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

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

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

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results

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.

Applicant's summary and conclusion

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).