4,8-Dimethyl-2,5,7,10-tetraoxaundecane

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2016 to 01 November 2016

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,8-dimethyl-2,5,7,10-tetraoxaundecane

Cas Number:

59039-15-5

Molecular formula:

C9H20O4

IUPAC Name:

4,8-dimethyl-2,5,7,10-tetraoxaundecane

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Lambiotte & Cie - 1604211010R
- Purity, including information on contaminants, isomers, etc.: 99.97%

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 16-EKIN-043
- Production date: 25 October 2016
- Delivery date: 26 October 2016
- Date of initiation of testing: 25 October 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS 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. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT solution
- Incubation time: tissues were incubated for 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: without a reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Histology scoring: 21.8
- IC 50 determination: 1.7 mg/ml
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: Absence of bacteria, fungus and mycoplasma

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability after 15 minutes exposure followed by the 42-Hour post-exposure incubation period is less than 50%.

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 (26.3 µl/cm²)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10µl
- Concentration (if solution): Unknown

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10µl
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 1: 102.4%
replicate 2: 102.4%
replicate 3: 98.4%

Value:

101.1

Negative controls validity:

valid

Remarks:

replicate 1: 104.1% replicate 2: 97.3% replicate 3: 98.7%

Positive controls validity:

valid

Remarks:

replicate 1: 7.2% replicate 2: 2.6% replicate 3: 5.7%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

UN GHS Not classified for Irritation (category 3 can not be determined)

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply:
EU DSD & CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

Introduction
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM 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 colorimetric 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.
Method
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-labeled 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 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 nm.
Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).
Results
The relative mean viability of the test item treated tissues was 101.1% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.
Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.
Conclusion
The test item was classified as non-irritant. The following classification criteria apply:
EU DSD and CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).