Hexanamide, N-(2-hydroxyethyl)-3,5,5-trimethyl-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 September 2012 to 08 February 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD (2010), In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, OECD Guidelines for the Testing of Chemicals No. 439, OECD, Paris.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ECVAM Scientific Advisory Committee (ESAC) Statement on the Validity of In Vitro Tests for Skin Irritation (2007).

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ECVAM Skin Irritation Validation Study. Validation of the EpiSkin® Irritation Test – 42 Hours Assay for the Prediction of Acute Skin Irritation of Chemicals, L’Oreal Recherche, January 2005.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ECVAM Skin Irritation Validation Study (SIVS). Performance Standards for Applying Human Skin Models to In Vitro Skin Irritation Testing (2007).

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD (2010), In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, OECD Guidelines for the Testing of Chemicals No. 439, OECD, Paris.

Deviations:

no

Principles of method if other than guideline:

in vitro skin irritation using the EpiSkin model.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-hydroxyethyl)-3,5,5-trimethylhexanamide

EC Number:

800-884-5

Cas Number:

1154308-86-7

Molecular formula:

C11 H23 N1 O2

IUPAC Name:

N-(2-hydroxyethyl)-3,5,5-trimethylhexanamide

Test material form:

other: liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- SkinEthic Episkin

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Surface area of the EpiSkin was ca 0.38 cm2.
- Application rate was ca 26.3 μL/cm2.

Duration of treatment / exposure:

15 minutes.

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 replicates

Test animals

Species:

other: in vitro

Test system

Details on study design:

TEST SITE
- Area of exposure: 0.38 cm2; RATE OF 26.3 μL/cm2.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Rinsed with 25 mL PBS and blotted dry on tissue paper.
- Time after start of exposure: 15 mins

INITIAL ASSESSMENT OF TOXICITY
Prior to the conduct of the irritation assay, a preliminary test was conducted to assess the intrinsic ability of the test item to reduce methylthiazoldiphenyl-tetrazolium bromide (MTT) to formazan. The substance coating additives did not reduce MTT to formazan.

PROCEDURE OF THE MAIN TEST
The dermal irritation potential was assessed by applying 10 μL of the substance coating additives to the exposed surface of three EpiSkin tissues for 15 min. The surface area of the EpiSkin was 0.38 cm2, therefore the application rate was 26.3 μL/cm2. After the 15 min exposure period, the test item was washed from the surface of the EpiSkin using Dulbecco’s phosphate-buffered saline (PBS). The EpiSkin tissues were then incubated for a recovery period of 42 h in a humidified incubator, set to maintain temperature and CO2 levels of 37°C and 5%, respectively. Following incubation, the tissues were transferred to assay medium containing MTT (0.3 mg/mL) and returned to the incubator for 3 h. Biopsies of the EpiSkin membranes were removed, added to acidified isopropanol, and refrigerated for ca 68 h in order to extract the formazan. The formazan production (cell viability) was assessed by measuring the optical density of the extracts at a wavelength of 550 nm. Three replicates of the positive control, aqueous sodium dodecyl sulphate (SDS, CAS 151-21-3) solution (5%, w/v), and the negative control, PBS, were tested in parallel to demonstrate the efficacy of the assay. The viability of each individual EpiSkin tissue was calculated as a percentage of the mean negative control viability (defined as 100%).

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

9.56

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Basis: mean mean of 3 replicates. Time point: 42 h. Reversibility: no data. Remarks: Reduced viability of cells in the EpiSkin® model.

Applicant's summary and conclusion

Interpretation of results:

other: Category 2 (irritant) based on CLP criteria

Conclusions:

Under the conditions of the in vitro EpiSkin® study, the test substance was demonstrated to be an irritant to skin

Executive summary:

A study was conducted to determine dermal irritation potential of the test substance, in an EpiSkin® in vitro irritation test, according to OECD 439, in compliance with GLP. Prior to the conduct of the irritation assay, a preliminary test was conducted to assess the intrinsic ability of the test substance to reduce methylthiazoldiphenyl-tetrazolium bromide (MTT) to formazan. The test substance did not reduce MTT to formazan. The dermal irritation potential was assessed by applying 10 μL of the test substance to the exposed surface of three EpiSkin tissues for 15 min. The surface area of the EpiSkin was 0.38 cm2, therefore the application rate was 26.3 μL/cm2. After the 15 min exposure period, the test substance was washed from the surface of the EpiSkin using Dulbecco’s phosphate-buffered saline (PBS). The EpiSkin tissues were then incubated for a recovery period of 42 h in a humidified incubator, set to maintain temperature and CO2 levels of 37°C and 5%, respectively. Following incubation, the tissues were transferred to assay medium containing MTT (0.3 mg/mL) and returned to the incubator for 3 h. Biopsies of the EpiSkin membranes were removed, added to acidified isopropanol, and refrigerated for ca 68 h in order to extract the formazan. The formazan production (cell viability) was assessed by measuring the optical density of the extracts at a wavelength of 550 nm. Three replicates of the positive control, aqueous sodium dodecyl sulphate (SDS) solution (5%, w/v), and the negative control, PBS, were tested in parallel to demonstrate the efficacy of the assay. The viability of each individual EpiSkin tissue was calculated as a percentage of the mean negative control viability (defined as 100%). Exposure to the test substance resulted in a mean EpiSkin viability of 9.56% ± 3.26% of the negative control value. Exposure to the positive control, aqueous SDS solution (5%, w/v), resulted in a mean EpiSkin viability of 13.36% of the negative control value. Under the conditions of the in vitro EpiSkin® study, the test substance was demonstrated to be an irritant to skin (Blackstock, 2013).