Reaction product of Fatty acids, C18 alkyl with amines, polyethylenepoly-tetraethylenepentamine fraction

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

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-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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

High moecular weight adducts of Fatty acids, C16-18 sat. C18 unsat., linear, dimers, and trimers, with Amines, polyethylenepoly-, triethylenetetramine fraction

IUPAC Name:

High moecular weight adducts of Fatty acids, C16-18 sat. C18 unsat., linear, dimers, and trimers, with Amines, polyethylenepoly-, triethylenetetramine fraction

Constituent 2

Reference substance name:

Higher molecular weight adducts of C18 Fatty acids linear (unsat & sat) with amines,polyethylenepoly-, tetraethylenepentamine fraction

IUPAC Name:

Higher molecular weight adducts of C18 Fatty acids linear (unsat & sat) with amines,polyethylenepoly-, tetraethylenepentamine fraction

Constituent 3

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

Constituent 4

Reference substance name:

lower molecular weight adducts of C18 Fatty acids linear (unsat & sat) amines,polyethylenepoly-, tetraethylenepentamine fraction

Molecular formula:

for example : C26H53N5O, C26H55N5O...

IUPAC Name:

lower molecular weight adducts of C18 Fatty acids linear (unsat & sat) amines,polyethylenepoly-, tetraethylenepentamine fraction

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report):TOFA DimerFA TEPA PAA
- Physical state:Yellow-red, transparent, viscous liquid
- Analytical purity: 9% free amine
- Storage condition of test material:Room temperature (15°C to 30°C)
-Sponsor batch: BB000649V1

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 vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean value, 3 minute exposure

Value:

66

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean value, 1 hour exposure

Value:

48

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Other effects / acceptance of results:

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.

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 EpiDerm^TM test, in accordance with OECD Test Guideline 431 and EU Method B.40 and in compliance with GLP.

Duplicate EpiDerm^TM 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 EpiDerm^TM.