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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:
weight of evidence
Study period:
2009-09-08 to 2009-11-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study with acceptable restrictions Minor deviations from the guideline OECD 431 (2015) occurred: - according to the guideline complete information for the specific RhE model used including e.g. viability, barrier function, morphology, and tissue quality control should be stated. This is missing in this study report. - colour interference test was not conducted - acceptability criteria are different from guideline - MTT and isopropanol concentrations used were not stated
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
adopted 2004-04-13
Deviations:
yes
Remarks:
, MTT concentration used was not stated; supporting information for the specific skin model used incl. its validity was not stated
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
adopted 2008
Deviations:
yes
Remarks:
, MTT concentration used was not stated; supporting information for the specific skin model used incl. its validity was not stated
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-10-22

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Physical state: solid/ brown, dark
- Storage condition of test material: room temperature

Test animals

Details on test animals and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.

Test system

Vehicle:
other: highly de-ionized water
Controls:
other: Control (50 µL highly de-ionised water) skin cultures were used.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μL of the test item

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL of the vehicle
Duration of treatment / exposure:
3 minutes and 1 hour
Observation period:
not applicable
Number of animals:
Number of skin tissue replicates per dose and per exposure time:
Test item: duplicates
Negative control: duplicates
Positive control: duplicates
Details on study design:
THREE DIMENSIONAL HUMAN EPIDERMIS MODEL:
- EpiDerm™ 200 kit were obtained from MatTek Corporation, Ashland MA, USA
- EpiDermTM tissues (surface 0.6 cm²) 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.

DIRECT MTT REDUCTION:
- approximately 25 μL bulk volume of the test item was added to 0.9 mL of the MTT solution.
- mixture was incubated in the dark at about 37 °C for 55 to 65 minutes.
- negative control (50 μL of vehicle) was tested concurrently.
- if the MTT solution colour or, in case of water-insoluble test items the border to the water-phase, turned blue / purple, the test item was presumed to directly reduce MTT.
- in case that direct MTT reduction occurred, one freeze-killed control tissue per exposure time was treated with, each, the test article and the negative control, in the same way as described below.

CORROSION TEST:
- tissues were kept in the refrigerator.
- at least 1 hour but not more than 1.5 hours before test-item application, tissues were transferred to 6-well plates with assay medium and preconditioned in the incubator at 37 °C.
- preincubation medium was replaced with fresh medium before application.
- two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator) and test material, negative control (50 μL highly de-ionized water) and positive control (50 μL 8 n potassium hydroxide) were used.
- for the test item, the vehicle was applied first followed by the test material.
- control tissues were concurrently applied.
- tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application.
- tissues were incubated in MTT solution for 3 hours.
- after incubation, tissues were washed with PBS
- formazan produced by the tissues was extracted with isopropanol over night at room temperature.
- 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.

DATA EVALUATION.
- Principle: the OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the negative control (percent of control) is used for evaluating whether or not a test material is corrosive.
- Calculation of individual and mean optical densities: the individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way is calculated.
- Tissue viability: the quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 negative control value in percent for each exposure time.

EVALUATION OF RESULTS:
Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 minutes treatment compared to the negative control tissues. A chemical is considered as "corrosive" or "non-corrosive" according to table 1 in the field "Any other information on materials and methods incl. tables" below.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: relative tissue viability(%)
Basis:
mean
Time point:
other: after 3 minutes incubation
Score:
123
Reversibility:
no data
Irritation parameter:
other: relative tissue viability(%)
Basis:
mean
Time point:
other: after 60 minutes incubation
Score:
104
Reversibility:
no data
Irritant / corrosive response data:
3 minute exposure: the relative viability increased (123 %) after exposure to the test item (threshold for corrosivity: 50 %)
1 hour exposure: the relative viability increased (104 %) after exposure to the test item (threshold for corrosivity: 15 %)
The test item is not considered to be corrosive to the skin.

Any other information on results incl. tables

HISTORICAL DATA

Table 1: Historical range of negative control (OD570)

Exposure time

Historical period

Mean OD

Standard deviation

Mean + 2 Standard deviation

Mean – 2 Standard deviation

3 minutes

Mar 2008 – Nov 2009

1.891

0.257

2.40

1.38

60 minutes

Mar 2008 – Nov 2009

1.897

0.230

2.36

1.44

Table 2: Historical range of positive control (OD570)

Exposure time

Historical period

Mean OD

Standard deviation

Mean + 2 Standard deviation

Mean – 2 Standard deviation

3 minutes

Mar 2008 – Nov 2009

0.408

0.110

0.63

0.19

60 minutes

Mar 2008 – Nov 2009

0.189

0.051

0.29

0.09

Table 3: Viability (%)

Exposure time

Historical period

Mean %

Standard deviation

Mean + 2 Standard deviation

Mean – 2 Standard deviation

3 minutes

Mar 2008 – Nov 2009

21.84

5.21

32.26

11.42

60 minutes

Mar 2008 – Nov 2009

9.97

2.11

14.18

5.75

RESULTS

Table 4: Result of corrosion test with the test item

Test article

 

Exposure: 3 minutes

Exposure: 1 hour

tissue 1

tissue 2

mean

tissue 1

tissue 2

mean

Negative control

mean OD570

1.2662

1.4622

1.3642

1.6587

1.7942

1.7264

viability [% of negative control]

92.8

107.2

100

96.1

103.9

100

Test item

mean OD570

1.6172

1.7452

1.6812

1.7767

1.8022

1.7894

viability [% of negative control]

118.5

127.9

123

102.9

104.4

104

Positive control

mean OD570

0.3322

0.3452

0.3387

0.2282

0.1947

0.2114

viability [% of negative control]

24.3

25.3

25

13.2

11.3

12

The test item is not able to reduce MTT directly.

Applicant's summary and conclusion

Interpretation of results:
other: not corrosive to the skin
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The substance is not corrosive to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as corrive to skin.
Executive summary:

In this in vitro study the substance was tested for its skin corrosive potential according to the OECD guideline 431 (2004) by the means of the human skin model test using EpiDerm™. Duplicates of EpiDerm™ tissues were exposed to either the substance (25 µL) with the vehicle (25 µL), the positive control (8 n potassium hydroxide; 50 µL) or the negative control (highly de-ionised water; 50 µL) for each of two different exposure periods: 3 minutes and 1 hour. Afterwards the test and the control items were rinsed off the tissues, and a 3 hour incubation period with MTT solution followed.

After incubation, tissues were washed and the formazan produced by the tissues was extracted with isopropanol overnight. This was followed by the measurement of the optical density (OD570).

After an exposure period of 3 minutes the relative tissue viability increased to 123 % (threshold for corrosivity: 50 %). After an exposure period of 1 hour the relative tissue viability increased to 104 % (threshold for corrosivity: 15 %). Therefore, the substance was not considered to be corrosive. The controls confirmed the validity of the study.

According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is not corrosive to the skin.