Reaction mass of N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethyldocosan-1-aminium chloride and N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethylicosan-1-aminium chloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date:16 June 2016. Experimental completion date: 17 June 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

please see any other information on materials and methods section

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

please see any other information on materials and methods section

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethyldocosan-1-aminium chloride and N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethylicosan-1-aminium chloride

EC Number:

947-079-6

IUPAC Name:

Reaction mass of N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethyldocosan-1-aminium chloride and N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethylicosan-1-aminium chloride

Test material form:

other: paste

Specific details on test material used for the study:

Identification: Methoxycinnamidopropyl Behendimonium Chloride
Physical state/Appearance: Off-white paste
Batch: INV-1603010
Purity: 29.37% (solids content, % by mass)
Expiry Date: 01 February 2017
Storage Conditions: Room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Details on animal used as source of test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier: MatTek
Date received: 14 June 2016
EpiDermTM Tissues (0.63cm2) lot number: 23341
Assay Medium lot number: 060916ZSA

Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Dye Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:
Approximately 135 mg of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
If the MTT solution containing the test item turns blue relative to the control, the test item was presumed to have reduced the MTT.

Assessment of Color Interference with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
Approximately 135 mg of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 oC, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

Main Test
Pre-Incubation
The assay medium was pre-warmed 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.

Application of Test Item and Rinsing
Before pre-incubation was complete, a 24 well plate was prepared for use as a “holding plate” for both the 3 Minute and 60 Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24 well plate was prepared for the MTT loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 Minute exposure period was returned to the incubator, while the other was being dosed for the 60 Minute exposure. For the 60 Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 0.05 ml of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60 Minute exposure period.
When dosing for the 60 Minute exposure period was complete, the same procedure was repeated for the 3 Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS 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. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60 Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3 Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

Absorbance/Optical Density Measurements
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Negative Control: 50 µL of sterile distilled water. The negative control item was used as supplied.
Positive Control: 50 µL of 8.0 N Potassium Hydroxide. The positive control item was used as supplied.
Test Item: 0.05 ml

Duration of treatment / exposure:

60 minutes and 3 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes (Mean)

Value:

87.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive to skin

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute (mean)

Value:

99.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive to skin

Other effects / acceptance of results:

Direct MTT Reduction
The MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.

Assessment of Color Interference with the MTT endpoint
The solution containing the test item was a white color. This color was considered not to have the potential to cause color interference with the MTT endpoint.

Quality Criteria
The mean OD562 for the negative control treated tissues was 1.834 for the 3 Minute exposure period and 1.766 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 4.6% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.

In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	MeanOD562of individual tissues	Mean OD562of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.826	1.834	0.011	0.6	100*
		1.841
	60 Minutes	1.747	1.766	0.027	1.5
		1.785
Positive Control	3 Minutes	0.093	0.103	0.013	na	5.6
		0.112
	60 Minutes	0.083	0.081	0.004	na	4.6
		0.078
Test Item	3 Minutes	1.415	1.609	0.274	17.0	87.7
		1.802
	60 Minutes	1.819	1.765	0.076	4.3	99.9
		1.711

OD= Optical density

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

na=  Not applicable

Relative mean % tissue viability = (mean OD562 of test item/ mean OD562 of negative control) x 100

Coefficient of variation = (standard deviation/ mean OD562 of duplicate tissues) x 100

Applicant's summary and conclusion

Interpretation of results:

other: Non-corrosive to the skin.

Conclusions:

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

Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item 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.

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 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).

Results

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

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	5.6	87.7
60 minute	100*	4.6	99.9

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

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

Conclusion

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