are within a defined historical acceptance range. Historical data and the quality certificate of the supplier of the test kit demonstrating its robustness can be found in "Attached background material". Furthermore, tissue viability has been checked by the supplier in an MTT QC assay (Acceptance criteria: OD (540-570) [1.0-3.0], Result: OD 2.019 ± 0.08)
- Barrier function: Concurrent positive controls were used in each run to demonstrate that barrier function and resulting tissue sensitivity of the tissues are within a defined historical acceptance range. Historical data and the quality certificate of the supplier of the test kit demonstrating its robustness can be found in "Attached background material". The ET50 should be between 4.0 hours and 8.7 hours after treatment with 1% Triton X-100 (QC batch release criteria). Furthermore, tissue viability has been checked by the supplier in an ET-50 assay (Acceptance criteria: ET-50 [4.77-8.72 hours], Result: 5.62 hours)
- Contamination: The cells used to produce EpiDerm™ tissues were screened for potential biological contaminants. These included HIV-1 virus, Hepatitis B virus, Hepatitis C virus, bacteria, yeast and funghi. No contaminations were observed.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues: 2
- Method of calculation used: True viability = Viability of treated tissue – Interference from test chemical = ODtreated viable tissue - ODkilled tissue, where ODkilled tissue = (mean ODtreated killed tissue - mean ODuntreated killed tissue)

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be a skin irritant if the viability after 3 hours exposure is less than or equal 50%.
- The test substance is considered to be non-irritant to skin if the viability after 3 hours exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL (undiluted)

NEGATIVE CONTROL
- Amount applied: 30 µL (undiluted)

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 %

Duration of treatment / exposure:

60 minutes (35 minutes at 37°C and 25 minutes at room temperature)

Duration of post-treatment incubation (if applicable):

42 hours (incubation medium was changed after 24 hours)

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three runs

Value:

80.04

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not indicated
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent showed purple colour.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes, see "Attached background material"

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (mean OD: 1.840)
- Acceptance criteria met for positive control: Yes (mean OD: 0.059)
- Acceptance criteria met for variability between replicate measurements: Yes (SD: 6.2)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is non-irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This GLP compliant in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test (OECD guideline 439). The test item reduced MTT (pre-test for direct MTT reduction), but it did not change colour when mixed with deionised water (pre-test for colour interference). Consequently, additional tests with freeze-killed were necessary. The test item, the negative control (DPBS), and the positive control (5% SDS) were applied to triplicate tissue, respectively. The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours, the tissues were treated with the MTT solution for 3 hours followed by about 2.5 hours of extraction of the colourant from the cells. The amount of extracted colourant was determined photometrically at 570 nm. After treatment with the negative control, the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues. Treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 3.20% thus ensuring the validity of the test system. The standard deviations between the three tissue percentage viability values of each group (test item, positive and negative controls) in the main test were below 18 (threshold according OECD 439: ≤ 18), thus ensuring the validity of the study. Compared to the negative control the mean relative viability was reduced to 80.04% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.