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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:
4 February 2013 to 9 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) succinate
EC Number:
220-836-1
EC Name:
Bis(2-ethylhexyl) succinate
Cas Number:
2915-57-3
Molecular formula:
C20H38O4
IUPAC Name:
1,4-bis(2-ethylhexyl) butanedioate
Test material form:
other: colourless liquid
Details on test material:
- Name of test material (as cited in study report): Bis(2-ethylhexyl) Succinate
- Storage condition of test material: In a sealed container, at room temperature, in the dark

In vitro test system

Test system:
human skin model
Source species:
human
Cell source:
other: reconstructed human epidermal model that is grown from human-derived epidermal keratinocytes
Details on animal used as source of test system:
The model is a three-dimensional reconstructed human epidermis model consisting of human-derived epidermal keratinocytes.
Multiple layers of viable epithelial cells are present under a functional stratum corneum. The stratum corneum is multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevent the passage of material around the stratum corneum to the viable model tissue. The skin model was supplied free of contamination with bacteria, mycoplasma and fungi.

Pre-Test
-Assessment of Direct Test Item Reduction of MTT
In order to assess the potential non-specific reduction of the test material, 40 µL of test material was added to 0.3 mL of 1.0 mg/mL MTT and the colour change was assessed after three hours. There was no change in colour therefore the test material did not interact with MTT.

Main Test
Application of Test and Control Substances
On the day of receipt EpiDerm™ tissues were placed in a refrigerator. The next day, at least one hour before starting the assay, the tissues were transferred to 6-well plates with the assay medium, which was replaced immediately before the test was started. The test was performed on a total of four tissues per test material, negative and positive control, out of which two were used for a 3 minute application and two tissues were used for a 1 hour application. A volume of 40mL of the undiluted test material was added into the MILLICELL insert on top of the Epi-200 tissues. Further tissues were concurrently treated with 40mL distilled water (negative control) and with 40mL 8N potassium hydroxide (positive control). After the 3-minute or 1-hour contact periods, the tissues were washed with phosphate buffered saline (PBS) to remove residual material. The rinsed tissues were kept in 24-well plates (holding plates) until all tissues were dosed and rinsed.

Cell Viability Measurements Once all tissues had been rinsed, they were transferred to wells containing 300mL of 1 mg/mL MTT-medium and were incubated for 3 hours (37°C). After incubation, the tissues were washed with PBS and any resultant colour was extracted with 2 mL isopropanol overnight. The optical density of each resultant extract was determined spectrophotometrically at 570 nm and cell viability was calculated for each tissue as a percentage of the mean of the negative control tissue. Skin corrosivity potential of the test material was classified according to the remaining cell viability obtained after test article treatment with either of the two treatment times.

Data Evaluation
The OD values obtained for each test sample were used to calculate the percentage viability relative to the negative control, which is arbitrarily set at 100 %. The prediction of corrosivity associated with the EpiDerm™ model is:
1. The test material is considered to be corrosive to skin if the viability after a three minute exposure is less than 50 %, or if the viability after three minutes exposure is greater than or equal to 50 % and the viability after one hour is less than 15% .
2. The test material is considered to be non-corrosive to skin if the viability after a three minute exposure is greater than or equal to 50 % and the viability after a one hour exposure is greater than or equal to 15 %.
Justification for test system used:
The test has been scientifically validated and granted regulatory approval for the identification and classification of the corrosive potential of chemicals.
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
A volume of 40mL of the undiluted test material was added into the MILLICELL insert on top of the Epi-200 tissues.
Duration of treatment / exposure:
2 replicates: 3 minute exposure
2 replicates: 1 hour exposure
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2 replicates for each exposure duration (3 minute and 1 hour) and type (positive control, negative control, and treatment)

Test animals

Details on test animals or test system and environmental conditions:
EpiDerm™: Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum supplied by MatTek Corporation, Ashland, Massachusetts, USA.

Test system

Amount / concentration applied:
40 µL mg of the test material was applied topically.
Duration of treatment / exposure:
3 and 60 minutes.
Observation period:
3 hours
Number of animals:
Not applicable

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
3 minute exposure
Value:
13.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
1 hour exposure
Value:
16.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: OD570
Run / experiment:
Mean / 1 hour exposure
Value:
1.67
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: Max score 1.821

Any other information on results incl. tables

Skin viability after a three minute or one hour exposure to the test material was 93 % and 89 %, respectively.

Skin viability after a three minute or one hour exposure to the positive control article was 18 % and 11 %, respectively, demonstrating appropriate performance of the assay.

Table 1: Three Minute Exposure Period

Substance

OD570

Mean

Tissue mean

% viability

% survival

Negative

2.007

2.015

2.041

2.021

1.854

16.5

100

Negative

1.669

1.686

1.705

1.687

Test material

1.597

1.593

1.625

1.605

1.732

13.6

93

Test material

1.0853

1.862

1.860

1.859

Positive

0.354

0.342

0.361

0.352

0.330

12.7

18

Positive

0.307

0.304

0.311

0.307

 

 

Table 2: One Hour Exposure Period

Substance

OD570

Mean

Tissue mean

% viability

% survival

Negative

1.957

1.924

1.923

1.935

1.879

5.7

100

Negative

1.821

1.794

1.857

1.824

Test material

1.525

1.511

1.519

1.518

1.670

16.6

89

Test material

1.827

1.810

1.826

1.821

Positive

0.177

0.184

0.174

0.178

0.200

19.3

11

Positive

0.225

0.209

0.230

0.221

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study, the test material was considered not to be corrosive to the skin in the in vitro skin model EpiDerm™.
Executive summary:

The potential of the test material to be corrosive to skin was investigated in vitro, in a GLP study which was conducted in accordance with the standardised guidelines OECD 431 and EU Method B.40 bis.

During the study, duplicate

EpiDerm™ inserts were treated with test material, distilled water (negative control) and 8N potassium hydroxide (positive control) for 3 minutes and 1 hour. At the end of the treatment period, the tissues were washed with phosphate buffered saline and cell viability was assessed using the MTT assay. The skin corrosivity potential was classified according to the remaining cell viability obtained after test material treatment with either of the two treatment times.

Skin viability after a three minute or one hour exposure to the test material was 93 % and 89 %, respectively.

Skin viability after a three minute or one hour exposure to the positive control article was 18 % and 11 %, respectively, demonstrating appropriate performance of the assay.

Under the conditions of the study, the test material was considered not to be corrosive to the skin in the in vitro skin model EpiDerm™.