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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
Study period:
28th June 2012 to 8th November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to GLP and relevant testing guidelines.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

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
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
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and tetraethylenepentamine
EC Number:
500-289-8
EC Name:
Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and tetraethylenepentamine
IUPAC Name:
Reaction product of Fatty acids, C18 alkyl with amines, polyethylenepoly-tetraethylenepentamine fraction
Constituent 2
Reference substance name:
TOFA_DimerFA_TEPA_PAA
IUPAC Name:
TOFA_DimerFA_TEPA_PAA
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): TOFA_DIMERFA_TEPA_PAA
- Physical state: Yellow liquid with a brown hue
- Analytical purity: 100%
- Lot/batch No.: BB000649V1
- Expiration date of the lot/batch: 31 March 2013
- Storage condition of test material: When not in use the test article was stored in a sealed container in the dark, at 15 to 25°C.

Test animals

Species:
other: Not applicable - in vitro study
Strain:
other: Not applicable - in vitro study
Details on test animals or test system and environmental conditions:
Not applicable - study conducted using the in vitro EpiDerm test system.

Test system

Type of coverage:
not specified
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
In order to assess the potential non-specific reduction of the test article, 40 μL of test article was added to 0.3 mL of 1.0 mg/mL MTT.

During the EpiDerm test, approximately 50 mg of test article was applied to each tissue.

Further tissues were concurrently treated with 40 μL distilled water (negative control) and with 40 μL 8N potassium hydroxide (positive control).
Duration of treatment / exposure:
3 minutes and 1 hour.
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
In order to assess the potential non-specific reduction of the test article, 40 μL of test article was added to 0.3 mL of 1.0 mg/mL MTT and the colour change was assessed after three hours.
An increase in optical density was noted over the control (untreated MTT solution) therefore additional controls were included to enable assessment of the potential impact of the non-specific reduction of MTT by residual test article following the wash-off procedure.
To evaluate whether residual test article was binding to the tissue, a functional check (using freeze-killed control tissue) was performed. Freeze-killed tissues were prepared by placing untreated EpiDerm constructs in the -20°C freezer overnight. The freeze-killed tissues were allowed to thaw once at room temperature and placed back in the freezer until required.

On the day of receipt EpiDermTM tissues were placed in a refrigerator. Ninety minutes before starting the assay, the tissues were transferred to 6-well plates containing the assay medium. The test was performed on a total of four tissues per test article, 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. Sufficient test article was applied to cover the outer layer of the tissue. The cryovials containing the test article were weighed both prior to and post treatment and an average of approximately 50 mg of test article was applied to each tissue.
Further tissues were concurrently treated with 40 μL distilled water (negative control) and with 40 μL 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.

Once all tissues had been rinsed, they were transferred to wells containing 300 μL of 1 mg/mL MTT-medium and were incubated for 3 hours at 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 article was classified according to the remaining cell viability obtained after test article treatment with either of the two treatment times.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: Skin viability
Basis:
mean
Time point:
other: 3 minute exposure
Remarks on result:
other: 66% skin viability
Irritation parameter:
other: Skin viability
Basis:
mean
Time point:
other: 1 hour exposure
Remarks on result:
other: 48% skin viability
Irritant / corrosive response data:
Skin viability after a three minute or one hour exposure to the test article was 66% and 48%, respectively.
Skin viability after a three minute or one hour exposure to the positive control article was 22% and 12%, respectively, demonstrating appropriate performance of the assay.
Other effects:
No additional information.

Any other information on results incl. tables

Three-minute exposure period

Test substance

OD540

Mean

Tissue Mean

Difference between initial and freeze-killed tissues

% variability

% survival

Negative

1.961

1.996

2.025

1.994

1.922

1.272

-7.8

100

Negative

1.847

1.842

1.859

1.849

Test article

1.508

1.465

1.506

1.493

1.467

-3.6

66

Test article

1.430

1.449

1.444

1.441

Test article#

0.212

0.205

0.202

0.206

0.195

11.0

Test article#

0.177

0.191

0.183

0.184

Positive

0.358

0.375

0.377

0.370

0.415

-24.304

22

Positive

0.466

0.454

0.460

0.460

# Freeze-killed tissues

One-hour exposure period

Test substance

OD540

Mean

Tissue Mean

Difference between initial and freeze-killed tissues

% variability

% survival

Negative

2.037

2.141

2.148

2.109

1.903

0.923

-24.2

99

Negative

1.685

1.701

1.705

1.697

Test article

1.421

1.302

1.246

1.323

1.326

0.5

48

Test article

1.344

1.306

1.339

1.330

Test article#

0.424

0.414

0.405

0.414

0.404

5.1

Test article#

0.398

0.396

0.387

0.393

Positive

0.182

0.199

0.204

0.195

0.238

-44.606

12

Positive

0.278

0.274

0.292

0.282

# Freeze-killed tissues

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: not specified
Conclusions:
Under the conditions of this study and based on the results, the test article, TOFA_DimerFA_TEPA_PAA, was not corrosive to skin in the in vitro skin model EpiDerm.
Executive summary:

The potential of TOFA_DimerFA_TEPA_PAA to cause skin corrosion was evaluated using the in vitro EpiDermTM test, in accordance with OECD Test Guideline 431 and EU Method B.40 and in compliance with GLP.

Duplicate EpiDermTM inserts were treated with TOFA_DimerFA_TEPA_PAA, 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 (PBS) and cell viability was assessed using the MTT assay. The skin corrosivity potential was assessed 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 article was 66% and 48%, respectively. Skin viability after a three minute or one hour exposure to the positive control article was 22% and 12%, respectively, demonstrating appropriate performance of the assay.

Under the conditions of this study, the test article, TOFA_DimerFA_TEPA_PAA, was not corrosive to skin in the in vitro skin model EpiDermTM.