Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine

Skin irritation / corrosion

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-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

TETA – Fatty acids adducts (Mw 600-1000Da)

IUPAC Name:

TETA – Fatty acids adducts (Mw 600-1000Da)

Constituent 2

Reference substance name:

High molecular weight adducts of Fatty acids, C18-unsatd dimers and trimers with amines, polyethylenepoly-, triethylenetetramine fraction

IUPAC Name:

High molecular weight adducts of Fatty acids, C18-unsatd dimers and trimers with amines, polyethylenepoly-, triethylenetetramine fraction

Constituent 3

Reference substance name:

lower molecular weight adducts of Fatty acids, C18-unsatd dimers with amines, polyethylenepoly-, triethylenetetramine fraction

IUPAC Name:

lower molecular weight adducts of Fatty acids, C18-unsatd dimers with amines, polyethylenepoly-, triethylenetetramine fraction

Constituent 4

Reference substance name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

EC Number:

292-587-7

EC Name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

Cas Number:

90640-66-7

IUPAC Name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): TOFA_DimerFA_TETA_PAA
- Physical state: Yellow liquid with a brown hue.
- Analytical purity: 100%
- Lot/batch No.: BB001030V1
- Expiration date of the lot/batch: 30 May 2013
- Storage condition of test material: When not in use the test article was stored in a sealed container, at room temperature 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

Results

Results 1

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

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

negative control

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

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)