IPEMA

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-11-12 to 2019-12-25

Reliability:

1 (reliable without restriction)

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:

June 18, 2019

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenoic acid, 2-methyl-, 3-methyl-3-buten-1-yl ester

Cas Number:

156291-88-2

Molecular formula:

C9H14O2

IUPAC Name:

2-Propenoic acid, 2-methyl-, 3-methyl-3-buten-1-yl ester

additive 1

Reference substance name:

Mequinol

EC Number:

205-769-8

EC Name:

Mequinol

Cas Number:

150-76-5

IUPAC Name:

(Polymerization inhibitor) 4-Methoxyphenol

In vitro test system

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Cell source:

other: donor - All cells used to produce EpiDermTM are purchased or derived from tissue obtained by MatTek Corporation from accredited lnstitutions, ln all cases, consent was obtained by these Institutions from the donor or the donor's legal next of kin, for use

Justification for test system used:

EpiDerm SCT kit is recommended in the test method

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm SCT kit
- Tissue batch number: 32116
- Production date: 2019-11-07
- Delivery date: 2019-11-11
- Date of initiation of testing: 2019-11-12

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3-minutes exposure at room temperature; at 60-minute exposure each plate was placed into the inbubator
- Temperature of post-treatment incubation (if applicable): room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure, each tissue twenty times with PBS(-).
Inside and outside the tissue inserts were wiped with a sterile cotton swab. The tissue inserts were placed into a 24-well plate (Corning) filled with 300 µL/well of fresh medium

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
PRE-TEST
- MTT concentration: 1mg/ml MTT solution (MTT medium)
- 50 µl test substance + 1 ml MTT medium
- Incubation time: 60 minutes

NYLON MESH
- 50µl of test substance was dropped onto a nylon mesh on a glass slide. After 60 minutes at room
temp., the corrosion of the nylon mesh was evaluated microscopically. Corrosion was not observed.

MAIN TEST
- MTT concentration: 1mg/ml MTT solution (MTT medium); 0.9 mL/well
- 50µl test substance or 50 µl control substance
- Pre-incubation time: 60 +- 5 minutes (0.9 mL/well of the medium)
- Incubation time (after rinsing) : 180+- 5 minutes (with 300 µL/well of MTT medium); extraction at room temperature for 2 hours or more using a plate shaker
- Spectrophotometer: Multimode Microplate Reader (FlUOstar Omega, BMG LABTECH) at 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA - OD:
3 min exposure:
Negative control: 2.004 ± 0.166
Positive control: 0.157 ± 0.049
60 min exposure:
Negative control: 1.951 ± 0.152
Positive control: 0.053 ± 0.020

NUMBER OF REPLICATE TISSUES: 2 (Duplicate tissue inserts were used for the test substance, negative control substance and positive control substance. Duplicate tissue inserts were used to check the tissue-binding of the test substance
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
Mean cell viability Category
<25% (3-minute exposure) Corrosive ( UN GHS Cat. 1A)
>=25%, <50% (3-minute exposure) Corrosive ( UN GHS Cat. 1B and 1C)
>=50% (3-minutes exposure) and <15% (60-minutes exposure) Corrosive ( UN GHS Cat. 1B and 1C)
>=50% (3-minutes exposure) and >=15% (60-minutes exposure) Non-Corrosive (UN GHS Cat. 2 or not classified)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µl

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µl
- Concentration (if solution): 8N KOH

Duration of treatment / exposure:

3 minutes
60 minutes

Duration of post-treatment incubation (if applicable):

180 minutes

Number of replicates:

2 tissue insert per treatment group

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

97.4

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-minutes

Value:

75.6

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes: The ODs in the negative control substance group in the 3-minute and 60-minute exposures were 1.848 and 1.689, respectively.
- Acceptance criteria met for positive control: Yes: The mean cell viability in the positive control substance group in the 60-minute exposure was 2.8%.
- Acceptance criteria met for variability between replicate measurements: The CVs between the two tissue inserts were = 30% for all substances which the cell viability was from 20% to 100%.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

It is concluded that IPEMA was "non-corrosive" (UN GHS category 2 or not classified) under the present test conditions