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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EU) 640/2012
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-dimethyldodecanamide
EC Number:
221-117-5
EC Name:
N,N-dimethyldodecanamide
Cas Number:
3007-53-2
Molecular formula:
C14H29NO
IUPAC Name:
N,N-dimethyldodecanamide
Details on test material:
- Name of test material (as cited in study report): N,N Dimethyldodecane-1-amide
- Test-substance No.: 13/0555-1
- Physical state: liquid
- Analytical purity: N,N-Dimethyldodecanamide: 95.9 area-%; dodecanoic acid (lauric acid): 2.26 area-%; (for details see project No. AU134560-1)
- Lot/batch No.: 0009565072
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor.
- Homogeneity: The test substance was homogeneous by visual inspection.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes

Test animals

Species:
other: in vitro
Strain:
other: in vitro

Test system

Type of coverage:
other: in vitro
Preparation of test site:
other: in vitro
Vehicle:
other: in vitro
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Corrosion test: Fifty microliter (50 μL) of the undiluted liquid test substance was applied. Irritation test: Thirty microliter (30 μL) of the undiluted liquid test substance was applied
- Concentration (if solution): undiluted
Duration of treatment / exposure:
3 min and 1 hour(s)
Observation period:
direct measuremet of MTT reduction in corrosion test whereas in the irritation test washed tissues were incubated (42h postincubation).
Number of animals:
in vitro test
Details on study design:
TEST SYSTEM
The EpiDerm TM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in viva. The EpiDerm TM tissues (surface 0.6 cm2)
are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm 0) and commercially available as kits (EpiDerm TM 200), containing 24 tissues on shipping agarose.
Skin model: Epi-200
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

TEST PROCEDURE

Corrosion test:
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used. In addition, one killed tissue per exposure time was treated with the test substance and NC, respectively, in order to detect direct MTT reduction. Fifty microliter (50 μL) of the undiluted liquid test substance was applied using a pipette. Control tissues were concurrently applied with 50 μL of de-ionized water (negative control, NC) or with 50 μL of 8 n potassium hydroxide (positive control, PC) or test substance (killed tissue control, KC). The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours. After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Irritation test:
Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted liquid test substance was applied using a pipette. A nylon mesh was placed carefully onto the tissue surface afterwards. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. 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 tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of two tissue values
Run / experiment:
Corrosiontest 3min exposure
Value:
103
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of two tissue values
Run / experiment:
Corrosiontest 1 h exposue
Value:
104
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of three tissue values
Run / experiment:
Irritationtest
Value:
3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

RESULTS

 

Corrosiontest

 

Table3:IndividualandmeanOD570 values,individualandmeanviabilityvalues

 

 

Exposure:3min

Exposure:1hour

Test

substance

 

tissue1

tissue2

KC

mean

tissue1

tissue2

KC

mean

 

 

NC

meanOD570

2.112

2.101

0.111

2.106

1.931

2.006

0.093

1.968

viability[%ofNC]

100.3

99.7

-

100

98.1

101.9

-

100

 

 

13/0555-1

meanOD570

2.143

2.196

0.144

2.170

2.008

2.068

0.117

2.038

viability[%ofNC]

101.8

104.3

-

103

102.0

105.1

-

104

 

 

PC

meanOD570

0.398

0.468

-

0.433

0.100

0.097

-

0.098

viability[%ofNC]

18.9

22.2

-

21

5.1

4.9

-

5

 

 

Irritationtest

 

Table4:IndividualandmeanOD570 values,individualandmeanviabilityvaluesandstandarddeviations

 

Test

substance

 

tissue1

tissue2

tissue3

mean             SD

 

NC

meanOD570

2.377

2.370

2.580

2.443

viability

[%ofNC]

97.3

97.0

105.6

100

4.88

 

13/0555-1

meanOD570

0.066

0.072

0.085

0.074

viability

[%ofNC]

2.7

2.9

3.5

3

0.39

 

PC

meanOD570

0.060

0.080

0.070

0.070

viability

[%ofNC]

2.5

3.3

2.9

3

0.41

 

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information
Conclusions:
Corrosion Test: The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 103%, and it was 104% after an exposure period of 1 hour.
Irritation test: The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 3%.
Based on the observed results it was concluded, that N,N Dimethyldodecane-1-amide shows a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.
Executive summary:

The potential of N,N Dimethyldodecane-1-amide to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™).

For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiDerm™ skin corrosion/irritation test showed the following results:

The test substance is able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing (performed with corrosion test, only).

Corrosion test: The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 103%, and it was 104% after an exposure period of 1 hour.

Irritation test:

The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 3%.

Based on the observed results it was concluded, that N,N Dimethyldodecane-1-amide shows a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.