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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
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):Pentaerythritol, olig. react. prod. with 1-chloro-2,3-epoxypropane, react. prod. with acrylid acid
- Physical state:liquid, viscous
- Analytical purity: 100% UVCB
- Lot/batch No.: Mischcharge aus G44/082/11 und G44/085/11

Test animals

Species:
other: EpiDerm TM Skin Corrosivity Tes
Strain:
other: in vitro

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): a single topical application of 50 μL (corrosion test) or 30 μL (irritation test)
Duration of treatment / exposure:
3 min and 1 hour(s)
Details on study design:
TEST SYSTEM
The EpiDermTM 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 multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Tissue model: Epi-200
Origin: 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.
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).
The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour afterstart 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.
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 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.

CONTROLS
Negative control (NC): De-ionized water (corrosion test); PBS, sterile (irritation test)
Positive control (PC): 8-n potassium hydroxide solution (Sigma-Aldrich, Munich, Germany) for the corrosion test
5% (w/v) sodium dodecyl sulfate (SDS, Sigma, Germany) in deionized water, sterile for the irritation test

Results and discussion

Any other information on results incl. tables

Corrosion test

 

 

Exposure: 3 min

Exposure: 1h

Test substance

 

Tissue 1

Tissue 2

mean

Tissue 1

Tissue2

mean

NC

mean OD570

 

1.882

1.948

1.915

1.840

1.838

1.839

Viability    [% of NC]

98.3

101.7

100

100.1

99.9

100

12/0027-1

mean OD570

 

1.894

1.782

1.838

1.816

1.850

1.833

Viability    [% of NC]

98.9

93.1

96

98.7

100.6

100

PC

mean OD570

 

0.330

0.376

0.353

0.139

0.193

0.166

Viability    [% of NC]

17.2

19.6

18

7.6

10.5

9

 

Irritation test

Test substance

 

Tissue 1

Tissue 2

Tissue 3

Mean

SD

NC

mean OD570

 

2.036

1.728

2.221

1.995

 

Viability    [% of NC]

102.0

86.6

111.3

100

12.49

12/0027-1

mean OD570

 

1.781

1.496

1.403

1.560

 

Viability    [% of NC]

89.3

75.0

70.3

78

9.88

PC

mean OD570

 

0.058

0.063

0.058

0.060

 

Viability    [% of NC]

2.9

3.2

2.9

3

0.15

Applicant's summary and conclusion