Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
other: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
Justification for read-across is given in Section 13 of IUCLID.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
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.
Vehicle:
other: minimally moistened with PBS
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM; reconstructed three-dimensional human epidermis (EPI-200).
- Source: MatTek Corporation, Ashland MA, USA.

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
- Temperature: 37 ± 1 °C.
- CO2 gas concentration: 5 %.
- Humidity: 90 - 95 %.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing steps: the test item was washed from the skin surface with phosphate buffered saline.
- Time after start of exposure: 60 min.

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 wavelength in a plate spectrophotometer.

DECISION CRITERIA: a chemical is considered as irritant when the tissue viability is less than or equal to 50 % after the exposure and post- treatment incubation period. If the tissue viability after exposure and post-treatment incubation period is above 50%, the test chemical may be considered as non-irritant.

ACCEPTANCE CRITERIA
Negative control (NC): the absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.
Positive control (PC): 5 % SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20 % is acceptable.
Tissue variability: For every treatment, 3 tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD is ≤ 20 %.
Control samples:
yes, concurrent no treatment
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µl bulk volume (about 15 mg).
- Concentration: 50 % (v/v).
- Area of exposure: 0.6 cm²

VEHICLE
- Amount(s) applied: 25 µl.
- Concentration: 50 %.
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 ± 4 h
Number of replicates:
the test was performed in triplicates for each treatment and control group.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
96
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean value of three replicates

Any other information on results incl. tables

Table: cell viability 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:
other: not classified as skin irritant according to the CLP Regulation (EC) No.1272/2008
Conclusions:
Not irritating to the skin; cell viability = 96 %.
Executive summary:

The irritation potential of the category member substance to the skin was assessed in-vitro according to the OECD Guideline 439. The substance was applied topically to a three-dimensional human skin model, comprising a reconstituted epidermis with a functional stratum cornneum. Irritation was identified by the ability of the substance to produce a decrease in cell viability after 1 hour of exposure and 42 hours post-treatment incubation, quantified by the formazane production. For this reason, the tissues, after the removal of the substance, were incubated with MTT medium and the optical density of the extracted formazan was measured at 570 nm. Negative and positive controls run in parallel; the % photometric absorption of test item and positive control was calculated by comparison with the negative control.

Cell viability of the tissues treated with the test substance was 96 % (mean value of three replicates). These values are laying above the threshold for irritation.

The category member substance is not considered to be irritating to the skin.