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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 to 18 August 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to relevant testing guidelines, with no deviations.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 68082-29-1
- Physical state: Yellow liquid with a brown hue
- Analytical purity: 100%
- Lot/batch No.: BB001030V1
- Expiration date of the lot/batch: 30th May 2013
- Storage condition of test material: When not in use the test article was stored in a sealed container, at 15 to 25ºC, in the dark.

In vitro test system

Test system:
other: Reconstructed Human Epidermis Test Method.
Remarks:
(EPI-200)
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Details on animal used as source of test system:
not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
A three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum was used. Human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells were present under a functional stratum corneum. The stratum corneum was 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. The magnitude of viability is quantified using MTT and measuring the optical density (OD) of the extracted (solubilized) dye from each tissue.
In order to assess the potential of tissue staining of the test article, 21 mg of the test article was added to 0.3 mL deionised water and the colour change was assessed after incubation for 60 minutes, at 37°C, 5% CO2. No staining was observed and the test article did not dissolve into the solution.

In order to assess mesh compatibility with the test article, a nylon mesh was placed on to a glass microscope slide and 30 mg of the test article was added. After exposure for 60 minutes, at 37°C, 5%CO2, the mesh was microscopically examined for any signs of interaction No interactions with the mesh were observed.

On the day of receipt EpiDermTM tissues were transferred to multi-well plates containing the appropriate medium and then incubated overnight. Three tissues per test article, negative control and positive control were treated by application of 30 μL of the negative control, 30 μL of the positive control and approximately 36 mg of test article onto the surface of the tissues. The tissues were incubated at 37°C, 5 % CO2 for a 35 minute period. The plates were then removed from the incubator and placed into a sterile hood until the 60 minute treatment period was complete for each tissue. Following treatment, substances were removed by washing the tissues. The tissues were then placed on the appropriate medium and incubated for 42 hours.
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 freezekilled tissues were allowed to thaw once at room temperature and placed back in the freezer until required.
At the end of the 42 hour incubation period, tissue viability was assessed by MTT
assay.
Once all tissues were rinsed, they were transferred to wells containing 300 μL of 1 mg/mL MTT-medium and were incubated for 3 hours (37°C, 5% CO2). After incubation, any resultant colour was extracted with 2 mL isopropanol, at room temperature, by shaking for 2 hours.
Upon completion of the extraction, each tissue was pierced using a hypodermic needle so that the extract could run through the tissue. Once drained, the tissue was discarded and the extract mixed by pipette. Two x 200 μL aliquots of each resultant extract were placed into a 96-well plate for spectrophotometric determination of optical density at 570 nm using extraction solution as a blank. Tissue viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
16 μL or 16 mg
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 replicates for negative control.
3 replicates for positive control
3 replicates for the test item + 3 test item replicates for Freeze Killed tissue

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test article
Value:
5.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
negative control
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
positive control
Value:
10.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
No other effects reported.

Any other information on results incl. tables

Optical density values

Test Substance

OD570

Mean

Corrected mean

Corrected mean minus freeze-killed tissues

% Relative survival

Mean

SD

CV

Negative

Negative

Negative

1.359

1.499

1.292

1.401

1.487

1.337

1.380

1.493

1.314

1.380

1.493

1.314

 

 

 

0.067

0.032

0.137

98.9

107.0

94.2

100.0

6.48

6.48

Test article

Test article

Test article

0.207

0.156

0.285

0.193

0.153

0.274

0.200

0.154

0.280

0.200

0.154

0.280

4.8

2.3

9.8

5.6

3.82

67.80

Test article #

Test article #

Test article #

0.127

0.118

0.140

0.133

0.120

0.139

0.130

0.199

0.139

0.130

0.199

0.139

 

 

 

 

Positive

Positive

Positive

0.179

0.142

0.125

0.175

0.143

0.129

0.177

0.143

0.129

0.177

0.143

0.129

12.7

10.2

9.3

10.7

1.75

16.37

Blank

0.000

0.000

 

 

 

 

 

 

 

Blank#

-0.005

-0.001

 

 

 

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test article, TOFA_DimerFA_TETA_PAA, was identified as a skin irritant in the in vitro skin model EpiDermTM SIT (EPI-200).
Executive summary:

This study was conducted to determine whether the test article TOFA_DimerFA_TETA_PAA, causes dermal irritation in the in vitro skin model EpiDerm™ SIT (EPI-200).

EpiDerm™ SIT (EPI-200) inserts were treated with test article, negative control (phosphate buffered saline (PBS)) and positive control (5% w/v sodium dodecyl sulphate (SDS)) for 60 minutes. At the end of the treatment period, the tissues were washed with PBS and cell viability was assessed using the MTT assay. The skin irritation potential was classified according to the remaining cell viability obtained after test article treatment.

The group mean viability for the test article was 5.6%, for the negative control was 100% and for the positive control was 10.7%.

The test article, TOFA_DimerFA_TETA_PAA, was considered to be irritant (GHS category 2) to the in vitro skin model EpiDerm™ SIT (EPI-200)