N,N'-(2-(4-(2-aminobenzamido)butyl)pentane-1,5-diyl)bis(2-aminobenzamide)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 08 July 2020 to 13 July 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-N-{6-[(2-aminophenyl)formamido]-2-{3-[(2-aminophenyl)formamido]propyl}hexyl}benzamide

EC Number:

951-985-7

Molecular formula:

C30H38N6O3

IUPAC Name:

2-amino-N-{6-[(2-aminophenyl)formamido]-2-{3-[(2-aminophenyl)formamido]propyl}hexyl}benzamide

Test material form:

solid: crystalline

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

Supplier: EpiSkin Laboratories, Lyon, France.
Date received: 07 July 2020
EpiSkinTM Tissues (0.38cm2)
Lot number: 20-EKIN-028
Maintenance Medium lot number: 20-MAIN3-018
Assay Medium lot number: 20-ESSC-018


Main Test

Application of Test Item and Rinsing (Day 1)

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm test item was then applied to the epidermal surface ensuring uniform covering. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls.

The plates were kept in the biological safety cabinet at room temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 hours.


MTT Loading/Formazan Extraction (Day 3)
Following the 42-hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer (-35 to -10 ºC) for possible inflammatory mediator determination.

2 mL of MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells with any excess maintenance medium from the bottom of the tissue insert removed by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C,5% CO2 in air. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Following mixing on a vortex mixer the tubes were refrigerated at 2 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.


Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.

For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using a Labtech LT-4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Approximately 10 mg (26.3 mg/cm) test item was then applied to the epidermal surface ensuring uniform covering.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

The post-exposure incubation period was 42-hour.

Number of replicates:

Tripilcate tissues were treated for the test item and positive and negative controls.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item

Value:

95.6

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:

ACCEPTANCE CRITERIA

The relative mean tissue viability for the positive control treated tissues was 2.8% relative to the negative control treated tissues and the standard deviation value of the viability was 0.5%. The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.845 and the standard deviation value of the viability was 9.8%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.8%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.  

 

Assessment of Colour Interference with the MTT endpoint

The solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study and under the experimental conditions reported, the test item was classified as non-irritant. The following classifications apply: EU CLP Not classified for Irritation. UN GHS Not classified for Irritation (category 3 cannot be determined).

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TM reconstructed human epidermis model. The test was designed to be compatible with the following guidelines:

 

• OECD Guideline for the Testing of Chemicals No. 439 In Vitro Skin Irritation: Reconstructed Human EpiDermis (RHE) Test Method (18 June 2019)
  
  


• Method B.46. in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 640/2012, of 06 July 2012 amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

 

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42-hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm.

 

 Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Results

The relative mean viability of the test item treated tissues was 95.6% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

 

Conclusion

In this study and under the experimental conditions reported, the test item was classified as non-irritant. The following classifications apply: EU CLP Not classified for Irritation. UN GHS Not classified for Irritation (category 3 cannot be determined).