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

Endpoint summary

Description of key information

Data summary of skin and eye irritation and corrosion studies.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

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.

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.

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.

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).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 02 June 2020 to 04 June 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008.

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

other: 25 µL of sterile water was added for wetting of the test item to increase tissue surface contact.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek MTT 100 kit from MatTek In Vitro Life Sciences Laboratories
- Tissue lot number: 30870
- Delivery date: 02 June 2020
- Date of initiation of testing: 02 June 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C, 5% CO2

PRE-INCUBATION
The assay medium was brought to room temperature before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3 Minute and 60 Minute exposure periods. EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24 well plate prepared for MTT-loading.
- Observable damage in the tissue due to washing:


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT 4500 microplate reader and LT-com analysis software
- Wavelength: 570nm


NUMBER OF REPLICATE TISSUES:
Duplicate for each treatment


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- As given in OECD 431 test guideline.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
- Concentration (if solution): 25 µL of sterile water was added for wetting of the test item to increase tissue surface contact.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 100%

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8.0N Potassium Hydroxide

Duration of treatment / exposure:

1 x 3 minute exposure period
1 x 60 minute exposure period

Duration of post-treatment incubation (if applicable):

None

Number of replicates:

Testing was conducted in duplicate for each tretment.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure period

Value:

98.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% tissue viability

Positive controls validity:

valid

Remarks:

3.5% tissue viability

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute Exposure Period

Value:

98.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% tissue viabilty

Positive controls validity:

valid

Remarks:

2.6% tissue viability

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.
- Colour interference with MTT: The solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause colour interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viabilty
	Negative Control	Positve Control	Test Item
3 minute	100*	3.5	98.3
60 minute	100*	2.6	98.9

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

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions used in the study the test item was considered to be non-corrosive to the skin.

Executive summary:

Introduction

The purpose of the study was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. The test method was designed to be meet the requirements of the following guidelines:

• OECD Guideline for the Testing of Chemicals No. 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (18 June 2019)
  
  
• 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, 18 December 2006, of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

 

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction.

At the end of the formazan extraction period triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD₅₇₀).

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viabilty
	Negative Control	Positve Control	Test Item
3 minute	100*	3.5	98.3
60 minute	100*	2.6	98.9

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

Conclusion

Under the experimental conditions used in the study the test item was considered to be non-corrosive to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase start and completion: 07 July 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes

Species:

cattle

Details on test animals or tissues and environmental conditions:

Source of Bovine Eyes
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.


Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.

The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Vehicle:

physiological saline

Remarks:

For the purpose of this study the test item was prepared as a 20% w/v solution in sodium chloride 0.9% w/v.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

.TEST MATERIAL
- Concentration (if solution): 0% w/v solution in sodium chloride 0.9% w/v

NEGATIVE CONTROL
The negative control item, sodium chloride 0.9% w/v, was used as supplied.

POSITIVE CONTROL
The positive control item, Imidazole, was used as a 20% w/v solution in sodium chloride 0.9% w/v.

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

Three replicates (corneas) per treatment (test substance, negative control, positive control).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS

QUALITY CHECK OF THE ISOLATED CORNEAS : Yes

NUMBER OF REPLICATES: Three per treatment

NEGATIVE CONTROL USED : Sodium Chloirde 0.9% w/v

SOLVENT CONTROL USED (if applicable): No

POSITIVE CONTROL USED: Imidiazole

APPLICATION DOSE AND EXPOSURE TIME : 240 minutes

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 washings with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red
- POST-EXPOSURE INCUBATION: No

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer (calibrated on the day of the test)
- Corneal permeability: passage of sodium fluorescein dye measured using microtiter plate reader at 492nm (without a reference filter)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As specified in the test guideline.

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Substance (mean of three replicates)

Value:

1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

in vitro irritancy score = 0.3

Positive controls validity:

valid

Remarks:

in vitro irritancy score = 92.4

The test item is classified according to the following criteria:

IVIS	UN GHS
≤ 3	No Category
>3 ≤ 55	No prediction can be made
> 55	Category 1

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the test, the test item is given No Category classification according to the UN GHS classification scheme.

Executive summary:

Introduction

The ability of the test item to induce eye damage was assessed using a method designed to meet the requirements of the following guidelines:

 

• OECD Guideline for the Testing of Chemicals No. 437 (updated 09 October 2017) “Bovine Corneal Opacity and Permeability Assay”


•  Method B.47 of Commission Regulation (EC) No. 440/2008  

Method

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).  Three corneas were used per treatment group.

 

Data Interpretation

The test item is classified according to the followign criteria:

 

IVIS	UN GHS
≤ 3	No Category
>3 ≤ 55	No prediction can be made
> 55	Category 1

 

Results

The In Vitro irritancy scores are summarised as follows:

 

Treatment	In Vitro Irritancy Score
Test Item	1.0
Negative Control	0.3
Positive Control	92.4

 

Conclusion

Under the conditions of the test, the test item is given No Category classification according to the UN GHS classification scheme.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The EPISKIN skin irritation test and Epiderm skin corrosion tests did not meet the criteria for classification for skin effects according to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures.

 

The BCOP test gave a 'no category' result for eye effects and therefore the substance is not classified for eye effects according to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures.