4,11-diamino-2-[3-(2-methoxyethoxy)propyl]-1H-naphth[2,3-f]isoindole-1,3,5,10(2H)-tetrone

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

yes

Remarks:

See below

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on REACH

Deviations:

yes

Remarks:

See below

Principles of method if other than guideline:

None

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4,11-diamino-2-[3-(2-methoxyethoxy)propyl]-1H-naphth[2,3-f]isoindole-1,3,5,10(2H)-tetrone

EC Number:

265-334-3

EC Name:

4,11-diamino-2-[3-(2-methoxyethoxy)propyl]-1H-naphth[2,3-f]isoindole-1,3,5,10(2H)-tetrone

Cas Number:

65059-45-2

Molecular formula:

C22H21N3O6

IUPAC Name:

4,11-diamino-2-[3-(2-methoxyethoxy)propyl]-1H,2H,3H,5H,10H-naphtho[2,3-f]isoindole-1,3,5,10-tetrone

Test material form:

solid: particulate/powder

Details on test material:

None

In vitro test system

Test system:

human skin model

Source species:

human

Justification for test system used:

Recommended by the OECD guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier: MatTek
Date received: 14 April 2015
EpiDermTM Tissues (0.5cm2) lot number: 21654
Assay Medium lot number: 040915TMA
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was placed into a refrigerator.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

two exposure periods of 3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

relative mean

Run / experiment:

60 minute exposure

Value:

105.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosion

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

relative mean

Run / experiment:

3 minute

Value:

108.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosion

Other effects / acceptance of results:

Direct MTT Reduction
The direct MTT reduction test was inconclusive due to the dark blue color of the test item. Therefore, an additional procedure using freeze killed tissues was performed to assess for the possibility of direct MTT reduction. An additional procedure was also performed using viable tissues to assess for the possibility of color interference. The results of the additional procedures showed a negligible degree of interference due to possible direct reduction of MTT or color interference. It was therefore considered unnecessary to use the results of the additional procedures for quantitative correction of results and reporting purposes.

Any other information on results incl. tables

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 2.4 % relative to the negative control treated tissues following the 3-minute exposure period. The positive control acceptance criterion was therefore satisfied. The mean OD562 for the negative control treated tissues was 2.004 for the 3-minute exposure period and 2.005 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

 

Mean OD₅₆₂ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item	Exposure Time	Mean OD562^1	Percentage Viability
Negative Control†	3 minutes	2.004	100*
	60 minutes	2.005	100*
Positive Control†	3 minutes	0.049	2.4^2
	60 minutes	0.048	2.4^3
Test Item	3 minutes	2.180	108.8^2
	60 minutes	2.122	105.8^3

*=   The mean viability of the negative control tissues is set at 100%

1=   Mean of EpiDerm^TMtissues tested in duplicate

2=   Viability expressed as a percentage of the 3 minute negative control tissues

3=   Viability expressed as a percentage of the 60 minute negative control tissues

†= Control data was shared with Harlan Laboratories Ltd. study number 41500303

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

The corrosivity potential of the test item was evaluated using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. The direct MTT reduction test was inconclusive due to the dark blue color of the test item. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT or cause color interference and could have given rise to a false negative result. Therefore, an additional procedure using freeze killed tissues was performed to assess for the possibility of direct MTT reduction. An additional procedure was also performed using viable tissues to assess for the possibility of color interference. At the end of the exposure period the test item was rinsed from each tissue before the tissues were taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The results of the additional procedures showed a negligible degree of interference due to possible direct reduction of MTT or color interference. It was therefore considered unnecessary to use the results of the additional procedures for quantitative correction of results or for reporting purposes. The relative mean viabilities of the test item treated tissues were as follows:

60 minute exposure: 105.8 %

3 minute exposure: 108.8 %

The quality criteria required for acceptance of results in the test were satisfied. The test item was considered to be non-corrosive to the skin.