N,N'-methylenedistearamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 Jun 2018 to 22 Jun 2018

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)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-methylenedistearamide

EC Number:

203-657-3

EC Name:

N,N'-methylenedistearamide

Cas Number:

109-23-9

Molecular formula:

C37H74N2O2

IUPAC Name:

N,N'-methylenedistearamide

In vitro test system

Test system:

other: EpiDerm™ Reconstructed Human Epidermis Model Kit

Cell type:

non-transformed keratinocytes

Cell source:

other: Human-derived cells

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number: 28626
- Delivery date: 19 June 2018
- Date of initiation of testing: 21 June 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT solution
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader and LT-com analysis software
- Wavelength: 570 nm (OD570)
- Details on the method for assessing direct MTT reduction or colour interference can be found in 'Any other information on results incl. tables'.

METHODS
Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction.
At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).
Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60-Minute positive control is < 15%.
- Reproducibility: In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

NUMBER OF REPLICATE TISSUES:
Duplicate tissues

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if 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 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%.
- In addition, if test the test substance is considered to be corrosive based on the previous criteria, and if viability after 3 minutes exposure is smaller than 25%, the test substance is classified as sub-category 1A.
- In addition, if test the test substance is considered to be corrosive based on the previous criteria, and if viability after 3 minutes exposure is greater than or equal to 25%, the test substance is classified as a combination of sub-categories 1B and 1C.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg of the test item (+ 25 μL of sterile water for wetting the test item)

NEGATIVE CONTROL: Sterile distilled water
- Amount applied: 50 μL

POSITIVE CONTROL: 8.0N Potassium Hydroxide
- Amount applied: 50 μL
- Concentration: 7.92M

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

3-Hour MTT incubation

Number of replicates:

Diplicate

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean viability for 3-minute experiment

Value:

107.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean viability for 60-minute experiment

Value:

97.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.
- Colour interference with MTT: The solution containing the test item was a white colour. This colour was attributed to the intrinsic colour of the test item itself. It was therefore unnecessary to run colour correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.901 for the 3-Minute exposure period and 2.082 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

other: GHS criteria not met for corrosivity

Conclusions:

Based on this GLP compliant study performed according to OECD 431, the test item was considered to be non-corrosive to the skin.

Executive summary:

The skin corrosion potential of the test substance was evaluated in a GLP compliant study performed according to OECD 431, using the EpiDerm™ Reconstructed Human Epidermis Model Kit. In this model, corrosion is directly related to cytotoxicity, measured by the reduction of MTT to formazan by viable cells. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The negative and positive controls were valid and coefficient of variation did not exceed 30%. All acceptance criteria were therefore satisfied. The relative mean viability for the 3-minute exposure experiment was 107.3% and for the 60-minute exposure experiment 97.6%. Based on the classification criteria, the test item was considered to be non-corrosive to the skin.