Long-chain alkenyl amide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 July 2016 - 22 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU method B.40 BIS (In Vitro Skin corrosion: Human Skin Model Test), adopted 31 May 2008

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

Reaction products of fatty acids, C16-18, C18 unsatd. with Amines, polyethylenepoly-, triethylenetetramine fraction and 3-(C9–C15, C12 rich, alk-1-enyl)dihydro-2,5-furandione

EC Number:

947-263-6

Cas Number:

68478-81-9

Molecular formula:

C36H68N4 - C76H136N4O6

IUPAC Name:

Reaction products of fatty acids, C16-18, C18 unsatd. with Amines, polyethylenepoly-, triethylenetetramine fraction and 3-(C9–C15, C12 rich, alk-1-enyl)dihydro-2,5-furandione

Test material form:

solid

Details on test material:

Appearance: Dark red-brown solid
Storage conditions: At room temperature

Specific details on test material used for the study:

No correction for purity was made.

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:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue lot number: 24306 kit J

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment at 37.0 ± 1.0°C (actual range: 36.4-37.3 °C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- 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
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2 replicates for the 3-minute exposure and 2 replicates for the 1-hour exposure, one negative control, one positive contol.

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 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 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

DECISION CRITERIA (see table 1):
A test item is considered corrosive in the 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: MTT reduction by the test item was checked prior to the study

Amount/concentration applied:

- Test material: an excess amount of test substance was applied directly on top of the skin tissue, after the skin was moistened with 25 μl Milli-Q water
- Positive control: 50 μl
- Negative control: 50 μl

Duration of treatment / exposure:

3-minute and 1-hour

Duration of post-treatment incubation (if applicable):

3 h with MTT

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

68

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the absolute mean OD570 fell within historical range of negative controls
- Acceptance criteria met for positive control: Yes, mean relative tissue viability following 3-minute exposure was 16% and following 1-hour exposure 8% (should be below 15% after 1 h exposure).
- Acceptance criteria met for variability between replicate measurements: Yes, coefficient of variability for the 3-minute exposure was 27% and for the 1-hour exposure 12% (should be ≤ 30%).
- Results from positive and negative controls fell withing the historical data range.

Any other information on results incl. tables

Table 2          Mean tissue viability in thein vitro skin corrosion test with X-19555

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	68	99
Positive control	16	8

Applicant's summary and conclusion

Interpretation of results:

GHS 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 test substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this study.