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

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Remarks:

(screen for acute oral toxicity)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 20 June 2016; Experimental completion date: 14 July 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:

other: OECD Guidance Document No. 129: Guidance Document on Using Cytotoxicity Tests to Estimate Starting Doses for Acute Oral Systemic Toxicity Tests. 20 July 2010

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ICCVAM-Recommended Test Method Protocol, BALB/c 3T3 NRU Cytotoxicity Test Method (2006)

Principles of method if other than guideline:

The basis of the NRU assay is that 3T3 cells will normally grow in culture, continuously dividing and multiplying with time. A cytotoxic chemical will interfere with this process resulting in a reduction of the growth rate reflected by a decreased cell number. The growth of 3T3 cells treated with a range of concentrations of the test item is compared with vehicle control cultures after 48 hours exposure. The cell numbers are then estimated using the neutral red uptake technique, which depends on the ability of viable cells to take up neutral red into their lysosomes. The amount of neutral red taken up by each treated culture is measured and is proportional to the number of viable cells present. The data are used to estimate the concentration producing 50% inhibition (IC50). Data from this in vitro test will be used for estimating the starting dose for acute oral systemic toxicity tests. The in vivo starting dose is an estimated LD50 value calculated by inserting the in vitro IC50 value into a regression formula.

GLP compliance:

yes (incl. QA statement)

Test type:

other: 3T3 NRU cytotoxicity test (screen for acute oral toxicity)

Limit test:

no

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:

Sponsor’s identification: Methoxy Cinnamidopropyl Behenyl Dimonium Chloride
Batch No.: INV-1603010
Purity: Solids content, 29.3%
Appearance: Pearly off-white paste
Expiry/retest date: February 2017
Storage conditions: Room temperature

Test animals

Species:

other: Not applicable

Administration / exposure

Route of administration:

other: test item in cell culture dilution medium

Vehicle:

other: cell culture dilution medium (containing 0.5% v/v ethanol)

Doses:

Range-finding test: 100 µg/mL to 10 pg/mL.
Main test: 80, 44.9, 25.2, 14.2, 8.0, 4.5, 2.5 and 1.4 µg/mL.
Main test repeated: 40, 22.5, 12.6, 7.1, 4.0, 2.2, 1.3 and 0.7 µg/mL.

Details on study design:

CELL CULTURES:
The cells used in this assay were from the permanent murine fibroblast cell line, BALB/c 3T3 cells, clone 31, obtained from the American Type Culture Collection (ATCC). They were certified to be mycoplasma free. The passage number of the cells used in the cytotoxicity assays ranged from 85 to 95. The cells were routinely grown and subcultured in high glucose (4.5 g/L) Dulbecco’s Modified Eagles Medium containing 4 mM L-glutamine, supplemented with 10% newborn calf serum, 100 IU penicillin and 100 µg/mL streptomycin at 37°C ± 1°C in a humid atmosphere containing 5% carbon dioxide.

CELL CULTURE FROM FROZEN STOCKS:
Vials of 3T3 cells, stored frozen in cryotubes at -196°C under liquid nitrogen, in medium containing 5% dimethyl sulphoxide, were thawed rapidly at 37°C in a water-bath and the contents diluted with pre-warmed cell culture medium in tissue culture flasks. The medium was replaced when the cells had attached to the bottom of the flask (within 4 to 24 hours). The flasks were incubated until 50 - 80% confluent cell monolayers had been obtained.

CELL PASSAGE:
Actively growing cell stocks were maintained and expanded by subculturing (passage). When the cells had reached 50 – 80% confluence, the medium from each flask was removed, the cells washed with phosphate buffered saline (PBS) and harvested using trypsin-EDTA solution. Cultures were incubated at 37 ± 1°C until complete detachment and disaggregation of the cell monolayer had occurred. The cells were then resuspended in medium to neutralise the trypsin. The cells were resuspended and distributed into flasks containing fresh medium. This passage procedure was repeated to provide a sufficient number of cells for the test, and the cells were passaged at least twice before using the cells in the cytotoxicity test. The passages of 3T3 cells from frozen stock was limited to 18 passages.

PREPARATION OF TEST CELL CULTURES:
The cells from flasks of actively growing cultures were detached and disaggregated as described above. The number of viable cells in the prepared cell suspension was determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. The cell suspension was diluted with medium to give 3.0 x 10^4
viable cells/mL and 100 µL volumes pipetted into the relevant wells of sterile 96-well flat- bottomed microtitre plates, according to the format shown in illustration section. Some wells received 100 µL cell culture medium with no cells. These plates were incubated for 24 ± 2 hours at 37 ± 1°C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach.

VC1 and VC2 were the left and right vehicle control wells, which contained cells and cell culture medium. VCb wells were VC blanks that contained routine cell culture medium but not cells.

C1 – C8 were the eight test item or positive control concentrations. C1 was the highest concentration and C8 was the lowest. Each concentration had six replicate wells.
Cxb were blank wells that contained test item or positive control, but not cells.

