Alkenes, C15-18-branched and linear, maleated, hydrolyzed

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-11-25 to 2008-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

The study was carried out according to EU guideline B46 "In vitro skin irritation: reconstructed human epidermis model test" and complies with GLP. The EPISKIN model is a validated model and suitable to derive classification.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN reconstituted 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 is based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (witin the mitochondia of viable cells) in the test material treated tissues relative to the negative controls.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(6E)-3,13-diethylpentadec-6-ene; (6E)-tetradec-6-ene; (7E)-hexadec-7-ene; (8E)-nonadec-8-ene; 2-[(6E)-3,13-diethylpentadec-6-en-8-yl]butanedioic acid; 2-[(8E)-nonadec-8-en-7-yl]butanedioic acid; 2-[(8E)-tetradec-8-en-7-yl]butanedioic acid; 2-[(9E)-hexadec-9-en-8-yl]butanedioic acid

EC Number:

700-363-1

Cas Number:

1335203-20-7

IUPAC Name:

(6E)-3,13-diethylpentadec-6-ene; (6E)-tetradec-6-ene; (7E)-hexadec-7-ene; (8E)-nonadec-8-ene; 2-[(6E)-3,13-diethylpentadec-6-en-8-yl]butanedioic acid; 2-[(8E)-nonadec-8-en-7-yl]butanedioic acid; 2-[(8E)-tetradec-8-en-7-yl]butanedioic acid; 2-[(9E)-hexadec-9-en-8-yl]butanedioic acid

Details on test material:

- Name of test material (as cited in study report): Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes
- Lot/batch No.: PO709-4739

In vitro test system

Test system:

human skin model

Test animals

Species:

other: In vitro: reconstituted human epidermis model

Strain:

other: EPISKIN reconstituted human epidermis model

Details on test animals or test system and environmental conditions:

Triplicate tissues were treated with 10 µl of the test material for an exposure period of 15 minutes at room temperature. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours at 37ºC, 5% CO2 in air. 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-labelled micro tubes and stored in a freezer at-14 to -30ºC 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. The tubes were refrigerated at 1 to 10ºC until day 6 to allow for the extraction. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 ul samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm using the Anthos 2001 microplate reader.

Test system

Type of coverage:

other: Not relevant

Preparation of test site:

other: Not relevant

Vehicle:

other: Not relevant

Controls:

not required

Amount / concentration applied:

10 µl of the test material was applied to the epidermis surface and triplicate tissues, treated with either 10 ul of PBS ( negative controls) or 10 ul of 5% w/v SDS (positive controls).

Duration of treatment / exposure:

Treatment period of 15 minutes, followed by a post-exposure incubation period of 42 hours.

Observation period:

Not relevant.

Number of animals:

Not relevant.

Details on study design:

The relative mean tissue viability after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control tissues. The relative mean viabilities were calculated as the ratio between the mean optical density at 540 nm of the test material and the mean optical density at 540 nm of the negative control. Classification of irritation potential is based upon the relative tissue viability following the 15 minute exposure period. If the mean tissue viability is ¿ 50%, the test material is classified as Irritant (R38) and if it is >50%, the test material is non-irritating.

The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ¿ 40% relative to the negative control treated tissues, and the Standard Deviation (SD) value of the % viabillity is ¿20%.

The assay establishes the acceptance criterion for an acceptable test if the mean OD540 for the negative control treated tissues was ¿0.6, and the SD value of the % viability is ¿20%.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 89.8

Remarks on result:

other:

Remarks:

Basis: other:. Time point: 15 minute exposure followed by a 42 hour post exposure period. Remarks: Non-Irritating.

Other effects / acceptance of results:

The relative mean viability of the test material treated tissues was 89.9% after a 15 minute exposure, and the test material was considered to be non-irritant.

Any other information on results incl. tables

For this in vivo test, if the MTT solution containing the test substance turns blue/purple, the test substance is presumed to have reduced the MTT. As the MTT solution did not turn blue/purple, this indicated that the test material did not directly reduce MTT.

The relative mean tissue viability for the positive control treated tissues was less than or equal to 40% relative to the negative control treated tissues and the SD value of the % viability was less than or equal to 20%. The positive control acceptance criterion was therefore satisfied. The mean OD₅₄₀ for the negative control treated tissues was greater than or equal to 0.6 and the SD value of the % viability was less than or equal to 20%. The negative control acceptance criterion was therefore satisfied.

Table 1: Mean OD₅₄₀values and % viabilities for the negative control material, positive control material and test material

Material	OD540of tissues	Mean OD540of triplicate tissues	± SD of OD540	Relative individual tissue viability	Relative mean % viability	± SD of % viability
Negative control material	0.770	0.783	0.012	98.3	100*	1.56
	0.794			101.4
	0.784			100.1
Positive control	0.065	0.073	0.020	8.3	9.3	2.61
	0.058			7.4
	0.096			12.3
Test material	0.584	0.703	0.107	74.6	89.8	13.66
	0.792			101.1
	0.733			93.6

 * = The mean viability of the negative control tissues is set at 100%.

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean viability of the test material treated tissues was 89.9% after a 15 minute exposure, and the test material was considered to be non-irritant since the mean tissue viability is >50%. On this basis, the test material does not warrant any classification according to Regulation (EC) No 1272/2008.

Executive summary:

Triplicate EPISKIN reconstituted human epidermal tissues were treated with 10 ul of DCI-30.n for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. At the end of the post exposure period, each tissue was taken for MTT-loading. After MTT loading , each epidermis was extracted with formazan crystals out of the MTT-loaded tissues and optical density was then measured at 540 nm.

The relative mean viability of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes treated EPISKIN tissues was 89.9% after a 15 minute exposure and therefore Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes was considered to be non-irritant since the mean tissue viability is >50%.The results of the negative and positive controls demonstrated the validity of the test. On this basis, the test material does not warrant any classification according to Regulation (EC) No 1272/2008.