[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April to May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Amides, C16-18 (even numbered), N-C16-18 (even numbered) alkyl

Molecular formula:

R1-NH-C=O-R2, with R1=C16H33 and C18H37 & R2 = C15H31 and C17H35

IUPAC Name:

Amides, C16-18 (even numbered), N-C16-18 (even numbered) alkyl

Test material form:

solid

Details on test material:

- Chemical registery number : 955-212-4
- Chemical name: Amides, C16-18 (even numbered), N-C16-18 (even numbered) alkyl

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agreed by each registrant.

In vitro test system

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Justification for test system used:

In accordance with the OECD Testing Guideline 439

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™
- Delivery date: 30 April 2013

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing OPBS with Ca++ and Mg++ Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml MTT solution
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Classification of irritation potential is based upon relative mean tissue viability following the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period. Test item is classified as non-irritant if the relative mean tissue viability is > 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Approximately 1 0 mg of the test item was applied to the epidermis surface
Triplicate tissues treated with 10 µI of DPBS served as the negative controls and triplicate tissues treated with 10 µI of SDS 5% w/v served as the positive controls

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Three

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

109.2

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an OECD 439 study, conducted according to GLP, the relative mean viability of the test material with the treated tissues (using the EPISKIN system, in vitro) was 109.2% after a 15-Minute exposure, therefore, is considered not irritating to skin.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed
human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory
mediator IL-1a in the culture medium retained following the 42-Hour post-exposure incubation period is also determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μI samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 109.2% after the 15-Minute exposure period.

The quality criteria required for acceptance of results in the test were satisfied.

The test item was considered to be Non-Irritant (NI).