4,4'-methylenebis[N-sec-butylaniline]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 February 2018 - 23 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

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: EU method B.40 BIS (In Vitro Skin corrosion: Human Skin Model Test).

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-methylenebis[N-sec-butylaniline]

EC Number:

226-122-6

EC Name:

4,4'-methylenebis[N-sec-butylaniline]

Cas Number:

5285-60-9

Molecular formula:

C21H30N2

IUPAC Name:

4,4'-methylenebis[N-sec-butylaniline]

Test material form:

liquid

Details on test material:

Appearance: Slight yellow liquid
Storage conditions: At room temperature protected from light

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on animal used as source of test system:

EpiDerm™ Reconstructed Human Epidermis (Lot no.: 27958, kit F and G,)
- All cells used to produce Epiderm™ are purchased or derived from tissue obtained by MatTek Corporation from acredited institutions.
- Cells are screened for potential biological contaminants (HIV-1, Hepatitis B, Hepatitis C, bacteria, yeast and fungi)

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue lot number: 27958, kit F and G
- Surface: 0.6 cm^2
- The model consists of keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment at 37.0 ± 1.0°C. (actual range 35.0 - 37.3°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2 replicates per exposure duration, one negative control, one positive contol.

ACCEPTANCE OF RESULTS:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

DECISION CRITERIA
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

DATA INTERPRETATION
See Table 1 (Any other information on methods inc. tables)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: concurrent control for MTT reduction by test item

Amount/concentration applied:

50 μL of the undiluted test item was applied on top of the skin tissues.

Duration of treatment / exposure:

3-minute and 1-hour

Number of replicates:

2 replicates per exposure duration (4 total), one negative control, one positive contol.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

9.1%

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

7.9%

Other effects / acceptance of results:

- OTHER EFFECTS:
- 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, mean relative tissue viability following 1-hour exposure was 7.9%.
- Acceptance criteria met for variability between replicate measurements: Yes

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro skin corrosion test was conducted according to OECD 431 guideline and GLP principles. It is concluded that this test is valid and that the test substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

In an in vitro skin corrosion test using a human skin model ( EpiDerm Skin Model) performed according to OECD 431 and GLP principles, the influence of the test substance on the viability of human skin was tested. 50 μL test substance was applied directly on top of 0.6 cm² cultured skin. After 3-minute and 1-hour treatments the substance was removed and the viability of the cells was tested by reduction of MTT. Viability of unexposed skin was set at 100%. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with test item compared to the negative control tissues was 104% and 91%, respectively. The positive control had a mean cell viability of 7.9% after 1 hour exposure and no color interference or direct reduction by MTT was observed in the test system. Since the mean relative tissue viability was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, it can be concluded that the test item is not corrosive in the in vitro skin corrosion test.