2-Butenedioic acid (E)-, di-C12-18-alkyl esters

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - non official national or international study, well documented. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The potential of didodecyl fumarate to cause ocular irrritation was assessed by a single topical application of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcularTM). The study was performed according to the methods described in the following publications: MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1a of February 10, 2010 and Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing In Vitro Eye Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter Laboratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of Toxicology, March 2009.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Didodecyl fumarate

EC Number:

219-280-2

EC Name:

Didodecyl fumarate

Cas Number:

2402-58-6

Molecular formula:

C28H52O4

IUPAC Name:

didodecyl but-2-enedioate

Details on test material:

- Name of test material (as cited in study report): Didodecyl fumarate
- Physical state: solid
- Analytical purity: 93.8 area-%
- Lot/batch No.: 0008043725

Test animals / tissue source

Species:

human

Strain:

other: EpiOcularTM; reconstructed three-dimensional human cornea model (OCL-200)

Details on test animals or tissues and environmental conditions:

TEST SKIN MODEL
- Source: MatTek Corporation, Ashland MA, USA

TEST METHOD
The model represents a reconstructed three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratiozytes used to model the human corneal epithelium. Irritant materials are identified by their ability to induce cytotoxicity (= loss of viability) at the surface of the EpiOcularTM tissue. The decrease in cell viability is determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
Upon receipt, tissues were transferred into 6-well plates containing 1 mL assay medium per well and preincubated in a humidified incubator for 16 to 24 hours (37 ± 1 °C, 5% CO2) before use. After the pre-incubation the tissue were pre-treated with 20 µL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1
- CO2 gas concentration (%): 5
- Humidity: 90-95%

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent control tissues treated with the water served as negative controls, positive controls were exposed to 50 µL methyl acetate

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL bulk volume (about 22 mg)

NEGATIVE CONTROL:
- 50 µL of sterile de-ionized water

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: 50 µL methyl acetate

Duration of treatment / exposure:

90 minutes

Observation period (in vivo):

Not applicable. Post-treatment period: 18 hours

Number of animals or in vitro replicates:

Not applicable. The test was performed in duplicates for each treatment and control group.

Details on study design:

TEST SITE
- Area of exposure: 0.6 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the tissue three times with phosphate buffered saline (PBS). In order to remove residual test substance, washed tissue were immediatly immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion). After 12 minutes of post-soak immersion, each tissue was dried and transferred to fresh 6-well plates filled with pre-warmed medium.
- Time after start of exposure: 90 min
- Post-treatment incubation period: 18 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed 18 h after the incubation period. Therefore, tissues were incubated in 300 µL MTT solution for 3 h at 37 ± 1 °C and 5% CO2. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissue in isopropanol. The optical density was measured at 570 nm wave length in a plate spectrophotometer.

Results and discussion

In vitro

Results

Irritation parameter:

other: cell viability (%)

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Results:

test substance		tissue 1	tissue 2	mean	SD
NC	mean OD570	1.646	1.393	1.52
	viability [% of NC]	108.3	91.7	100	16.6
test item	mean OD570	1.133	1.349	1.241
	viability [% of NC]	74.6	88.7	82	14.2
PC	mean OD570	0.43	0.344	0.387
	viability [% of NC]	28.3	22.6	25	5.7

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not irritant
DSD: not irritant