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EC number: 948-778-9 | 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:
- 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
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)
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate
- EC Number:
- 948-778-9
- Molecular formula:
- C14O8H19N2Na3
- IUPAC Name:
- trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate
- Test material form:
- liquid
- Details on test material:
- Active ingredient (%): 48.9% dry substance
Purity (%): 95.50 %
Stability: 5 years
Sterilization: None
Solubility: Soluble in water
Storage: Room Temperature
1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 23ADB/50
- Expiration date of the lot/batch: 21/11/2023
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature
PRE-TEST
Optical properties of test item or its action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability for this reason Pre-tests have been performed to allow identification potential direct MTT reducers and/or interfering and define, if additional controls need to be used to detect and correct for potential interference from such test item.
Assessment for non-coloured test item
In 6-well plates, 50 ul of test sample has been added to 2 ml isopropanol, and incubated for 3 hours at
room temperature; at the end of incubation period a visual observation has been performed and any
change in colour has been recorded.
No colour change has been observed neither in water nor isopropanol.
Assessment of Direct Reduction by MTT
The test sample has been put in contact with MTT solution to detect non-specific reduction of MTT.
In a 6-well plate, 50 ul of test sample have been added to 1 ml of MTT solution 1.0 mg/ml and
incubated at 37±1°C, 5±1% CO2 for 180±15 minutes.
MTT solution in contact with the test substance has caused change in colour.
The test sample is presumed to have not the potential to stain the tissue, and has been considered no interacting with the MTT measurement this no additional controls need to be performed.
TEST
Pre-treatment
Before the beginning of the test, the tissues have been pre-treated with 20 ul of DPBS without Ca2+
and Mg2+ for 30±2 minutes at 37±1°C, 5±1% CO2, protected from light.
Test animals / tissue source
- Species:
- human
- Details on test animals or tissues and environmental conditions:
- - Justification of the test method and considerations regarding applicability
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
EpiOcularTM from MatTek Corporation, is a 3D system consists of a non-keratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively, but not cornified, cells.
The tissue is prepared in inserts with a porous membrane through which the nutrients pass to the cells. The ability to expose the tissue topically is essential to model the same kind of progressive injury expected in vivo.
Potential biological contaminants have been assessed by the system supplier.
The following contaminants have not been detected:
HIV-1 virus
Hepatitis B virus
Hepatitis C virus
Bacteria, yeast and other fungi
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 µl
- Concentration (if solution): n/a
VEHICLE
- Amount(s) applied (volume or weight with unit): n/a test sample have been tested neat
- Concentration (if solution): n/a - Duration of treatment / exposure:
- 30±2 minutes at 37±1°C, 5±1% CO2
- Duration of post- treatment incubation (in vitro):
- At the end of the treatment, the test sample and the controls have been removed from the tissues and rinsed with 100 ml of DPBS for 3 times without Ca2+ and Mg2+.
Each tissue have been transferred in a 12 well plate with 5 ml of Assay Medium and incubated for 12±2 minutes at room temperature, then transferred in 6-well plate containing 1 ml of Assay Medium and incubated for 120±15 minutes at 37±1°C, 5±1% CO2. - Number of animals or in vitro replicates:
- 2 replicates, 3 repetition each
- Details on study design:
- - Details of the test procedure used
CULTURE MEDIA AND REAGENTS
The validity of culture media and reagent has been assessed before starting the analyses.
EpiOcularTM (MatTek Corporation)
Assay Medium (OCL-200-ASY) (MatTek Corporation)
Dulbecco’s Phosphate buffer solution (DPBS) (MatTek Corporation)
MTT thiazolyl blue tetrazolium (MTT-100-CON) (MatTek Corporation)
MTT diluent (MTT-100-DIL) (MatTek Corporation)
Isopropyl alcohol (IPA) (MTT-100-EXT) (MatTek Corporation)
Methyl Acetate (MatTek Corporation )
Ultrapure Water (Eurospital))
SOLUTIONS
MTT SOLUTION: MTT thiazolyl blue tetrazolium 5 mg/ml has been diluted with MTT diluent up to 1 mg/ml.
