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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: 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
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Details on 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 are commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (25 min), 37°C (35 min)
- Temperature of post-treatment incubation (if applicable): 37°C

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength: 570 nm
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 ul

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 ul

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 ul

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 ul
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 tissues
Value:
3
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Test substance identification

 

Tissue 1

Tissue 2

Tissue 3

Mean

SD

CV [%]

NC

Mean OD570

1.995

2.178

2.097

2.090

 

 

Viability [% of NC]

95.5

104.2

100.3

100.0

4.4

4.4

Test substance

Mean OD570

0.057

0.061

0.068

0.062

 

 

Viability [% of NC]

2.7

2.9

3.2

3.0

0.3

8.9

PC

Mean OD570

0.052

0.051

0.049

0.051

 

 

Viability [% of NC]

2.5

2.5

2.4

2.4

0.1

2.6

NC, negative control

PC, positive control

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

In vitro test system

Test system:
human skin model
Source species:
human
Details on test system:
THREE-DIMENSIONAL HUMAN EPIDERMIS MODEL
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 are commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application. 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.
25 μL de-ionized water was applied first. Thereafter, a bulk volume of ca. 25 μL of the solid ground test material was applied with a sharp spoon and homogeneously distributed with the water.
Control tissues were concurrently treated with 50 μL of de-ionized water (NC) or with 50 μL of 8 N potassium hydroxide (PC).
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, 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 4 microtiter wells filled with isopropanol for each microtiter plate.

DATA EVALUATION:
Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with de-ionized water. A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 ul of solid ground material

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 ul

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 ul

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 ul
Duration of treatment / exposure:
3 min or 1 hour
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure
Value:
86.5
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure, experiment 1
Value:
21.1
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
variations in results, therefore experiment was repeated
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure, experiment 2
Value:
7.4
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid

Any other information on results incl. tables

Exposure period: 3 min

Test substance identification

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Mean OD570

1.986

2.031

2.008

 

 

Viability [% of NC]

98.9

101.1

100

1.6

1.6

Test substance

Mean OD570

1.825

1.651

1.738

 

 

Viability [% of NC]

90.0

82.2

86.5

6.1

7.1

PC

Mean OD570

0.237

0.251

0.244

 

 

Viability [% of NC]

11.8

12.5

12.1

0.5

4.1


 

Exposure period: 1 hour (Experiment 1)

Test substance identification

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Mean OD570

2.195

1.981

2.088

 

 

Viability [% of NC]

105.1

94.9

100.0

7.2

7.2

Test substance

Mean OD570

0.599

0.284

0.442

 

 

Viability [% of NC]

28.7

13.6

21.1

10.7

50.5

PC

Mean OD570

0.148

0.159

0.154

 

 

Viability [% of NC]

7.1

7.6

7.4

0.4

5.1

 

Exposure period: 1 hour (Experiment 2)

Test substance identification

 

Tissue 1

Tissue 2

Mean

SD

CV [%]

NC

Mean OD570

1.814

1.984

1.899

 

 

Viability [% of NC]

95.5

104.5

100.0

6.3

6.3

Test substance

Mean OD570

0.136

0.146

0.141

 

 

Viability [% of NC]

7.1

7.7

7.4

0.4

5.3

PC

Mean OD570

0.078

0.087

0.082

 

 

Viability [% of NC]

4.1

4.6

4.3

0.3

7.3

 NC, negative control

PC, positive control

 

Applicant's summary and conclusion