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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-01-21 to 2009-01-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

The study was carried out GLP. The SkinEthic test is currently under validation and results are showing that this model is suitable for this type of testing and to derive classification.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death. The experimental design of the study consists of a test for the direct reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) by the test material followed by the main test. For the main test, triplicate SkinEthic tissues were treated with 30 ul of the test material for 10 minutes. Triplicate tissues treated with 30 ul of Solution A serves as the negative control and triplicate tissues treated with 30 ul of 1% w/v Sodium Dodecyl Sulphate served as the positive control. At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues. Atfer extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD540). Data are presented in the form of % viability (MTT conversion relative to 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

Test animals / tissue source

Species:

other: In vitro: reconstituted human corneal model

Strain:

other: SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France)

Details on test animals or tissues and environmental conditions:

Pre-test
Prior to carrying out the definitive test, on arrival of the tissue, the tissue are prepared and incubated overnight at 37ºC, 5% CO2 in air. Before treatment, the age day 7 tissues were transfered from the arrival plates into the treatment plates containing the maintenance medium.

Main test
Triplicate tissues were treated with 30 µl of the test material for 10 minutes. Triplicate tissues were treated with 30 µl of solution A to serve as negative controls and triplicate tissues were treated with 30 µl of 1% w/v SDS to serve as positive controls. The plates were incubated at 37ºC, 5% CO2 in air durign the exposure time. Further to rinsing with Dulbecco`s Phosphate Buffered Saline (DPBS), the tissues were loaded with MTT and placed in an incubator for approximately three hours at 37ºC, 5% CO2 in air . At the end of the exposure, the tissues were visually examined and the degree of MTT staining evaluated. Further to extraction of MTT, the plate wrapped in aluminium foil were allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue. At the end of the extraction period, the optical density was measured at 540 nm using the Anthos 2001 microplate reader. Tissues were retained for possible tissue histopathology and stored at room temperature.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

For the main test, triplicate SkinEthic tissues were treated with 30 ul of the test material for 10 minutes. Test materials were applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

Duration of treatment / exposure:

The treatment period was 10 minutes.

Observation period (in vivo):

Not relevant.

Number of animals or in vitro replicates:

Not relevant.

Details on study design:

The negative control material, Solution A, was used as supplied and the positive control material, Sodium Dodecyl Sulphate (SDS) was prepared as a 1% w/v solution in sterile water.

Results and discussion

In vitro

Results

Irritation parameter:

other:

Value:

ca. 11.7

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The relative mean viability of the test material treated tissues after a 10 minute exposure was 11.7%. Therefore as the test was classified according to the following criteria: (1) If the % relative mean tissue viability was greater than or equal to 60% the test material was considered to be non-irritant and (2) If the % relative mean tissue viability was < 60% the test material was considered to be an irritant. According to the above criteria, the test material was considered to be irritant.

Any other information on results incl. tables

The relative mean viability of the test material treated tissues after a 10 minute exposure was 11.7%, and so the test material was considered to be irritant (as the % relative mean tissue viability was < 60%). However, it was considered unnecessary to carry out histopathology on the corneal tissues.

Table 1: Assessmentof eye irritation potential ¿ viability of RHC tissues

Material	Mean tissue viability	Relative mean % viability	± SD of % viability
Negative control material	1.099	1.054	100*
	1.008
Positive control	0.159	0.184	17.5
	0.209
Test material	0.117	0.123	11.7
	0.128

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

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The relative mean viability of the test material treated tissues after a 10 minute exposure was 11.7%, and so the test material was considered to be irritant (as the % relative mean tissue viability was < 60%). Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes is classified as Category 2 for serious eye damage with the signal word "Warning" and is assigned the hazard statement H318 "causes serious eye irritation" according to Regulation (EC) No 1272/2008.

Executive summary:

Triplicate SkinEthic reconstituted corneal epithelial tissues were treated with 30 ul of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes for an exposure period of 10 minutes.

The relative mean viability of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes treated SkinEthic reconstituted human corneal epithelial tissues after a 10 minute exposure was 11.7%, and so Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes was considered to be irritant (as the % relative mean tissue viability was < 60%). Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes is classified as Category 2 for serious eye damage with the signal word "Warning" and is assigned the hazard statement H318 "causes serious eye irritation" according to Regulation (EC) No 1272/2008.