2,5-furandione, dihydro-, mono- C18- alkenyl derivs. reaction products with potassium hydroxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 February 2017 - 17 February 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 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:

Potassium hydrogen 2-octadecenylsuccinate

EC Number:

296-086-4

EC Name:

Potassium hydrogen 2-octadecenylsuccinate

Cas Number:

92218-39-8

Molecular formula:

C22H40O4.K

IUPAC Name:

hydrogen potassium 2-octadec-1-en-1-ylsuccinate

Test material form:

solid

Details on test material:

Name as cited in report: X-19924
Appearance: thick amber paste
Storage: at room temperature

Specific details on test material used for the study:

Purity correction factor: 1.14
Stability at higher temperatures: stable at a maximum temperature of 60°C for a maximum duration of 30 minutes

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 (Lot no.: 24982 kit H and I)
- 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).

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue lot number: 24982 kit H and I
- Surface: 0.6 cm^2
- The model consists of keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

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: 36.6 - 37.0°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 per exposure duration (4 replicates per test item), 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 acceptance limits of OECD 431 (lower limite ≥0.8 and upper limit ≤2.8)
b) The mean relative tissue viability following 1-hour exposue 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

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:

Excess amount of the paste, spread directly on top of skin moistened with 25 µL Milli-Q water

Duration of treatment / exposure:

3-minute and 1-hour

Duration of post-treatment incubation (if applicable):

none

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

107

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(100%)

Positive controls validity:

valid

Remarks:

(29%)

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

90

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(100%)

Positive controls validity:

valid

Remarks:

(9%)

Other effects / acceptance of results:

- Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered to be non corrosive.

- OTHER EFFECTS:
- 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, mean relative tissue viability following 1-hour exposure was 9%.
- In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤12% for the test item and negative control. For the positive control, the Coefficient of Variation between tissue replicates at the 3-minute exposure was 39%. Since both individual tissues of the positive control were clearly positive it was concluded that the test system functioned properly.

Table 2 shows the results of individual OD measurements at 570 nm.

Any other information on results incl. tables

Table 2 Individual OD measurements at 570 nm.

	3-minute application (OD570)		1-hour application (OD570)
	A	B	A	B
Negative control Measurement 1 Measurement 2 Measurement 3	1.4908 1.4052 1.4427	1.4308 1.3742 1.4020	1.7312 1.7305 1.6864	1.5932 1.5681 1.5657
X-19924 Measurement 1 Measurement 2 Measurement 3	1.6368 1.6057 1.5798	1.4898 1.3770 1.4145	1.4855 1.4863 1.4874	1.4870 1.4816 1.5166
Positive control Measurement 1 Measurement 2 Measurement 3	0.5413 0.5453 0.5385	0.3585 0.3446 0.3422	0.1999 0.2067 0.2032	0.1843 0.1793 0.1789

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

An in vitro skin corrosion test was conducted with X-19924 according to OECD 431 guideline and GLP principles. It is concluded that this test is valid and that X-19924 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

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