PREPARATION OF TEST AND CONTROL ITEMS:
Positive control:
The positive control, sodium lauryl sulphate, was diluted with cell culture dilution medium (as cell culture medium but containing 5% NCS) to give the following concentrations: 80, 70, 60, 55, 50, 40, 30 and 20 µg/mL.

Test Item:
The test item was supplied as a paste formulation. A solubility test was conducted in which solubility in cell culture dilution medium, DMSO and ethanol was assessed. Vortexing, sonication and warming at 37°C were used to assist solubilisation. The test item was insoluble in culture medium when formulated at 100, 10 and 1 mg/mL. It formed a cloudy suspension in DMSO at 100 mg/mL and a slightly cloudy solution in ethanol at 100 mg/mL. It was found to be soluble in ethanol at 10 mg/mL. When the 10 mg/mL solution in ethanol was dosed into culture medium at 0.5% v/v to give a final concentration of 50 µg/mL it was found to be soluble. The highest final concentration of the test item in culture medium chosen for the rangefinder test was 100 µg/mL, from an initial formulation of 20 mg/mL in ethanol dosed at 0.5% v/v.

For the rangefinder test, a series of eight ten-fold dilutions in ethanol from 20 mg/mL to 2 ng/mL was prepared, giving final concentrations of 100 µg/mL to 10 pg/mL.

For the main test, a series of eight concentrations of the test item in a decimal geometric series using a dilution factor of 1.78 (= 4√10) was formulated in ethanol from 16 mg/mL to 283 µg/mL, giving final concentrations of 80, 44.9, 25.2, 14.2, 8.0, 4.5, 2.5 and 1.4 µg/mL. This main test was subsequently repeated using concentrations formulated in ethanol from 8 mg/mL to 141 µg/mL, giving final concentrations of 40, 22.5, 12.6, 7.1, 4.0, 2.2, 1.3 and 0.7 µg/mL.

TEST PROCEDURE:
After incubation of the 96 well plates, the cells formed a <50% confluent monolayer.

The cell culture medium was removed by careful inversion of the plate and blotting onto sterile paper towel to remove residual culture medium. The test item in cell culture dilution medium (containing 0.5% v/v ethanol) was added in 100 µL aliquots according to the plate layout. Cell culture dilution medium (containing 0.5% v/v ethanol) was added in 100 µL aliquots to the VC wells and appropriate blanks. The plates were incubated at 37 ± 1°C in a humidified atmosphere of 5% CO2 in air for 48 ± 0.5 hours.

After at least 46 hours, the plates were inspected using an inverted microscope and the condition of the cells or any precipitate noted. The plates were then returned to the incubator.

After incubation, the cell culture medium was removed by careful inversion of the plate and blotting onto sterile paper towel to remove residual culture medium. The cells were rinsed carefully with 250 µL per well pre-warmed Dulbecco’s Phosphate Buffered Saline (DPBS). This was removed by inversion of the plate and blotting onto sterile paper towel. Neutral red was added in 250 µL aliquots per well (neutral red prepared at 25 µg/mL in DMEM with 5% NCS, 4 mM L-glutamine, 100IU/mL penicillin and 100 µg/mL streptomycin). The neutral red was prepared from a stock solution and filtered before use using a 0.45 µm filter. It was stored at 37°C in a water bath before adding to the cells and used within 60 minutes of preparation. The cultures were incubated for three hours at 37 ± 1°C in a humidified atmosphere containing 5% CO2.

After three hours the cultures were examined briefly to assess neutral red uptake. The neutral red medium was removed and the plates rinsed with 250 µL per well warm DPBS before adding 100 µL/well of NR desorb solution (49% distilled water, 50% ethanol, 1% glacial acetic acid). The plates were then gently shaken on a microtiter plate shaker for 20 – 45 minutes. The plates were protected from light while shaking. The plates were allowed to sit for at least five minutes, after which time any bubbles remaining were ruptured. The absorbance value of each well was read using a plate reader with a 540 nm filter. The blank wells were used as a reference.

Results and discussion

Preliminary study:

The pH of the formulation of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride at the highest concentration, 100 µg/mL, was measured as 7.5.

Range finder test:
The results for the toxicity of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride in the rangefinder test, as determined by neutral red uptake were:
Methoxy Cinnamidopropyl Behenyl Dimonium Chloride produced a relative toxicity value that was 31% of the viablity of the vehicle control at the highest concentration, 100 µg/mL. All lower concentrations were non-toxic (≥85% relative toxicity). A visual assessment of the cell monolayers showed that the test item produced rounded cells and a small amount of precipitate at the highest concentration, 100 µg/mL, whereas all lower concentrations produced 70% cell confluency, the same as the vehicle control, with no precipitate. As the highest concentration of 100 µg/mL was insoluble, it was not used in the data analysis.
However, as the relative toxicity did not fall below 50% for the concentrations of 10 µg/mL and below, an IC50 value was not calculated.

The results for the toxicity of the positive control, sodium lauryl sulphate, as determined by neutral red uptake wer:
The IC50 was calculated to be 55.44 µg/mL, which lay within the historical control range of the laboratory. The coefficient of determination (R2) was 0.9316 which met the acceptance criterion of ≥ 0.85, and the required range of cytotoxicity values was observed.

