Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31/07/2017 - 17/11/2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

Method B.40b

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid

IUPAC Name:

Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid

Test material form:

solid: bulk

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0001230562
- Expiration date of the lot/batch: 08 June 2019
- Purity test date: 100% (UVCB)


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: Not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None

In vitro test system

Test system:

human skin model

Remarks:

Normal human epidermal keratinocytes cells (EpiDermTM Tissue)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Justification for test system used:

The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium.

This human skin model is advantageous for the study of dermal corrosivity - Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C for 3 mins and 60 mins.
- Temperature of post-treatment incubation (if applicable): 37 °C for 3 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1 washing stage, 2 blotting stages.
- Observable damage in the tissue due to washing: N/A
- Modifications to validated SOP: N/A

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570nm (OD570)
- Filter: N/A
- Filter bandwidth: N/A

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 runs per time point per type (i.e. test substance, positive control, negative control).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 0 mg of test substance

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 mg of test substance

Duration of treatment / exposure:

3 mins or 60 mins

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2 per time point (3 mins or 60 mins) per type (e.g. test substance, positive control, negative control).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative control (3 mins)

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative control (60 mins)

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive Control (3 mins)

Value:

4.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive Control (60 mins)

Value:

3.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 5

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item (3 mins)

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 6

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item (60 mins)

Value:

84.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: N/A
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: No

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin based on the results (98% cell viability at 3 mins and 84.1% cell viability at 60 mins).

Executive summary:

The skin corrosion study investigates the potential for Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid to cause skin corrosion in vitro. The study was performed to GLP standards and is compliant with the Guideline for the Testing of Chemicals No. 431 (In vitro skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method) and EU Method B.40 (In vitro skin corrosion: Transcutaneous Electrical Resistance Test (Ter)) with no deviations. Using a human skin model, EpiDerm tissue (the target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium) the test substance was assessed for its potential to cause in vitro skin corrosion based on a MTT colourmetric assay. The results shown that the test substance was non-corrosive over both 3 mins and 60 mins time period, as cell variability was only reduced to 98% and 84.1%, respectively. This experimentation was in the presence of a suitable negative and positive control which allowed the outcome of the test substance on cell viability to be validated (Cell viability for the negative control was 100 % (3 mins and 60 mins) and for the positive control was 4.6% (3 mins) and 3.9 % (60 mins)). The experiment also showed that the result was not influenced by a direct reduction or colour interference with MTT from the test substance.