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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
Study period:
19 - 22 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EU-Method B.40 BIS (in vitro skin corrosion: human skin model test)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany

Test material

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

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Source strain:
other: EpiDermTM; reconstructed three-dimensional human epidermis (EPI-200-SCT)
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST METHOD:
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.

ADAPTATION TO CELL CULTURE CONDITIONS:
Upon receipt, tissues were transferred into 6-well plates containing 900 µL pre-warmed assay medium per well and preincubated in a humidified incubator for at least 1 h (37 ± 1 °C, 5% CO2) before use.

INCUBATION CONDITIONS (INCUBATOR):
- Temperature (°C): 37 ± 1
- CO2 gas concentration (%): 5
- Humidity: maximum

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE :
- Model used: EpiDermTM EPI-200-SCT
- Tissue batch number(s): 25822
- Delivery date: 20 June 2017

TEMPERATURE USED FOR TEST SYSTEM :
- Temperature used during treatment / exposure: room temperature (3 min exposure) and 37 +/- 1 °C, 5.0 +/- 0.5% CO2 (1 h exposure)
- Temperature of post-treatment incubation (if applicable): 37 +/- 1 °C, 5.0 +/- 0.5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS :
-Volume and number of washing steps: thoroughly rinsed with Dulbecco's phosphate-buffered saline and blotted with sterile cellulose tissue
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

CELL VIABILITY MEASUREMENTS:
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, tissues were incubated in 300 µL pre-warmed MTT solution for 3 h at 37 ± 1 °C and 5% CO2. After aspiration of the MTT solution, tissues were washed 3 times in phosphate buffered saline followed by tissue drying. Extraction of the formazan product was carried out in 2 mL isopropanol. The optical density was measured at 570 nm wave length in a plate spectrophotometer.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE :
- MTT concentration: 5 mg/mL
- Incubation time: 3 h, 37 +/- 1 °C, 5 +/- 0.5 % CO2
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA:
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 25 mg

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8N
Duration of treatment / exposure:
3 and 60 min
Duration of post-treatment incubation (if applicable):
1 h
Number of replicates:
The test was performed in duplicates for each test or control group and treatment period (3 and 60 min).

Test animals

Species:
other: in vitro system

Test system

Type of coverage:
other: in vitro system

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
test item
Run / experiment:
3 min
Value:
99.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
test item
Run / experiment:
60 min
Value:
107.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
positive control
Run / experiment:
3 min
Value:
19.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
positive control
Run / experiment:
60 min
Value:
7.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no, as assessed in a suitable pre-experiment
- Colour interference with MTT: no, as assessed in a suitable pre-experiment

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD 1.8 (3 min) and 1.6 (1 h) (criterion: OD ≥ 0.8 and ≤ 2.8)
- Acceptance criteria met for positive control: yes, % tissue viability after 1 h: 7.8 (criterion: < 15%)
- Range of historical values if different from the ones specified in the test guideline: values for negative control and for positive control were within the range of historical data of the test facility

The values of the negative control met the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals, thus showing the quality of the tissues. The positive control has met the validity criterion too, thus ensuring the validity of the test system. For these reasons, the result of the test is considered valid.

Any other information on results incl. tables

Table 1: Absorbance values blank isopropanol (OD 570nm)

 

Replicate

1

2

3

4

5

6

 

Mean 0.038

Absorbance

0.038

0.037

0.039

0.039

0.038

0.038

Replicate

7

8

9

10

11

12

Absorbance

0.039

0.038

0.038

0.039

0.038

0.037

 

Table 2: Absorbance Values (OD 570nm) for negative control, test item and positive control

 

Incubation

Negative Control

Test Item

Positive Control

 

Tissue 1

Tissue 2

Tissue 1

Tissue 2

Tissue 1

Tissue 2

 

3 min

1.797

1.826

1.830

1.822

0.380

0.385

1.853

1.786

1.761

1.813

0.381

0.384

1.791

1.776

1.771

1.818

0.378

0.385

 

1 h

1.602

1.600

1.741

1.720

0.167

0.157

1.596

1.607

1.767

1.679

0.165

0.156

1.597

1.618

1.722

1.686

0.166

0.154

 

Table 3: Mean Absorbance Values of the 3 min Experiment

 

Designation

Negative Control

Test Item

Positive Control

Mean – blank (tissue 1)

1.776

1.749

0.342

Mean – blank (tissue 2)

1.758

1.780

0.347

Mean of the two tissues

1.767

1.764

0.344

RSD

0.7%

1.2%

1.0%

 

Table: 4: Mean Absorbance Values of the 1 h Experiment

 

Designation

Negative Control

Test Item

Positive Control

Mean – blank (tissue 1)

1.560

1.705

0.128

Mean – blank (tissue 2)

1.570

1.657

0.118

Mean of the two tissues

1.565

1.681

0.123

RSD

0.5%

2.0%

6.0%

 

  Table 5: % Tissue Viability

 

Test Item

Positive Control

Incubation

99.9 %

19.5 %

3 min

107.4 %

7.8 %

1 h

 

Table 6: Historical Data

 

Parameter

Optical Density Negative Control

Optical Density Negative Con- trol

% Tissue viability Positive Control

% Tissue viability Positive Control

Incubation Time

3 min.

1 h

3 min.

1 h

Mean

1.956

1.905

25.1 %

12 %

Standard Deviation

0.275

0.220

6.8 %

3.8 %

Range

1.197 - 3.077

1.377 - 2.571

9.6 - 57.3 %

4.1 - 24.2 %

Study17051601G820

 

1.767

 

1.565

 

19.5

 

7.8

Applicant's summary and conclusion

Interpretation of results:
other: not corrosive
Conclusions:
There is regulatory acceptance in the EU that a substance can be considered corrosive based on a positive result in the human epidermis model test (OECD 431). Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant. Therefore, the result obtained needs to be supported by additional data in order to conclude the hazard assessment and determination of the classification and labelling.