Potassium (2S)-4-carboxy-2-(tetradecanoylamino)butanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May 2012 to 11 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

potassium (2S)-4-carboxy-2-tetradecanamidobutanoate

EC Number:

700-938-7

Cas Number:

72716-26-8

Molecular formula:

C19H34NO5K

IUPAC Name:

potassium (2S)-4-carboxy-2-tetradecanamidobutanoate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Substance type: white to pale yellow powder, a slightly characteristic odour.
- Storage condition of test material: room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

Skin Samples
- Model: EPISKIN-SM.
- Source: SkinEthic, France, Batch No.:12-EKIN-015, Expiry Date: 14 April 2012.
- Quality control: All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). The EPISKIN kit received was checked for pH and to verity that the kit had not been exposed to temperatures above 40 °C during transport; the kit was found to be in good order.
- Storage: The EPISKIN kit was kept in their packaging at 37 °C and the assay medium supplied with the kit was stored at 2-8°C until test initiation.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg.

Duration of treatment / exposure:

15 minutes

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

88

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

CELL VIABILITY
- The viability results of the test material treated disks was 88 % of the negative control, since this is > 50 % of the negative control the test material is considered to be a non-irritant.

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues was 0.756. The positive control result showed a mean of 6.9 % viability. Each standard deviation value (SD) of the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

INDICATOR FOR POTENTIAL FALSE VIABILITY
> Possible direct MTT reduction with the test material: No colour change was observed after three hours of incubation of the test item in MTT solution. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability due the MTT interaction can be precluded.
> Colouring potential of the test material: As the test material is coloured, one additional chemical-treated tissue was used for the non specific OD evaluation. Optical Density (measured at 540 nm) of this tissue was determined as 0.034, Non Specific Colour % was calculated as 4.5 %, below the threshold of 50 %. Therefore additional data calculation was not necessary.

Any other information on results incl. tables

Table 1: Optical Density (OD) and the Calculated % Viability

Substance	Optical Density (OD)		Viability (%)
Negative Control	1	0.820	108
	2	0.659	87
	3	0.788	104
	Mean	0.756	100
	(SD)		11.15
Positive Control	1	0.040	5.3
	2	0.079	10
	3	0.041	5.4
	Mean	0.053	6.9
	(SD)		2.69
Test Material	1	0.721	95
	2	0.536	71
	3	0.746	99
	Mean	0.668	88
	(SD)		15.14

Applicant's summary and conclusion

Interpretation of results:

other: not irritating according to EU criteria

Conclusions:

Under the conditions of the test, the results indicate that the test material is not a skin irritant.

Executive summary:

The potential for the test material to cause skin irritation was predicted in vitro using the EPISKIN Model in a study conducted under GLP conditions and in line with OECD 439 and EU Method B.46. The test material was applied topically to the surface of the skin for 15 minutes, which was terminated by rinsing with PBS 1 x solution (0.9 %). Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. Positive and negative controls were run concurrently and tissue viability was expressed as a % relative to the negative control.

Under the conditions of the study, exposure to the test material resulted in a mean relative cell viability of 88 %. Since the cell viability was determined to be > 50 % of the negative control the test material was considered to be non-irritating.