N,N'-[bicyclo[2.2.1]heptane-2,6-diylbis(methylene)]bis(2-cyanoacetamide)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-28th January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

N/A

Cas Number:

N/A

IUPAC Name:

N/A

Details on test material:

LME 11311

Specific details on test material used for the study:

Identification:
LME 11311
Batch:
Ua05 (258)
Purity:
99%
Molecular Weight:
292.38 g.mol-1
Physical state/Appearance:
Yellow solid
Expiry Date:
01 November 2023
Storage Conditions:
Room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

Supplier:
EpiSkin Laboratories, Lyon, France

Date received:
22 January 2019

EpiSkinTM Tissues (0.38cm2) lot number:
19-EKIN-004

Maintenance Medium lot number:
19-MAIN3-003

Assay Medium lot number:
19-ESSC-003

Amount/concentration applied:

2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 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. 1 disc of the test item was then applied to the epidermal surface. 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. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center).

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.

Duration of post-treatment incubation (if applicable):

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

3.2.1 Negative Control
Information as provided by the Supplier.
Identification:
Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
Batch:
1754643
Purity:
>98%
Expiry Date:
01 February 2019
Storage Conditions:
Approximately 4 °C in the dark
Supplier:
Gibco
3.2.2 Positive Control
Information as provided by the Supplier.
Identification:
Sodium dodecyl sulphate
Batch:
SLBT3991
Purity:
99.5%
Expiry Date:
01 March 2020
Storage Conditions:
Room temperature
Supplier:
Sigma-Aldrich
3.2.3 Preparation of Negative and Positive Control Items and MTT
The negative control item, Dulbecco’s Phosphate Buffered Saline (DPBS), was used as supplied.
The positive control item, Sodium dodecyl sulphate (SDS), was prepared as a 5% w/v aqueous solution. The positive control was formulated within 2 hours of being applied to the test system.
A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

85.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Item	Mean OD570 of triplicate tissues		+- SD of OD570	Relative individual tissue viability (%)	Relative Mean Viability (%)	+- SD of Relative Mean Viability (%)
Negative Control Item	0.934		0.095	89.0 102.0 109.0	100	10.1
Positive Control Item	0.032		0.012	4.9 2.7 2.7	3.4	1.3
Test Item	0.800		0.039	83.8 82.8 90.5	85.7	4.2

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Method

Triplicate tissues were treated with discs of the test item for an exposure period of 15 minutes. At the end of the exposure period the test item discs were removed from each tissue, and the tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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-labeled 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-labeled 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 85.7% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was classified as non-irritant. The following classification criteria apply:

EU CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).