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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 - 08 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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted in 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Slovak National Accreditation 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

Test animals / tissue source

Species:

human

Strain:

other: EpiOcular™ Eye Irritation Test (EIT)

Details on test animals or tissues and environmental conditions:

- Description of the cell system used, justification of the test method and considerations regarding applicability:
The EpiOcular™ OCL-200 tissue construct is a nonkeratinized epithelium prepared from primary human keratinocytes. It was obtained from MatTek In Vitro Life Science Laboratories, Slovak Republic (OCL-200-EIT, Lot no. 30633). It models the cornea epithelium with progressively stratified, but not cornified cells. The ability to expose the tissue topically is essential to model the same kind of progressive injury expected in vivo. In this assay, the test substance is applied to the surface of the cornea epithelial tissue construct for a fixed period, removed, and the tissue allowed to express the possibly resulting damage. Relative tissue viability is determined against the negative control-treated constructs by the reduction of the vital dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). A concurrent positive control is used with each assay. Based on the depth of injury model, the EpiOcular eye irritation test is intended to differentiate those materials that are non-irritants (would not require classification according to GHS) from those that would require labelling as either eye irritant category 2 or causing eye damage category 1 according to GHS.
According to test guideline OECD 492, this test has a broad applicability range. It allows amongst others testing of solid substances, soluble or insoluble in water, to be applied directly to the tissue. The test substance FV-1295 is a solid and readily soluble in water; thus is is within the applicability domain of the test system described.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg of the test substance (powder) was applied

NEGATIVE CONTROL
- Amount(s) applied: 50 µL of water for injection
- Lot no.: 18B2001

POSITIVE CONTROL
- Amount(s) applied: 50 µL of Methyl acetate
- Lot no.: 021319ISC

Duration of treatment / exposure:

6 h ± 15 min at 37 °C

Duration of post- treatment incubation (in vitro):

25 min post-soak at room temperature + 18 h post-treatment incubation at 37 °C

Number of animals or in vitro replicates:

Two tissue replicates for each treatment and control group.

Details on study design:

- Details of the test procedure used
The EpiOcular™ EIT consists of a topical exposure of the test substance to the human reconstructed cornea model followed by a cell viability test.
On the day of testing, the tissues were pre-wetted with Dulbecco's Phosphate Buffered Saline (DPBS). The tissues were incubated at standard culture conditions for 30 ± 2 min. Subsequently, the negative and positive controls were tested by applying 50 µL topically on the EpiOcular tissues. The test substance (50 mg) was applied topically onto the tissue surface. Two tissues were used per treatment, negative and positive controls. The tissues were placed back into the culture medium after dosing and incubated at standard culture conditions for 6 h ± 15 min.
At the end of the exposure time, tissues were rinsed with DPBS and tranferred to assay medium for a 25 ± 2 min immersion incubation (post-soak) at room temperature.
At the end of the post-soak immersion, each insert was transferred to fresh assay medium and incubated for a further 18 ± 0.25 h at standard culture conditions (post-treatment incubation).
After the post-treatment incubation, the MTT assay was performed.
Cell viability was measured by enzymatic conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide] into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

- RhCE tissue construct used, including batch number
In vitro EpiOcular™ OCL-200 was used. EpiOcular EIT is a reconstructed ocular tissue model containing primary human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. It was obtained from MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic. Lot no. 30633, Keratinocyte strain 4F1188.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
To assess whether the test substance has the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement, 50 mg of the test substance were added to 1 mL of MTT solution (1.0 mg/mL MTT in DMEM) and incubated for 3 h at 37 °C. At the end of the exposure time the colour of treated MTT remained unchanged. The test substance did not reduce MTT directly.
Coloured test substances may interfere with the quantitative photometric MTT measurement if the colourant binds to the tissue and is extracted together with MTT. Therefore, the test substance was checked for its colourant properties. 50 mg of the test substance were added to 2 mL of isopropanol and incubated for three hours at room temperature. The OD of the test substance solution was < 0.08 (blank corrected). The test substance does not interact with the MTT measurement. In addition, 50 mg of the test substance was added to 1 mL of purified water. The mixture was incubated for 60 min at 37 °C. Subsequently to incubation, presence of staining was evaluated. The colour of the mixture was unchanged. Therefore, an additional test with freeze-killed tissues was not performed.

- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
The optical densities (ODs) were read in a 96-well plate spectrophotometer (MRX II, Dynex) using a wavelength of 540 nm without a reference filter.

- Description of the method used to quantify MTT formazan
At the end of the 18 h post-treatment incubation, tissues were transferred to MTT solution (1 mg/mL) and incubated for 180 min at 37 °C. After incubation, each tissue was transferred to isopropanol, so that no isopropanol was flowing into the insert. The plates were stored overnight at 2 – 8 °C in the dark. At the end of the non-submerged extraction, inserts and tissues were discarded without piercing and isopropanol was added into each well. The extract solution was mixed and two aliquots were transferred to the appropriate wells of a pre-labeled 96-well plate and the absorbance at 540 nm (OD) of each well was measured with a plate reader (spectrophotometer MRX II, Dynex).

