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

Endpoint summary

Description of key information

An in vitro skin corrosion test was conducted according to OECD 431 guideline and GLP principles and an in vitro skin irritation test, performed according to OECD guideline 439 and under GLP principles. The results show that 4,4’-methylenebis-N-sec-butylaniline is not corrosive and not irritant to the skin.

A Bovine Corneal Opacity and Permeability test (BCOP) was performed according to OECD guideline and GLP principles, since 4,4’-methylenebis-N-sec-butylaniline induced an IVIS < 3, the substance is concluded not to cause eye irritation or serious eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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

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 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.

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%

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

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April 2018 - 15 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model(TM)
- Tissue batch number(s): 18-EKIN-015
- Surface: 0.38 cm^2
- Expiration date: 16 April 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure to the test item: room temperature
- Temperature of incubations(°C): 36.6 - 37.3°C
- Humidity(%): 62 - 85

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: tissues were washed with phosphate buffered saline (1 washing step)
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE: the test substance was checked for possible direct MTT reduction and color interference in a previous skin corrosion test using EpiDerm as a skin model. It was concluded that the test substance did not interfere with the MTT endpoint.
- Incubation time: 3 hours
- Measurement method: TECAN Infinite M200 Pro Plate Reader (570 nm)
- MTT concentration: 0.3 mg/mL

NUMBER OF REPLICATE TISSUES: 3 for the test substance, the negative and the positive control each.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 single run

CELL VIABILITY MEASUREMENT: After incubation, tissues were dried and incubated with 2 mL MTT-solution for 3 hours. After incubation, epidermis was separated from the collagen matrix and both parts were extracted with 500 μL isopropanol. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

PREDICTION MODEL / DECISION CRITERIA (see Table 1)
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

ACCEPTABILITY CRITERIA
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
-Amount applied: 25 µL on top of skin tissues

Duration of treatment / exposure:

15 ± 0.5

Duration of post-treatment incubation (if applicable):

42 hours; + 3 hours with MTT

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates

Value:

102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability (%): 9.0

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the negative control tissues was within the laboratory historical control data range but the SD of the % viability was 1.1.
- Acceptance criteria met for positive control: yes, the mean relative tissue viability was 9.0% and the SD of the % viability was 2.3
- Acceptance criteria met for variability between replicate measurements: yes, the variability between tissue treated with the test item was 9.5%.

- Since the mean relative tissue viability for 4,4’-methylenebis-N-sec-butylaniline was above 50% is considered to be non-irritant.

In a previously performed skin corrosion study, the test item was found to be non-corrosive to the skin.

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin irritation test, performed according to OECD guideline 439 and under GLP principles, 4,4’-methylenebis-N-sec-butylaniline showed to be a non-irritant (mean tissue viability of 102%). Therefore, the substance does not need to be classified according to GHS and CLP criteria.

Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model), the influence of 4,4’-methylenebis-N-sec-butylaniline on the viability of human skin was tested. 25 µL of test substance was applied directly on top of 0.38 cm²cultured skin. After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 9% whereas the test substance showed cell viability of 102%. Since the mean relative tissue viability after exposure to the test substance was above 50%, no classification is required for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse Vitelco, 's-Hertogenbosch, The Netherlands.
- Storage, temperature, and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected an transported in physiological saline in a suitable container under cooled conditions.
- The eyes were checked for unacceptable defects and those exhibiting defects were discarded.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ±10 minutes in cMEM for opacity measurements and subsequently 90 ±5 minutes in sodium-fluorescein for permeability determinations

Number of animals or in vitro replicates:

3 replicates for the negative, positive, treatment group each.

Details on study design:

TREATMENT METHOD: The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or the test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32.0 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.
- POST-EXPOSURE INCUBATION: 120 ±10 minutes in cMEM for opacity measurements and subsequently 90 ±5 minutes in sodium-fluorescein for permeability determinations

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). OD490 values of less than 1.500 were used in the permeability calculation.
- Other: possible pH effects of the test substance on the corneas were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
a) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
b) The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

DECISION CRITERIA (see table 1):
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces IVIS >55 is defined as a corrosive or severe irritant (UN GHS: category 1);
For a test substance that induces an IVIS >3 and ≤55, no prediction on irritant potency can be made.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3 replicates

Value:

-0.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean IVIS: 74

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- The corneas treated with the test item showed opacity values between 2.5 and 7.4
- Permeability values were ranging from 0.005 to 0.021
- IVIS scores were 1.8, -0.7 and -1.8 (n=3)

OTHER EFFECTS:
- No pH effect of the test item was observed on the rinsing medium

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, results were within historical range (IVIS ranging from 0.8 to 1.2).
- Acceptance criteria met for positive control: yes, results were within historical range (IVIS ranging from 56 to 103).
The corneas were translucent after the 10 minutes of treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed according to OECD guideline and GLP principles, since 4,4’-methylenebis-N-sec-butylaniline induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed with 4,4’-methylenebis-N-sec-butylaniline according to OECD guideline 437 and GLP principles. 4,4’-methylenebis-N-sec-butylaniline was applied undiluted (750 µL/ cornea, n=3). The mean in vitro irritancy score of the positive control (Ethanol) was 74, and the mean in vitro irritancy score of the negative control (physiological saline) was 1.0 which were both within historical range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 4,4’-methylenebis-N-sec-butylaniline did not induce ocular irritation through both endpoints, having a mean in vitro irritancy score of -0.1 after 10 minutes of treatment. Therefore no classification is required for eye irritation or serious eye damage according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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 4,4’-methylenebis-N-sec-butylaniline 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.

In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model), the influence of 4,4’-methylenebis-N-sec-butylaniline on the viability of human skin was tested. 25 µL of test substance was applied directly on top of 0.38 cm² cultured skin. After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 9% whereas the test substance showed cell viability of 102%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test item is not irritant in the in vitro skin irritation test.

A Bovine Corneal Opacity and Permeability test (BCOP) was performed with 4,4’-methylenebis-N-sec-butylaniline according to OECD guideline 437 and GLP principles. 4,4’-methylenebis-N-sec-butylaniline was applied undiluted (750 µL/cornea, n=3). The mean in vitro irritancy score of the positive control (Ethanol) was 74, and the mean in vitro irritancy score of the negative control (physiological saline) was 1.0 which were both within historical range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 4,4’-methylenebis-N-sec-butylaniline did not induce ocular irritation through both endpoints, having a mean in vitro irritancy score of -0.1 after 10 minutes of treatment.

Justification for classification or non-classification

Based on the available data, 4,4’-methylenebis-N-sec-butylaniline was found to be not corrosive and not irritating to the skin, and not to cause adverse effects on the eye, therefore no classification is required for skin and eye irritation/corrosion according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).