1,1'-(methylenedi-p-phenylene)bismaleimide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 16 May to 18 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-(methylenedi-p-phenylene)bismaleimide

EC Number:

237-163-4

EC Name:

1,1'-(methylenedi-p-phenylene)bismaleimide

Cas Number:

13676-54-5

Molecular formula:

C21H14N2O4

IUPAC Name:

1-(4-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl]methyl}phenyl)-2,5-dihydro-1H-pyrrole-2,5-dione

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): BMI (1,1’-(methylenedi-p-phenylene)bismaleimide)
- Substance type: Organic
- Physical state: Light yellow or light brown powder
- Storage condition of test material: Room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Epidermal cells from adult donors

Justification for test system used:

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
- Tissue batch number: 12-EKIN-024
- Surface: 0.38 cm^2
- Expiration date: 18 June 2012

CHECK FOR REDUCTION OF MTT BY TEST SUBSTANCE
- The MTT reducing capability of the test substance was investigated by mixing approximately 10 mg of the test substance with 2 mL of 0.3 mg/mL MTT solution in duplicate. A control of 10 μL of distilled water, mixed with 2 mL of 0.3 mg/mL MTT solution was also included in duplicate.

CHECK FOR COLOR INTERFERENCE OF MTT BY TEST SUBSTANCE
- The test substance was evaluated for its colour or ability to become coloured in contact with water (simulating a tissue humid environment). Evaluation was achieved by mixing 10.1 mg of the test substance with 91 μL of distilled water in a transparent container to give 10 mg/90 μL. 100 μL of distilled water was included as a control. The solution was mixed for 15 minutes on a shaker. At the end of the shaking period the colour of the solution was assessed by eye.

CHECK FOR PH
An estimate of the pH of the test substance as a 10% suspension in distilled water was determined using pH indicator paper and recorded.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature
- Temperature of post-treatment incubation (if applicable): 42 ± 1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air + 3 hours ± 5 minutes with MTT .

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT :
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours ± 5 minutes at 37 ± 2°C
- Wavelength of OD measurements: 540 nm

VIABILITY MEASUREMENTS PREPARATION
AFTER all the tissues had been punched, the tissues (epidermis and collagen matrix) were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration).
The tissues were extracted by storing at 2-8 ºC, protected from light, for 48 - 70 hours. After formazan extraction, duplicate 200 μL aliquots of the extractant from each micro tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.

NUMBER OF REPLICATE TISSUES: triplicate tissues with the test item, negative and positive control.

PREDICTION MODEL / DECISION CRITERIA
If the mean tissue viability was equal to or less than 50% of the negative control value, the sample was classed as Irritant R38 (EU classification) or Category 2 (GHS classification).

ACCEPTANCE CRITERIA:
The OD from the negative control tissue in the MTT assay is an indicator of tissue viability after the shipping and storage procedure and under the specific conditions of the assay. The mean absorbance of the triplicate negative control values should be ≥0.6 and ≤1.5. The Standard Deviation (SD) value of the % viability should be <18.

The OD of the positive control is an indicator of the sensitivity of the tissues. The mean viability should be ≤40% of the negative control and the SD <18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 ± 2 mg (spread over the surface of the tissue using a curved spatula)
- The tissues were wetted with 5 μL of distilled water prior to application of the test material.

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hour ± 1 hours ; + 3 hours ± 5 minutes with MTT

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 experiments

Value:

91.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Negative control
The mean absorbance of the triplicate negative control values was 0.917 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 5.9 which was below the maximum value of 18.
Positive control
The percentage mean viability of the positive control was 10.9 ± 2.9 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18.

Results of individual reports are reported in "any other information"

Any other information on results incl. tables

The results of the assay are summarised in the table below.

Sample	Tissue viability as percentage of mean OD negative control				Prediction MTT endpoint
	Replicate Tissues			Mean ± SD
	a	b	c
Negative control	99.2	106.3	94.6	100.0 ± 5.9	Not applicable
Positive control	12.9	12.3	7.6	10.9 ± 2.9	Irritant
Test substance	96.8	92.0	86.3	91.7 ± 5.3	Not-irritant

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin irritation test, performed according to OECD guideline 439 and in accordance with GLP principles, BMI showed to be a non-irritant (tissue viability of 91.7± 5.3%). Therefore, the substance was predicted as non-irritant to the skin.

Executive summary:

In an in vitro skin irritation test, performed according to OECD guideline 439 and in accordance with GLP principles. The influence of BMI on the viability of human skin was tested. Ca. 10 mg 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. BMI showed to be a non-irritant (mean tissue viability of 91.7%). Therefore, the substance does not need to be classified according to regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).