- Description of evaluation criteria used
In accordance with OECD TG 492, the test substance is considered to be not irritating to eye if the tissue viability after 6 h exposure is > 60% relative to the negative control-treated tissue.
If the test substance-treated tissue viability is ≤ 60% relative to negative control-treated tissue viability, the test item is considered to be irritant.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
Positive control: Mean tissue viability of the present study (30.9%), after treatment with the positive control, is within the historical control data range of 17.3 - 44.8%.
Negative control: Mean optical density value of the present study (1.504) after treatment with the negative control is within the historical control data range of 1.074 - 1.560.

- Complete supporting information for the specific RhCE tissue construct used
The quality of the EpiOcular™ OCL-200 tissue was assessed by an MTT cell viability test. The determined OD (540 - 570 nm) was 2.05 ± 0.055 (acceptance criteria: 1.1 - 3.0).
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 0.3% Triton X-100. The ET-50 value was determined to be 15.57 min (acceptance criteria: 12.2 - 37.5 min).
The cells used to produce the EpiOcular™ OCL-200 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.

- Reference to historical data of the RhCE tissue construct
MatTek's acceptance criteria are based on a quality check database. The historical control average of 0.3% Triton X-100 cell viability are 24.9 min ± 6.3 min (mean ± SD), minimum 12.2 min, maximum 37.5 min.

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
Demonstration of proficiency is stated in amendment No. 1 to the study report. The technical proficiency is demonstrated by using proficiency chemicals for in vitro eye irritation testing. From all proficiency substances included in the proficiency study 100% of the results were categorized correctly regarding the expected prediction model. For all control substances historical data demonstrated the reliability and validity of those substances.

- Positive and negative control means and acceptance ranges based on historical data
historical control data (HCD) positive control: mean cell viability ± SD = 31.05% ± 8.31%; range 17.3 - 44.8%.
historical control data (HCD) negative control: mean optical density ± SD = 1.323 ± 0.161; range 1.074 - 1.560 OD.

- Acceptance criteria and acceptable variability between tissue replicates for positive and negative controls
The results are acceptable if the negative control OD is > 0.8 and < 2.5 and if the mean relative viability of the positive control is < 50% of the negative control viability.
The assay is considered valid if the difference of viability between the two relating treatments is < 20% in the same run (for positive and negative control tissues).

- Acceptable variability between tissue replicates for the test chemical
The assay is considered valid if the difference of viability between the two relating tissues of a single test substance is < 20% in the same run.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability

Remarks:

mean of 2 tissues

Run / experiment:

6 h exposure + 18 h post-exposure

Value:

35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Demonstration of proficiency is stated in amendment No. 1 to the final report. The laboratory states technical proficiency is demonstrated by using proficiency chemicals for in vitro skin irritation testing. From all proficiency substances included in the proficiency study 100% of the results were categorized correctly regarding the expected prediction model. For all control substances historical data demonstrated the reliability and validity of those substances.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time (values were 1.433 and 1.576 for tissue 1 and 2, respectively).
- Acceptance criteria met for positive control: Yes, mean viability of the tissue replicates treated with the positive control was < 50% compared to the negative control (values were 31.0% and 30.8% for tissue 1 and 2, respectively).
See Table 1 under "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: Results of MTT assay of EpiOcular tissue after 6 h exposure to FV-1295.

	Negative control H2O		Positive control Methyl acetate		Test substance FV-1295
Tissue replicate	1	2	1	2	1	2
OD540 (blank corrected)	1.468	1.600	0.469	0.461	0.466	0.596
	1.398	1.551	0.464	0.467	0.446	0.595
OD540 (mean)	1.433	1.576	0.467	0.464	0.456	0.596
OD540 (mean of replicates)	1.504		0.465		0.526
Mean tissue viability [%]	100.0		30.9		35.0
Difference of viabilities [%]	9.47		0.17		9.27

Applicant's summary and conclusion

Interpretation of results:

other: irritating potential

Conclusions:

The determined cell viability of tissue cultures treated with the test substance FV-1295 fulfilled the criteria for irritancy (< 60%). Therefore, under the conditions of the conducted test, the test substance is considered to possess an irritating potential towards human cornea in the EpiOcular™ model but the result is not conclusive with respect to classification of the test substance as eye irritant (Eye Irritant Cat. 2) or serious eye damage (Eye Damage Cat. 1). Based on the precautionary principle, the substance FV-1295 will be classified for causing serious eye damage category 1 (Eye Damage Cat. 1 according to Regulation (EC) No. 1272/2008).