Dimethyl sebacate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2012 to 08 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully GLP compliant and in accordance with current test guidelines

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:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl sebacate

EC Number:

203-431-4

EC Name:

Dimethyl sebacate

Cas Number:

106-79-6

Molecular formula:

C12H22O4

IUPAC Name:

1,10-dimethyl decanedioate

Test material form:

other: solid to liquid (melting point 23°C)

Details on test material:

Test article name: Dimethyl sebacate
CAS number: 106-79-6
Batch number: 120801
Purity: 99.08%
Expiry date: 31 August 2013
Receipt date: 11 October 2012
Storage: stored in a sealed container, at room temperature in the dark. The sub-sample of test article used in this study was stored with a dessicant from 24 October 2012.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: human derived epidermal keratinocyte

Cell source:

other:

Vehicle:

unchanged (no vehicle)

Amount/concentration applied:

30µl

Duration of treatment / exposure:

60 min

Test animals

Species:

other: Not applicable

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

A three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum was supplied by MatTek Corporation, Ashland, Massachusetts, USA. Human keratinocytes are used to construct the epithelium. Multiple layers of viable epithelial cells are present under a functional stratum corneum. The stratum corneum is multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevent the passage of material around the stratum corneum to the viable model tissue. The skin model is manufactured to defined quality assurance procedures (certified ISO 9001) and supplied free of contamination with bacteria, mycoplasma and fungi.

Test system

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL of test system
- Concentration (if solution): Not applicable

VEHICLE
- Amount(s) applied (volume or weight with unit): 30 µL of positive and negative control
- Concentration (if solution): Not applicable
- Lot/batch no. (if required): Not applicalbe
- Purity: The positive control substance was 5% w/v aqueous Sodium Dodecyl Sulphate (SDS) in PBS.

Duration of treatment / exposure:

35 minutes at 37°C and 5% carbon dioxide. The plates were then removed from the incubator and placed into a sterile hood until the 60 minute treatment period was complete for each tissue. Following treatment, substances were removed by washing the tissues. The tissues were then placed on the appropriate medium and incubated for 42 hours. The protocol stated that the incubation period would be 24 hours. This deviation from protocol was considered not to have affected the integrity or outcome of the study as this was an error in the protocol and the correct incubation period was used for this test system

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

At the end of the 42-hour incubation period, tissue viability was assessed by MTT assay. Following rinsing, tissues were placed on 0.3 mL of MTT solution (1 mg/mL) and incubated for 3 hours. Once complete, the tissues were removed from the MTT solution and any resultant colour formed in the tissues by the MTT assay was extracted.
Extraction was achieved by flooding the tissue with 2 mL isopropanol, sealing the plate to avoid any evaporation occurring and then shaking at 150 rpm for 2 hours.
Upon completion of the extraction, each tissue was pierced using a hypodermic needle so that so that the extract could run through the tissue. Once drained, the tissue was discarded and the extract mixed by shaking at 150 rpm for 15 minutes. Two 200 µL aliquots of each resultant extract was placed into a 96-well plate for spectrophotometric determination of optical density at 570 nm using extraction solution as a blank.
Tissue viability was calculated for each tissue as a percentage of mean of the negative control tissues.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

90.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Table1 Cell viability measurements

Substance	Tissue replicate	OD570		Mean	Corrected mean	Relative survival
		Aliquot 1	Aliquot 2			%	Mean	SD	CV
Negative control	A	1.272	1.324	1.298	1.298	90.4	100.0	14.732	14.732
	B	1.679	1.678	1.679	1.679	117.0
	C	1.164	1.494	1.329	1.329	92.6
Test article	A	1.161	1.427	1.294	1.294	90.1	90.4	26.198	28.969
	B	0.817	1.031	0.924	0.924	64.4
	C	1.629	1.723	1.676	1.676	116.8
Positive control	A	0.077	0.096	0.087	0.087	6.1	5.1	1.023	20.119
	B	0.084	0.065	0.075	0.075	5.2
	C	0.055	0.060	0.058	0.058	4.0
Blank		0.000	0.000

 

The data in these tables was computer generated. In this system individual and derived figures are rounded. Thereby recalculation of derived values from the individual data will, in some instances, yield minor variations.

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: not specified

Conclusions:

The test article, Dimethyl Sebacate, was considered not to be irritant to the in vitro skin model EpiDerm SIT (EPI-200).

Executive summary:

This study was conducted to determine whether the test article, Dimethyl Sebacate, causes dermal irritation in the in vitro skin model EpiDerm^TM SIT (EPI-200).

EpiDerm^TM SIT (EPI-200) inserts were treated with test article, negative control (phosphate buffered saline (PBS)) and positive control (5% w/v sodium dodecyl sulphate (SDS)) for 60 minutes. At the end of the treatment period, the tissues were washed with PBS and cell viability was assessed using the MTT assay. The skin irritation potential was classified according to the remaining cell viability obtained after test article treatment.

The group mean viability for the test article was 90.4%, for the negative control was 100% and for the positive control was 5.1%.

The test article, Dimethyl Sebacate, was considered not to be irritant to the in vitro skin model EpiDerm^TM SIT (EPI-200).