Isoleucine, N-(1-oxohexadecyl)-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-22 to 2009-09-30

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline for the testing of chemicals, draft proposal for a new guideline, "In Vitro Irritation: Human Skin Model": reconstructed Human Epidermis (RHE) Test Method (20 March, 2099, version 6, 2nd circulation)

Deviations:

not specified

Principles of method if other than guideline:

The prediction model of Skinethic irritation assay is described below.
The test item is considered to be irritant to skin (R38), if the mean relative viability after 42 minutes exposure and 42 hours recovery is less or equal to 50% of the negative control (PBS).

1-Acceptance criteria:
Negative control : Data meet the acceptance criteria if the mean OD570 value of the 3 tissues is >= 1.2. The standard deviation value is considered as valid if it is <= 18%, according to the performance standard (ECVAM SIVS, 2007)
Positive control: Data meet the acceptance criteria if the mean viability, expressed as % of the negative control is <40% and the standard deviation value is <=18%
Batch acceptance criteria: all test item data from one batch are considered as valid if both negative and positive controls data fulfill the above criteria requirements.

II-MTT interference
Preliminary contact experiment was perform with all test item and MTT solution. The test item did not induce a change in colour of the MTT solution to blue or purple appearance. The test item did not interfere with MTT.

III-MTT viability testing
Data were valid, in accordance to the above criteria, Test item did not induced a decrease of viability below 50%.
Negative control OD : 1,669-1,631-1,694
Positive control OD : 0,027-0,019-0,023
Test Item OD : 1,898-1,439-1,798
Viability : 102,81%
SD : 14,51
IL-1alpha mean release : 57,9 pg/mL
Folowwing the irritation prediction model, test item is not irritant

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

LCA09019

IUPAC Name:

LCA09019

Constituent 2

Reference substance name:

DL-Isoleucine, N-(1-oxohexadecyl)

IUPAC Name:

DL-Isoleucine, N-(1-oxohexadecyl)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): LCA09019
- Substance type: Lipoamino acid
- Physical state: White powder
- Analytical purity: 100% ES
- Storage condition of test material: Room temperature

Test animals

Species:

other: not applicable (in vitro)

Strain:

other: not applicable (in vitro)

Test system

Type of coverage:

other: not applicable (in vitro)

Preparation of test site:

other: not applicable (in vitro)

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable (in vitro) : Epidermis negative control: concurrent no traitment Epidermis positive control : SDS 5% in water

Amount / concentration applied:

16 mg (+/- 2 mg)/Epidermis

Duration of treatment / exposure:

42minutes (+/- 1 minute)

Observation period:

42hours (+/-1h) after the end of exposure

Number of animals:

not applicable (in vitro)
3 Epidermis/condition

Details on study design:

I-MTT interference of the test item:
In order to assess if the test item interferes with MTT, a preliminary contact experiment was performed. A 24-well plate was filled with 300 µl of MTT (1mg/mL). 16 mg of the test item was added and incubated at 37°C for 3hours. Whether the MTT solution colour has become blue or purple, which means that the product interact with MTT, it would has been necessary to evaluate, the part of OD due to the non specific reduction of MTT, by using killed epidermis.

II- Tissue treatment:
The day before testing, the Skinethic RHE reconstructed epidermis were placed onto 1 mL of maintenance medium in 6-well plates. 10µL of distilled water were spreaded to the epidermis surface, in order to improve further contact between the solid test item and the epidermis. The 16mg (+/-2mg) of the test item were applied to the epidermis surface without addition of a nylon mesh, on 3 in vitro reconstructed human epidermal tissues. Tissues were then incubated for 42 minutes (+/- 1 minute) at room temperature, in 300µL of maintenance medium. For each test item, a single 24-well plate was used, to avoid cross-contamination between tissues. At the end of the incubation period, the tissues were rinsed with 25 mL of PBS, transferred in a new 24-well plate and allowed to recover for 42h at 37°C. Negative and positive control compounds were applied in parallel (PBS and SDS 5%). After 42 h (+/- 1 hour) of recovery, the cell viability was evaluated using the MTT test (n=3). Culture medium was collected for ELISA.

III-MTT assay procedure
In a 24-well plate, a volume of 300 µL of MTT solution at 1 mg/mL in assay culture medium was added to all test wells containing tissue or blank and incubated for 3 h (+/- 5 minutes) at 37°C (5%CO2). After incubation, the MTT solution was discarded and tissues were placed on absorbent parper. The epidermis were then placed in a new 24-well plate containing 800 µL of isopropanol. 700 µL of isopropanol were then added on the top of the tissue, which were then incubated at room temperature for 2h (+/- 5 minutes). A volume of 3X200 µL aliquots of blue MTT extraction solution was transferred from each well to corresponding wells of a 96-well plate. The Optical Density at 570 nm was determined spectrophotometrically using a microplate reader. The mean OD570 of the untreated control tissues exposed to PBS was set to represent 100% of viability and the results were expressed as a percentage of these control.

IV-IL-1alpha Quantification:
Tissue supernatants were harvested 42 hours after test item application. They were stored at -20°C untill analysis. Culture medium samples were processed for IL-1alpha detection by using ELISA, according to the kit manufacturer's procedure (Pierce).

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Viability-MTT

Value:

> 100

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 42 hours. Reversibility: other: non applicable. (migrated information)

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the irritation prediction model, the test item is not irritant

Executive summary:

Dermal acute irritation is a local, reversible inflammatory response of normal living skin to direct injury caused by the application of an irritant substance.The assesment of the irritant potential of test items in vitro is done using validated in vitro skin irritation assay such aas Skinethic RHE assay. It discriminates irritants (R38) and non-irritant chemicals, on the basis of reduction in viability under 50% after application of test item. The protocol used in this study is based on the OECD guideline for the testing of chemicals, draft proposal for a new guideline, "in vitro skin irritation: Human Skin Model Test".

After 42 minutes of application of test item and reference on the top of the epidermis and recovery for 42 hours, tissue viability was determined by MTT viability testing. In these conditions, test item did not decrease the viability of the epidermis, suggesting that according to the irritation prediction model, the test item is not irritant