The mean corrected absorbance of the left (VC1) and the mean corrected absorbance of the right (VC2) columns differed by 6% for the positive control and 4% for Methoxy Cinnamidopropyl Behenyl Dimonium Chloride which were both ≤15% from the mean corrected absorbance of all VCs. Therefore, the acceptance criteria were met.

Effect levels

Dose descriptor:

other: Relative toxicity (%)

Effect level:

69 other: %

Based on:

test mat.

Remarks on result:

other: IC50 not determined

Other findings:

Main Test:
As a slight precipitate was observed at the highest concentration, 100 µg/mL, in the rangefinding test, the highest concentration was reduced to 80 µg/mL in the main test. However, this initial main test was not reported as the mean corrected absorbance of the left (VC1) and the mean corrected absorbance of the right (VC2) columns differed by 28% for the positive control and 27% for Methoxy Cinnamidopropyl Behenyl Dimonium Chloride, which were both >15% from the mean corrected absorbance of all VCs, so this acceptance criterion was not met.

The main test was, therefore, repeated. However, as a precipitate was observed at the highest concentration, 80 µg/mL, and a very small amount of precipitate was observed at the second highest concentration, 44.9 µg/mL, the highest concentration was reduced to 40 µg/mL for the repeat test.

The results for the toxicity of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride in the main test, as determined by neutral red uptake, are shown in the table "Toxcity Data for Methoxy Cinnamidopropyl Behenyl Dimonium Chloride - Main Test". Methoxy Cinnamidopropyl Behenyl Dimonium Chloride produced a relative toxicity value that was 69% of the viablity of the vehicle control at the highest concentration, 40 µg/mL. All lower concentrations were non-toxic (≥82% relative toxicity). A visual assessment of the cell monolayers showed that the test item produced 70% cell confluency, the same as the vehicle control, at all concentrations, with no precipitate. As the relative toxicity did not fall below 50%, an IC50 value was not calculated.

The results for the toxicity of the positive control, sodium lauryl sulphate, as determined by neutral red uptake, are shown in Table "Toxicity Data for the positive control - Main Test". The IC50 was calculated to be 62.92 µg/mL, which lay within the historical control range of the laboratory. The coefficient of determination (R2) was 0.9680 which met the acceptance criterion of ≥ 0.85, and the required range of cytotoxicity values was observed.

The mean corrected absorbance of the left (VC1) and the mean corrected absorbance of the right (VC2) columns differed by 2% for the positive control and 2% for Methoxy Cinnamidopropyl Behenyl Dimonium Chloride which were both ≤15% from the mean corrected absorbance of all VCs. Therefore, the acceptance criteria were met.

Any other information on results incl. tables

Table: Toxicity Data for Methoxy Cinnamidopropyl Behenyl Dimonium Chloride – Main Test              

Concentration of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride (µg/ml)	Relative Toxicity (%)
0	100
0.7	96
1.3	86
2.2	86
4.0	87
7.1	85
12.6	87
22.5	82
40	69

 

Table: Toxicity Data for the Positive Control – Main Test                                                                                      

Concentration of SLS (µg/ml)	Relative Toxicity (%)
0	100
20	106
30	97
40	95
50	81
55	74
60	57
70	18
80	4

 

SLS = sodium lauryl sulphate

IC₅₀= 62.92 µg/mL

Historical Control Data

n	Date	IC50
1	26 May 16	62.65
2	09 Jun 16	51.67
3	10 Jun 16	52.78
4	16 Jun 16	57.85
5	17 Jun 16	57.46
	Mean	56.48
	SD	4.41

 

Laboratory Historical Data Range (Mean +/- 2.5 x SD): 45.46 to 67.50

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

It was concluded that it was not possible to calculate an IC50 value for Methoxy Cinnamidopropyl Behenyl Dimonium Chloride as the highest soluble concentration, 40 µg/mL, produced a relative toxicity value of 69%.

Executive summary:

The aim of this study was to evaluate the cytotoxicity of Methoxy Cinnamidopropyl Behenyl Dimonium Chloride, using a Neutral Red Uptake (NRU) assay in 3T3 cells.

The growth of 3T3 cells treated with a range of concentrations of the test item was compared with vehicle control cultures after 48 hours exposure both visually and using neutral red uptake.

Methoxy Cinnamidopropyl Behenyl Dimonium Chloride was demonstrated to be slightly toxic at the highest soluble concentration, 40 µg/mL, giving 69% viability relative to the vehicle control, as assessed by neutral red uptake.

The IC₅₀ value of the positive control, sodium lauryl sulphate, was calculated to be 55.44 µg/mL for the rangefinding test and 62.92 µg/mL for the main test, which both lay within the historical control range of the laboratory.

It was concluded that it was not possible to calculate an IC₅₀ value for Methoxy Cinnamidopropyl Behenyl Dimonium Chloride as the highest soluble concentration, 40 µg/mL, produced a relative toxicity value of 69%.