2-Butenedioic acid (2E)-, di-C14-16-alkyl esters

Skin irritation / corrosion

Administrative data

Endpoint:

skin 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 - Guideline Study. 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:

according to guideline

Guideline:

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

Version / remarks:

adopted July 22, 2010

Deviations:

no

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

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

Species:

human

Strain:

other: EpiDermTM; reconstructed three-dimensional human epidermis (EPI-200)

Details on test animals or test system and environmental conditions:

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

TEST METHOD
The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous and granular layers, and a multi-layered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
Upon receipt, tissues were transferred into 6-well plates containing 900 µL assay medium per well and preincubated in a humidified incubator for at least 1 h (37 ± 1 °C, 5% CO2) before use.

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

Test system

Type of coverage:

other: open - in vitro system

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

other: minimally moistened with PBS

Controls:

other: concurrent control tissues treated with the vehicle served as negative controls, positive controls were exposed to 5% SDS

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL bulk volume (about 15 mg)
- Concentration (if solution): 50% (v/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL
- Concentration (if solution): 50%

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: SDS, 5% (v/v)

Duration of treatment / exposure:

60 minutes

Observation period:

Not applicable. Post-treatment incubation period: 42 ± 4 h

Number of animals:

Not applicable. The test was performed in triplicates 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 skin surface with phosphate buffered saline.
- Time after start of exposure: 60 min
- Post-treatment incubation period: 42 ± 4 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed about 42 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

Results 1

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: mean value of the solvent controls (PBS). Time point: 60 min. Reversibility: other: not applicable. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

4

Remarks on result:

other:

Remarks:

Basis: other: mean value of positive controls (5% SDS). Time point: 60 min. Reversibility: other: not applicable. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

96

Remarks on result:

other:

Remarks:

Basis: other: mean value of the test item. Time point: 60 min. Reversibility: other: not applicable. (migrated information)

Any other information on results incl. tables

Results:

test substance		tissue 1	tissue 2	tissue 3	mean	SD
NC	mean OD570	2.404	2.415	2.401	2.407
	viability [% of NC]	99.9	100.3	99.8	100	0.29
test item	mean OD570	2.281	2.049	2.589	2.306
	viability [% of NC]	94.8	85.1	107.6	96	11.27
PC	mean OD570	0.096	0.09	0.105	0.097
	viability [% of NC]	4	3.7	4.4	4	0.32

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified