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Diss Factsheets

Administrative data

Description of key information

In in vitro eye irritation and corrosion studies (OECD 492 and OECD 438) test item was not considered as irritant and not corrosive to the eyes (GLP, Rel. 1).

Based on the QSAR results from Danish QSAR Database and profiling results from OECD QSAR Database, the query substance was predicted as mild irritant to the skin.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation / corrosion, other
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
17/04/2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
QMRF is provided as model output. The model doesn't provide any QPRF report
Justification for type of information:
1. SOFTWARE
Danish QSAR Database

2. MODEL (incl. version number)
Three individual models incorporated into Danish QSAR Database were used:
Case Ultra, SciQSAR and Leadscope

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Nc1c(C)cc(Cc2cc(C)c(N)c(C)c2)cc1C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Please refer to the attached QMRFs for each of the three QSAR models.

5. APPLICABILITY DOMAIN
The substance falls within the applicability domain of the applied models. Please refer to the attached QMRFs.

6. ADEQUACY OF THE RESULT
As the substance falls within the applicability domain of the applied models, the final prediction was considered as reliable.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Please refer to the attached QMRF reports to get further information about the model development and validation approach of each of the three models integrated into the Danish QSAR Database.
GLP compliance:
no
Remarks on result:
positive indication of irritation
Interpretation of results:
other: QSAR result
Conclusions:
Based on the battery prediction derived using the three models incorporated into Danish QSAR Database (Leadscope, Case Ultra and SciQSAR), the query substance was predicted as non-irritant to the skin.
Executive summary:

Based on the battery prediction derived using the three models incorporated into Danish QSAR Database (Leadscope, Case Ultra and SciQSAR), the query substance was predicted as non-irritant to the skin. The query substance falls within the applicability domain of all the three models, therefore the prediction can be considered as reliable.

Endpoint:
skin irritation / corrosion, other
Type of information:
(Q)SAR
Remarks:
Profiling results using OECD QSAR Toolbox v4.2
Adequacy of study:
weight of evidence
Study period:
17/04/2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
Profiling results are based on triggering of structural alerts for the query substance
Justification for type of information:
1. SOFTWARE
OECD QSAR Toolbox v4.2

2. MODEL (incl. version number)
OECD QSAR Toolbox v4.2

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Nc1c(C)cc(Cc2cc(C)c(N)c(C)c2)cc1C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Please refer to the user guide of OECD QSAR Toolbox v4.2 to get further information about the profilers used.

5. APPLICABILITY DOMAIN
Please refer to the user guide of OECD QSAR Toolbox v4.2 to get further information about the applicability domain of the profilers used.

6. ADEQUACY OF THE RESULT
The triggered alerts hints the possibility of skin irritation. Please refer to the attached Data matrix of the profiling results derived using OECD QSAR Toolbox v4.2.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The query substance was provided as input for profiling results with OECD QSAR Toolbox v4.2. Following Skin Irritation relevant profilers were applied:
a) Skin Irritation/Corrosion Inclusion rules by BfR
b) Skin Irritation/Corrosion Exclusion rules by BfR
GLP compliance:
no
Irritation parameter:
other: Skin Irritant
Remarks:
Alert for aromatic amines
Remarks on result:
positive indication of irritation

OECD QSAR Toolbox v4.2 triggered following alert for the query substance:

Aromatic amines

Based on this result, the substance was predicted as skin irritant.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Based on the structural alert triggered (aromatic amines) using OECD QSAR Toolbox v4.2, the query substance was predicted as skin irritant (Category 2 based on GHS criteria).
Executive summary:

The query substance was provided as input to the profiling using OECD QSAR Toolbox v4.2 for the two skin irritation relevant profilers. Based on a triggered alert for skin irritation, the query substance was predicted as skin irritant (Category 2 based on GHS criteria).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline 438 without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
dated 26 July 2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
dated 08 December 2010
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
27 Avril 2017
Specific details on test material used for the study:
• Sponsor’s identification: 4,4'-méthylène bis (2,6-diméthylaniline)
• Synonym: DURCISSEUR DX
• Chemical name: 4-[(4-amino-3,5-dimethylphenyl)methyl]-2,6-dimethylaniline

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 160 209 0004 (Lot Isochem)
- Expiration date of the lot/batch: 01 October 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay.
- Characteristics of donor animals (e.g. age, sex, weight): The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).
- Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 09 August 2017 at 8:30 am.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. The eyes were enucleated at Phycher on 09 August 2017 at 09:45 am.
- Indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: None
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, as supplied during 10 seconds, to the cornea such that the entire surface of the cornea was evenly covered with the test item
Duration of treatment / exposure:
10 seconds
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
- The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus were at a controlled temperature between 32.2 °C and 32.7 °C.
- After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.
- Once all eyes had been examined and approved, the eyes were incubated between 45 and 56 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES
- 1, 3 and 3 eyes for negative, positive controls and test item, respectively.

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: Sodium hydroxide

APPLICATION DOSE AND EXPOSURE TIME
- Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, as supplied during 10 seconds, to the cornea such that the entire surface of the cornea was evenly covered with the test item.

REMOVAL OF TEST SUBSTANCE
- After exposure, the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD
- Treated corneas were evaluated before the pre-treatment and at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

METHODS FOR MEASURED ENDPOINTS:
- All observations of the cornea and measurement of corneal thickness were performed using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. 1. For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm.
- The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which was determined only at pretreatment and 30 minutes after exposure to the test item) were determined at each of the above time points.

SCORING SYSTEM:
- Mean corneal swelling (%): Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope.
Corneal swelling (%) = ((corneal thickness at time t - corneal thickness at time = 0) / (corneal thickness at time = 0)) x 100
The mean percentage of corneal swelling for all tested eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for the test item.
- Mean maximum opacity score: Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all tested eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item
0: No opacity
0.5: Very faint opacity
1: Scattered or diffuse areas; details of the iris clearly visible
2: Easily discernible translucent area; details of the iris are slightly obscured
3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4: Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment: The mean fluorescein retention value for all tested eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item
0: No fluorescein retention
0.5: Very minor single cell staining
1: Single cell staining scattered throughout the treated area of the cornea
2: Focal or confluent dense single cell staining
3: Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA:
Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for the test item.
Once each endpoint was evaluated, ICE classes were assigned based on a predetermined range. Interpretation of corneal thickness, opacity, and fluorescein retention using four ICE classes was done according to the table 7.3.2/1, 7.3.2/2, 7.3.2/3.
Irritation parameter:
cornea opacity score
Run / experiment:
mean of three runs
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
mean of three runs
Value:
0.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Run / experiment:
mean of three runs
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OCULAR REACTIONS:
The ocular reactions observed in eyes treated with the test item were:
- maximal mean score of corneal opacity: 0.0, corresponding to ICE class I;
- mean score of fluorescein retention: 0.7, corresponding to ICE class II;
- maximal mean corneal swelling: 2%, corresponding to ICE class I.

The combination of the three endpoints for the test item was 2 x I,1 x II.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.
- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

Table 7.3.2/5: Individual and average values for evaluation of corneal lesions after treatment

 

Endpoint measured

Eye No.

Time (min)

0

30

75

120

180

240

Corneal opacity

4

0

0

0

0

0

0

5

0

0

0

0

0

0

6

0

0

0

0.0

0.0

0

Mean

0

0

0

0.0

0.0

0.0

ICE class

I

Fluorescein retention

4

0.5

1

-

-

-

-

5

0.5

0.5

-

-

-

-

6

0.5

0.5

-

-

-

-

Mean

0.5

0.7

-

-

-

-

ICE class

II

Corneal thickness

4

0.57

0.57

0.57

0.57

0.57

0.57

5

0.56

0.58

0.58

0.58

0.58

0.58

6

0.55

0.56

0.56

0.56

0.56

0.56

Corneal swelling (%)

4

-

0

0

0

0

0

5

-

4

4

4

4

4

6

-

2

2

2

2

2

Mean

-

2

2

2

2

2

ICE class

I

Combination of the 3 Endpoints

2 x I; 1 x II

CLASSIFICATION

No category

Interpretation of results:
GHS criteria not met
Conclusions:
In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that test item 4,4'-méthylène bis (2,6-diméthylaniline) does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No Category). No signal word and hazard statement are required.
Executive summary:

An ex vivo eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, as supplied during 10 seconds, to the cornea such that the entire surface of the cornea was evenly covered with the test item. Then the eyes were rinsed four times with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 0.0, corresponding to ICE class I;

- mean score of fluorescein retention: 0.7, corresponding to ICE class II;

- maximal mean corneal swelling: 2%, corresponding to ICE class I.

The combination of the three endpoints for the test item was 2 x I, 1 x II.

