Dimethyl sebacate

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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 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

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.

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.

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

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.

 

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).

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

1/In vivo skin irritation study :

There is an in vitro skin irritation study performed according to OECD 439 guideline.

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

EpiDerm^TMSIT (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%.

As the mean viability was above the cut-off value of 35% whatever the exposure duration, the test item was considered to be non corrosive to the skin.

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

2/In vitro eye irritation study:

A bovine corneal opacity and permeability (BCOP) assay was conducted. Three corneas were treated by application of 750 µL of the undiluted test article onto the anterior surface of the cornea followed by a 10 minute incubation at 32°C ± 1°C. A volume of 750 µL of the negative or positive control was similarly applied to further groups of three corneas.

The corneas were then washed and the anterior chamber filled with pre-warmed EMEM (without phenol red). The corneas were then incubated for an additional 2 hours at 32°C ± 1°C. At the end of the post-incubation period the corneas were assessed for opacity. Permeability was determined by the amount of sodium fluorescein dye that penetrated all corneal layers. The media in the anterior chamber was replaced with 4 mg/mL sodium fluorescein, while the posterior chamber was filled with fresh EMEM. The corneas were then incubated for 90 minutes ± 5 minutes at 32 ± 1°C after which the media in the posterior chamber was assessed for presence of sodium fluorescein by measuring the optical density at 490 nanometers using a spectrophotometer.

The mean opacity reading for the test article was -0.7 and the mean group corrected optical density for the test article was -0.158. The test article produced an IVIS score of -3.04 and was considered not to be corrosive or severely irritating to the eye. The in vivo test was therefore conducted.

3/In vivo eye irritation study

An in vivo eye irritation study with Dimethylsebacate was therefore performed according to OECD guideline 405 and GLP . In this study two animals had corneal opacity with a reversibility for only one animal at day 6 and the corneal opacity was also observed for the second animal until day 22. As corneal opacity was observed in 2 out of 3 tested animal and has not fully reversed within an observation period of day 22, the test item was considered to be eye irritant caterogy 1.

 

Justification for selection of skin irritation / corrosion endpoint:
The study was perfomed according to OECD 439 and is GLP compliant.

Justification for classification or non-classification

Skin irritation: The results of the in vitro skin irritation test performed according to OECD guideline 439 did not trigger classification for Dimethylsebacate according to GHS criteria.

Eye irritation: The results of the in vivo eye irritation study performed according to OECD guideline 405 showed corneal opacity in 2 out 3 animals with absence of reversibility after 22 of observation in one of these animals, therefore, classification as eye irritant category 1 is warranted according to GHS criteria.