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EC number: 812-548-5 | CAS number: 1621424-03-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 020
- 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
- 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
Constituent 1
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).
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