The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that test item 4,4'-méthylène bis (2,6-diméthylaniline) does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No Category). No signal word and hazard statement are required.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-25 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 492 without any deviation
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
• Sponsor’s identification: 4,4'-méthylène bis (2,6-diméthylaniline)
• Synonym: DURCISSEUR DX
• Chemical name: 4-[(4-amino-3,5-dimethylphenyl)methyl]-2,6-dimethylaniline

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 160 209 0004 (Lot Isochem)
- Expiration date of the lot/batch: 01 October 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
Species:
human
Details on test animals or tissues and environmental conditions:
- Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation]
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg
- Concentration: Undiluted
Duration of treatment / exposure:
6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions)
Duration of post- treatment incubation (in vitro):
- Post-exposure immersion period: 25 minutes at room temperature
- Post-exposure incubation period: 18 h at standard culture conditions
Number of animals or in vitro replicates:
Test item, negative and positive controls were applied on duplicate tissues.
Details on study design:
MAIN TEST
- Pre-incubation of the tissues:
The 0.60 cm² Reconstructed human Cornea-like Epithelia (EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 27020) were received on 23 January 2018.
The same day, the tissues in their well shipping container were equilibrated to room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium (MatTek Corporation, batch No. 012218ISA) and incubated during 20 hours and 27 minutes at standard culture conditions.

After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 3941117). The tissues were incubated at standard culture conditions for 30 minutes.

- Treatment and post-treatment incubation of the tissues:
The test item was applied as supplied, during 6 hours at standard culture conditions, at the dose of 50 mg to the entire surface of 2 living RhCE tissue replicates.

In the same experimental conditions, a positive control (Methyl acetate - MatTek Corporation, batch No. 032817ISA), and a negative control (distilled water - VWR - Batch No.1007123) were carried out. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 RhCE tissue replicates during 6 hours at standard culture conditions.

After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 3941117). The rinsed tissues were checked for any coloration and noted to be of comparable colour with the negative control treated tissues (whitish).
This rinsing step was followed by a 25-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue.
The RhCE constructs were then incubated for 18 hours post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.

- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.

The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at standard culture conditions.
The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 2 hours at room temperature in the dark.
The concentration of formazan was measured by determining the OD (Optical Density) at 570 nm, just after dilution of the extractions in isopropanol (1:2).
The OD at 570 nm was measured in triplicate samples of formazan extracts.
The measured OD are proportional to the number of living cells.

The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
Irritation parameter:
other: % mean viability of the tissues
Run / experiment:
Main test
Value:
94.31
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test item 4,4'-méthylène bis (2,6-diméthylaniline) was 94.31%, versus 33.28% in the positive control (Methyl acetate). Refer Tables 7.3.2/1 for more details.

Table 7.3.2/1: Main test - Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

Tissue

OD

Mean OD/disc (#)

Mean OD/product

Viability %

Mean viability %

Difference of viability %

Negative control

1

0.305

0.369*

0.616

74.92

100.00

50.16

0.402

0.401

2

0.665

0.616

125.08

0.589

0.594

Positive control

1

0.167

0.177

0.205

28.73

33.28

8.93

0.177

0.187

2

0.224

0.232

37.66

0.231

0.240

Test item

1

0.755

0.654

0.581

106.17

94.31

23.70

0.607

0.600

2

0.523

0.508

82.47

0.494

0.506

*: aberrant value, not taken into account for the calculation of cell viability

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test substance does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and signal word are required.
Executive summary:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

As the test item was a solid, it was administered directly applied on the entire surface of the epidermis (corresponding to 50 mg). The test substance was applied to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with PBS at room temperature, a 25-minute post-exposure immersion period at room temperature and a 18-hour post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay.

The mean percent tissue viability of the RhCE replicates treated with the test substance was 94.31% versus 33.28% in the positive control (Methyl acetate).

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test substance does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and signal word are required.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Based on the results derived using three individual models from Danish QSAR Database, the substance fell within their applicability domains and was predicted as non-irritant to the skin. However, with the profiling results with OECD QSAR Toolbox v4.2, an alert for aromatic amines was triggered. Combining both results, the consensus result predicted the query substance as skin irritant (mild irritant to the skin).

Eye irritation:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

As the test item was a solid, it was administered directly applied on the entire surface of the epidermis (corresponding to 50 mg). The test substance was applied to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with PBS at room temperature, a 25-minute post-exposure immersion period at room temperature and a 18-hour post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay.

The mean percent tissue viability of the RhCE replicates treated with the test substance was 94.31% versus 33.28% in the positive control (Methyl acetate).

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test substance does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and signal word are required.

Anex vivoeye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, as supplied during 10 seconds, to the cornea such that the entire surface of the cornea was evenly covered with the test item. Then the eyes were rinsed four times with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 0.0, corresponding to ICE class I;

- mean score of fluorescein retention: 0.7, corresponding to ICE class II;

- maximal mean corneal swelling: 2%, corresponding to ICE class I.

The combination of the three endpoints for the test item was 2 x I, 1 x II.

The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that test item 4,4'-méthylène bis (2,6-diméthylaniline) does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No Category). No signal word and hazard statement are required.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, the registered substance is classified as skin irritant category 2 (H315), and is not classified as eye irritant according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).