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Diss Factsheets
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EC number: - | CAS number: -
- 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:
- 2020
- 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 18 June 2019
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Unknown impurities
- IUPAC Name:
- Unknown impurities
- Reference substance name:
- Water
- EC Number:
- 231-791-2
- EC Name:
- Water
- Cas Number:
- 7732-18-5
- Molecular formula:
- H2O
- IUPAC Name:
- Dihydrogen oxide
- Reference substance name:
- pentasodium 4-amino-3-(2-{4-[2-(2,4-disulfonatophenyl)diazen-1-yl]-3-methylphenyl}diazen-1-yl)-5-hydroxy-6-[2-(4-nitro-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate
- Cas Number:
- 2259360-19-3
- Molecular formula:
- C29H17N8Na5O18S5
- IUPAC Name:
- pentasodium 4-amino-3-(2-{4-[2-(2,4-disulfonatophenyl)diazen-1-yl]-3-methylphenyl}diazen-1-yl)-5-hydroxy-6-[2-(4-nitro-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate
- Test material form:
- solid: particulate/powder
- Details on test material:
- Batch XMEF 1476
impurity 1
impurity 2
Constituent 1
Test animals / tissue source
- Species:
- other: in vitro test with human cornea cells
- Strain:
- other: three-dimensional human cornea model tissue model
- Details on test animals or tissues and environmental conditions:
- - Justification of the test method and considerations regarding applicability:
Eye irritation is generally defined as "the production of reversible changes in the eye". The potential for chemical induced eye irritation is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. It was usually determined in vivo in the Draize rabbit eye irritation test as described in OECD guideline 405. In a pre-validation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential.
A limitation of the Test Guideline OECD 492 is that it does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B), as defined by UN GHS. For these purposes further testing with other suitable test methods is required.
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.
The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazol 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, 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.
The technical proficiency of the test system according to OECD Guideline 492 guideline recommended proficiency substances was demonstrated.
Test system
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg (corresponds to according to Guideline a minimum of 83.3 mg/cm²)
- positive control: 50 µL
- negative control: 50 µL - Duration of treatment / exposure:
- 6 hours at standard culture conditions (5% CO2, 37°C, 95% humidity) followed by a post-soak immersion period of about 25 min in fresh medium
- Duration of post- treatment incubation (in vitro):
- 18 hours (37°C, 5% CO2, 95% humidity)
- Number of animals or in vitro replicates:
- each 2 tissues for the test item, positive and negative controls
- Details on study design:
- - RhCE tissue construct used, including batch number: The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier.
RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.
- Doses of test chemical and control substances used: 50 mg of the solid test item, 50 µL negative control (deionized water) and positive control (neat methyl acetate), respectively, were applied to the EpiOcular™ tissue surface in duplicate.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods for solid test items: 37 ± 2°C; 6 hours exposure, 25 min. post-soak immersion, 18 hours post-treatment incubation
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): The optical pre-experiment (color interference pre-experiment) to investigate the test item’s color change potential in water or isopropanol led to a change in color. Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed purple color. Therefore, an additional test with freeze-killed tissues was necessary.
Since the OD of the test item in deionised water or isopropanol at 570 nm after blank correction was > 0.08 and the test item interfered with MTT, Non-Specific Killed Controls (NSKC) were necessary in the main experiment additionally.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): each 2 tissues for the test item, positive and negative controls
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: For interpretation of cell viability results the cut-off value distinguishing classified (irritant) from non-classified substances as given in OECD TG 492 was used:
- The test chemical is identified as not irritant and not requiring classification according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.
- The test chemical is identified as irritant and potentially requiring classification according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%.
- Positive and negative control means and acceptance ranges based on historical data: The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.8
- mean relative viability of the positive control (PC) is < 50 % (relative to negative control)
- the difference of viability between the two replicates is < 20 %.
Results and discussion
In vitro
Results
- Irritation parameter:
- other: mean percent tissue viability
- Run / experiment:
- mean of two tissue cultures
- Value:
- 88.77
- Vehicle controls validity:
- not examined
- Remarks:
- no vehicle used
- Negative controls validity:
- valid
- Remarks:
- 100 % final cell viability
- Positive controls validity:
- valid
- Remarks:
- 5.30 % final cell viability
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
Any other information on results incl. tables
According to the conducted pre-check the test items were demonstrated to exert optical interferences directly affecting the test results that are not related to cytotoxic effects on tissue cells. Therefore killed controls and colour controls had to be conducted.
Colour of test item | MTT Reduction | Colour reaction in isopropanol | Colour reaction in water | Killed control test to be conducted | Colour control test to be conducted | Non specific Killed control test to be conducted |
black | + | + | + | yes | yes | yes |
+ positive reaction, - negative reaction
As the final test item-treated tissue viability was > 60% relative to negative control, the test item was characterized as NOT having eye irritating properties (UN GHS No Category):
Compound | Final cell viability [%] | Category |
Test item | 88.77 | No category |
Positive control | 5.30 | Cat. 1 / 2 |
Negative control | 100.00 | No category |
All assay acceptance criteria were met.
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Remarks:
- not irritant
- Executive summary:
This in vitro study was performed to assess the eye irritation potential of the registered substance by means of the Human Cornea Model Test following OECD TG 492. The test item proved to be an MTT reducer and to dye water and isopropanol in the color interference pre-experiment. The OD of the test item in isopropanol at 570 nm after blank correction was > 0.08. Therefore, additional tests with freeze-killed tissues, viable tissues and Non-specific Killed Control (NSKC) tissues (without MTT addition) had to be performed.
Each 50 mg of the solid test item or 50 µL of the negative control (deionised water) and of the positive control (methyl acetate), were applied to each duplicate tissue for 6 hours. Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.
After treatment with the test item the mean relative viability value was 88.77% compared to the mean value of the negative control. This value is above the threshold for irritancy of ≤ 60%. Therefore, the test item does not need to be classified according UN GHS.
In conclusion, it can be stated that in this study and under the experimental conditions reported, the registered substance does not need to be classified for eye irritation according UN GHS.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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