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Skin irritation / corrosion

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Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 August 2011 - 14 November 2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to guideline
other: EU Method B.46 (In vitro dermal irritation)
according to guideline
other: OECD Guideline 439 (In vitro dermal irritation)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
dodecane-12-lactam, manufacturing of, by-products from, distillation residues
EC Number:
dodecane-12-lactam, manufacturing of, by-products from, distillation residues
Test material form:
Details on test material:
- Name of test material: Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues
- Physical state: brown solid
- Lot/batch No.: AVRIL 2011
- Expiry date: 30 April 2012
- Composition of test material: UVCB
- Storage condition: at room temperature.

In vitro test system

Test system:
human skin model
Source species:
Cell type:
non-transformed keratinocytes
unchanged (no vehicle)
Details on test system:
EpiskinTM Model Kit (0.38 cm2 tissues) supplied by SkinEthic Laboratories, Lyon, France.
Medium and Incubation T°C: 37°C
Dates of experimental phase: from 13 September 2011 to 14 November 2011
Amount/concentration applied:
10 mg +/- 2 mg
Duration of treatment / exposure:
15 (+/- 1) minutes exposure period and a 42-hour recovery period, followed by rinsing.
Duration of post-treatment incubation (if applicable):
MTT-loading after a 42 h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells.
Number of replicates:
Not applicable.
Triplicate tissues for each timepoint and tested substance (test item, negative control, positive control)

Test animals

other: reconstructed human epidermis
other: not applicable
Details on test animals or test system and environmental conditions:
At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Excess D-PBS was removed by blotting the bottom of the tissue culture insert with absorbent paper. If necessary the epidermal surface was gently swept with a cotton-bud to remove excess D-PBS (without damaging the epidermis). If the test material was not removed, this was noted in the study file. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well and the plates were incubated at 37°C, 5% CO2 in a humidified incubator for 42 (± 1) hours.

Name: Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution.

Name: Phosphate-Buffered Saline (PBS).

- Optical density (OD) was measured between 540 and 595 nm.
Relative mean viability (%) = 100 x mean OD(test item) / mean OD(negative control)

Interpretation: see below

Results and discussion

In vitro

Irritation / corrosion parameter:
other: other: relative mean viability
Run / experiment:
15 min
Remarks on result:
no indication of irritation
Basis: mean. Time point: 15 min exposure + 42h expression. Reversibility: no data not applicable. Remarks: 100% = control. (migrated information)

Applicant's summary and conclusion

Interpretation of results:
not irritating
Migrated information Criteria used for interpretation of results: EU
In vitro, the test item was non-irritant to the skin.
Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item using the EpiskinTM reconstituted human epidermis model.

The study design is based on international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46) and thestudy was conducted in compliance with CIT’s standard operating procedures and the principles of Good Laboratory Practice.

Preliminary tests were performed to detect the ability of the test item to directly reduce MMT as well as its coloring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in one main test. The test item, the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature during 15 (± 1) minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 (± 1) hours at 37°C, 5% CO2in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay.

Relative viability values were calculated for each tissue and expressed as percentages of the negative control tissues viability which was set at 100% (reference viability).

In the preliminary test, the test item was presumed to have direct MTT reducing properties since the MTTsolution containing the test item changed color (brown color) when compared to the negative control. As a result, additional controls were performed on water-killed tissues in parallel to the main test.

The test item was found to have a coloring potential in the preliminary test since the water solution containing the test item changed color. As a result, additional controls were used in parallel to the main test for the evaluation of the non specific OD.

In the main test, following a 15-minute exposure and a 42-hour recovery period, the true relative mean viability of the tissues treated with the test item was 99.2%. This value being > 50%, the test item is therefore considered as non-irritant to skin.