N,N'-[1,4-phenylenebis(methylene)]bis{N,N-dimethyl-3-[(prop-2-enoyl)amino]propan-1-aminium} dichloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 - 22 Nov 2019

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted in 2019

Deviations:

yes

Remarks:

Demonstration of appropriate morphology of the tissues is not provided by the RhE model developer/vendor.

GLP compliance:

yes (incl. QA statement)

Remarks:

Slovak National Accrediatation Service, Bratislava, Slovak Republic

Test material

Test material information

Constituent 1

Reference substance name:

{[4-({dimethyl[3-(prop-2-enamido)propyl]azaniumyl}methyl)phenyl]methyl}dimethyl[3-(prop-2-enamido)propyl]azanium dichloride

EC Number:

812-548-5

Cas Number:

1621424-03-0

Molecular formula:

C24H40Cl2N4O2

IUPAC Name:

{[4-({dimethyl[3-(prop-2-enamido)propyl]azaniumyl}methyl)phenyl]methyl}dimethyl[3-(prop-2-enamido)propyl]azanium dichloride

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm SIT (EPI-200), MatTek In Vitro Life Science Laboratories, Slovak Republic

Justification for test system used:

The EpiDerm model is a three-dimensional reconstructed human epidermal (RhE) model, comprised of non-transformed human-derived epidermal keratinocytes, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The test substance is applied topically onto the tissue and subsequently cytotoxicity is measured. Irritant substances are identified by their ability to decrease cell viability (as determined by using the MTT reduction assay) below defined threshold levels. The EpiDerm skin irritation test allows discrimination between substances requiring classification and labelling (EU GHS category 1 or 2) and nonirritants according to the EU GHS classification system.

Vehicle:

unchanged (no vehicle)

Remarks:

Shortly before application of the test substance the tissue surface was moistened with DPBS to improve contact of the tissue surface with the test item.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ skin irritation test (SIT) (EPI-200), MatTek In Vitro Life Science Laboratories, Slovak Republic
- Tissue batch number(s): Lot no. 30836; Keratinocyte strain: 00267
- Production date: 20 Nov 2019 (Certificate of Analysis from MatTek)
- Delivery date: 18 Nov 2019
- Date of initiation of testing: 19 Nov 2019

Comment on the production date and experimental starting date: The tissue supplier states that the production date for the tissues produced by MatTek IVLSL is Monday. The lab performs preQC and if the tissues do not fulfil the quality control criteria the tissues are not shipped to the customers. To obtain the real life data for the tissues shipped to the customers, MatTek mimics the shipment conditions and therefore performs the quality controls on Tuesdays with quality control measurements on Wednesdays. Therefore the certificates of analysis for the tissues are issued on Wednesdays. This is the standard procedure for RhE tissues currently produced at MatTek IVLSL.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: After dosing, all plates were transferred to the humidified incubator at 37 °C for 35 ± 1 min. After 35 min, all plates were removed from the incubator and placed into the sterile hood (room temperature) until the period of 60 min incubation was completed.
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Each tissue was rinsed with sterile DPBS, filling and emptying the tissue insert 15 times to remove any residual test material, then was submerged 3 times in 150 mL of DPBS. Finally, the tissues were rinsed once from inside and once from outside with sterile DPBS and each insert was blotted on sterile blotting paper and transferred to new 6-well plates prefilled with 0.9 mL of fresh assay medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL MTT solution, 0.3 mL/well
- Incubation time: 3 h
- Spectrophotometer: Dynex MRX II
- Wavelength: 540 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS
- Viability: The quality of the EpiDerm™ SIT tissue was assessed by an MTT cell viability test. The OD (540 - 570 nm) was 2.002 ± 0.169 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) following application of 100 µL 1% Triton X-100. The ET-50 value was determined to be 4.85 h (acceptance criteria: 4.77 - 8.72 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of HIV-1 virus, Hepatitis B virus, Hepatitis C virus, bacteria, yeast and other fungi. No contamination was detected.

MTT DIRECT INTERFERENCE
Tests for interference of the test substance with the MTT assay were performed. First, the test substance was incubated for 60 min in purified water. The colour of the mixture was unchanged. It was concluded that the test substance does not have the potential to stain tissue. Second, the test substance was incubated for 60 min in MTT medium. The colour of MTT solution remained unchanged. It was concluded that the test substance did not reduce MTT directly.
Since the test substance did not directly reduce the MTT solution, an additional functional check was not performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: Single experiment.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean tissue viability after 60 min exposure is less than or equals 50%.
- The test substance is considered to be non-irritant to skin if the mean tissue viability after 60 min exposure is > 50%.

DEMONSTRATION OF TECHNICAL PROFICIENCY TESTING:
Demonstration of proficiency is stated in amendment No. 1 to the final report. The laboratory demonstrates technical proficiency by the non-classified substances (UN GHS no Category) and classified substances (UN GHS Category 2) indicated by the OECD 439 (version 25 Jul 2015) in vitro skin irritation test.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg

VEHICLE
- The test substance was applied without vehicle. Shortly before application of the test substance the tissue surface was moistened with 25 µL of sterile DPBS to improve contact of the tissue surface with the test substance.

NEGATIVE CONTROL
- Amount applied: 30 µL of DPBS

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% SDS

Duration of treatment / exposure:

60 min (35 min at 37 °C and 25 min at RT)

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

Triplicates for each treatment and control group were used.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

60 min exposure

Value:

98.8

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:

- OTHER EFFECTS:
- Direct-MTT reduction: The test substance did not directly reduce MTT.
- Colour interference with MTT: The test substance did not change the colour, when mixed with deionised water and thus passed the colour interference pre-test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8. The mean OD of the negative control was 1.334.
- Acceptance criteria met for positive control: Yes, the mean viability of the tissue replicates treated with the positive control for 60 min was < 20%. The mean viability of tissues treated with the positive control was 4.2%.
- Acceptance criteria met for variability between replicate measurements: Yes, the SD calculated from individual % tissue viabilities of three identically treated replicates was 5.49, i.e. < 18%. The coefficient of variation was 5.56.

Any other information on results incl. tables

Table 1. Skin irritation potential of FV-1295 after 60-minute exposure in reconstructed human epidermal model EpiDerm SIT

Test item	Tissue No.	OD - blank corrected	OD - mean	SD of OD	Cell viability (%)	Mean cell viability (%)	SD of cell viability	CV (%)	Classification
Negative control DPBS	1	1.312	1.334	0.025	98.3	100	1.91	1.91	Non-irritant
	2	1.329			99.6
	3	1.362			102.1
Positive control 5 % SDS	1	0.058	0.056	0.002	4.3	4.2	0.15	3.57	Irritant
	2	0.054			4.0
	3	0.056			4.2
FV-1295	1	1.397	1.318	0.073	104.7	98.8	5.49	5.56	Non-irritant
	2	1.306			97.9
	3	1.252			93.8

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

The determined viability of FV-1295-treated tissue culture fulfilled the criteria for non-irritancy.