Reaction mass of (S)-2-[(R)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide and (S)-2-[(S)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Dec 2018 - 07 Dec 2018

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:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (S)-2-[(R)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide and (S)-2-[(S)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide

Cas Number:

943986-70-7

Molecular formula:

C11H20N2O2

IUPAC Name:

Reaction mass of (S)-2-[(R)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide and (S)-2-[(S)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide

Test material form:

solid

Details on test material:

- Appearance: Off-white solid
- Storage condition of test material: At room temperature protected from light

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on animal used as source of test system:

EpiDerm™ Reconstructed Human Epidermis
- All cells used to produce Eipderm™ are purchased or derived from tissue obtained by MatTek Corporation from acredited institutions.
- Cells are screened for potential biological contaminants (HIV-1, Hepatitis B, Hepatitis C, bacteria, yeast and fungi)

Justification for test system used:

Recommended test system in international guidelines (OECD and EC)

Vehicle:

other: the skin was moistened with 25 µL Milli-Q water (Millipore Corp.) to ensure close contact of the test item to the tissue

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue lot number: 29642, kit J and K

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure (3 minutes): room temperature
- Temperature used during treatment / exposure (1 hour): in a controlled environment at 37.0 ± 1.0°C

REMOVAL OF TEST MATERIAL
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h (at 37.0 ± 1.0°C)
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2 replicates per exposure duration

ACCEPTANCE OF RESULTS:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should be within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be < 15%.
c) In the range of 20 – 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

DECISION CRITERIA
A test item is considered corrosive in the in vitro 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 item 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 item is decreased below 15%.

A test item 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

yes, concurrent MTT non-specific colour control

other: concurrent control for MTT reduction by test item

Amount/concentration applied:

TEST MATERIAL
30.6 to 52.0 mg

Duration of treatment / exposure:

3-minute and 1-hour

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

98

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

32

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Color interference in aqueous conditions: No
- Direct-MTT reduction: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes (4.1%)
- Acceptance criteria met for variability between replicate measurements: Yes (≤ 21%)

Applicant's summary and conclusion

Interpretation of results:

other: GHS and CLP criteria not met

Conclusions:

The in vitro skin corrosion test was conducted according to OECD 431 guideline and GLP principles.
It is concluded that this test is valid and that the substance is not corrosive in the in vitro skin corrosion test.

Executive summary:

In an in vitro skin corrosion test using a human skin model (EpiDerm Skin Model), the influence of the substance on the viability of human skin was tested according to OECD 431 (2016) and GLP principles. The substance was applied directly to 0.6 cm2 cultured skin (30.6 to 52.0 mg, in presence of 25 μL Milli-Q water). Negative and positive controls were included. After 3 -minute and 1 -hour treatments the substance was removed and the viability of the cells was tested by reduction of MTT. Viability of unexposed skin was set at 100%. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the substance compared to the negative control tissues was 98% and 32%, respectively. The positive control had a mean cell viability of 4.1% after 1 hour exposure. The acceptability criteria of the study were met. Since the mean relative tissue viability was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, it can be concluded that the substance is not corrosive in the in vitro skin corrosion test.