Condensation products of N-(2-aminoethyl)octadecanamide and N-(2-aminoethyl)hexadecanamide with sebacic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 March to 23 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Condensation products of N-(2-aminoethyl)octadecanamide and N-(2-aminoethyl)hexadecanamide with sebacic acid

EC Number:

945-989-8

IUPAC Name:

Condensation products of N-(2-aminoethyl)octadecanamide and N-(2-aminoethyl)hexadecanamide with sebacic acid

Test material form:

solid: crystalline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Kyoeisha Chemical Co., Ltd
- Lot No.of test material: 6021874
- Expiration date of the lot/batch: 18 February 2019
- Purity test date: 3 October 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (Ambient). In original container as supplied by the Sponsor. Container was kept tightly closed and away from heat or sunlight
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable, the test item was administered as supplied
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, cornea surface wetted with sterile water and test item added

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: obtained from human derived non-transformed epidermal keratinocytes

Justification for test system used:

This study addresses the human health endpoint skin irritation. It uses reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin. The use of reconstructed human epidermis (RhE) is recommended by the OECD and other regulatory authorities. SkinEthicTM RHE model has been validated and is part of the OECD validated reference methods (VRMs) and is a recommended model for conducting in vitro skin irritation studies. The results of the study are believed to be of value in predicting the potential of inducing skin irritation by the test item in humans.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The SkinEthicTM RHE model
- Tissue batch number: 18-RHE-029
- Production date:
- Shipping date:
- Delivery date:
- Date of initiation of testing: 10 March 2018 (Experimental start)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Incubated at room temperature for 42 minute exposure.
- Temperature of post-treatment incubation (if applicable): After washing and drying, tissues were incubated in a 6-well plate containing 2 mL growth medium at 37 ± 1°C in 5 ± 1% CO2 in a humidified incubator for 42 hours (± 60 min).

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After exposure, tissues were rinsed and then dried with cotton buds. The test item was removed by rinsing 25 times in a constant stream of DPBS at 5-8 cm distance from the insert to remove test item from the epidermal surface. Mesh (applied on negative and positive control tissues) was removed by washing. The bottom of tissue inserts were dried on a sterile absorbent paper (Kim wipes) for 1-2 seconds. The surface of the stratum corneum was gently swept up using both ends of a cotton tip (5-6 turns per end). After washing, inserts were transferred to holding plates (at room temperature) containing 300 µL maintenance medium.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 180 minutes at 37 ± 1°C in 5 ± 1% CO2 in a 95% humidified incubator.
- Spectrophotometer: SynergyHT Microplate Reader
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not spacified
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: yes, cell viability in tissues of the negative control, test item and positive control was compared against laboratory historical data.
- Barrier function: Not specified
- Morphology: obtained from human derived non-transformed epidermal keratinocytes which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin,
- Contamination: None
- Reproducibility: within acceptable parameters

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (weight with unit): 16 ± 2 mg of test item/0.5 cm2

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL/0.5 cm2 sterile dulbecco’s phosphate buffered saline (DPBS)

POSITIVE CONTROL
- Amount(s) applied (volume): 16 µL/0.5 cm2 of 5% sodium dodecyl sulphate (5% aq.)
- Concentration (if solution): 5% aq

Duration of treatment / exposure:

Incubated at room temperature for 42 minute exposure.

Duration of post-treatment incubation (if applicable):

After washing and drying, tissues were incubated in a 6-well plate containing 2 mL growth medium at 37 ± 1°C in 5 ± 1% CO2 in a humidified incubator for 42 hours (± 60 min).

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

One experiment

Value:

100

Negative controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

one experiment

Value:

110.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

one experiment

Value:

1.5

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: The test item did not produce direct MTT reduction when compared to the concurrent negative control (Maintenance medium).
- Colour interference with MTT: The results of the color interference test showed no interference in optical density due to the test item.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, the classification for Reaction mass of N, N’-bis [2-(octadecanoylamino) ethyl] decanediamide and N, N’-bis [2-(hexadecanoylamino) ethyl] decanediamide is as follows:

Globally Harmonized System of Classification and : No Category (Non Skin Irritant)
Labelling of Chemicals