The MTT SOLUTION has been prepared at use, sheltered from light, and discarded at the end of the study.
EQUIPMENT
The validity of instruments and equipment has been assessed before starting the analyses.
Laminar flow filtered work area (Flow)
CO2 incubator (Flow)
Chronometer (Oregon Scientific)
Microplate reader Mod EL800 (Bio-Tek)
- RhCE tissue construct used, including batch number :
EpiOcularTM from MatTek Corporation, is a 3D system consists of a non-keratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively, but not cornified, cells.
The tissue is prepared in inserts with a porous membrane through which the nutrients pass to the cells. The ability to expose the tissue topically is essential to model the same kind of progressive injury expected in vivo.
- Doses of test chemical and control substances used :
Assay Sample
50 µl of the test sample have been tested neat.
Controls
Positive control: 50 µl of neat Methyl Acetate have been used as positive control.
Negative control: 50 µl of ultrapure water have been used as negative control.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
Treatment
The test sample, the negative and positive control have been topically applied on tissues replicates for 30±2 minutes at 37±1°C, 5±1% CO2, to respect the exposure times the applications have been performed at intervals of not less than 1 minute.
Post treatment incubation
At the end of the treatment, the test sample and the controls have been removed from the tissues and rinsed with 100 ml of DPBS for 3 times without Ca2+ and Mg2+.
Each tissue have been transferred in a 12 well plate with 5 ml of Assay Medium and incubated for 12±2 minutes at room temperature, then transferred in 6-well plate containing 1 ml of Assay Medium and incubated for 120±15 minutes at 37±1°C, 5±1% CO2.
MTT TEST
-After the post treatment, the tissues have been treated for 180±10 minutes at 37±1°C, 5±1% CO2 with 300 µl of MTT SOLUTION 1 mg/ml and then submerging in 2 ml of Isopropanol for 2 hours at room temperature with gentle agitation (about 120 rpm), for formazan extraction.
A 96-well plate has been prepared by transferring 3 replicates of 200 ul of extracts from positive control, negative control and sample, to read the optical density (OD) at 570 nm at the microplate reader. Isopropanol has been used as blank.
Optical density measurements
The absorbance is measured on a microplate reader using a 570 nm wavelength using a GEN5
software (Biotek).
ACCEPTABILITY CRITERIA
Negative control: mean OD570nm of the tissue of negative control should be >0.8 and <2.5 .
Positive control: mean viability of the tissue replicates exposed for 30 min with the positive control, expressed as % of the negative control, should be <50%.
Difference of viability: the difference of viability between two tissue replicates should be <20%.
INTERPRETATION OF RESULTS
The OD values obtained with the replicate tissue extracts is used to calculate the mean percent tissue viability normalised to the negative control, which is set at 100%.
The percentage tissue viability cut-off value for identifying test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) is given in Table below for Prediction Models according to UN GHS classification.
Results should thus be interpreted as follows:
- The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) the established percentage tissue viability cut-off value, as shown in Table for Prediction Models according to UN GHS classification
In this case no further testing in other test methods i s required.
- If the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to the established percentage tissue viability cut-off value, no prediction can be made, as shown in Table for Prediction Models according to UN GHS classification.
Table: Prediction Models according to UN GHS classification
Tissue No Category No prediction can be made
EpiOcularTM EIT Mean tissue viability>60% Mean tissue viability≤60%
Results and discussion
In vitro
Results
- Irritation parameter:
- other: viability vs negative control
- Value:
- 87.5
- Vehicle controls validity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: the mean percent tissue viability after exposure and post-exposure incubation is >60%, the established percentage tissue viability cut-off value, for Prediction Models according to UN GHS classification
Any other information on results incl. tables
RESULTS
Optical Density |
Tissue 1 |
Tissue 2 |
||||
Repetition 1 |
Repetition 2 |
Repetition 3 |
Repetition 1 |
Repetition 2 |
Repetition 3 |
|
Blanks |
0,041 |
0,041 |
0,041 |
0,041 |
0,041 |
0,041 |
Negative control (NC) |
2,464 |
2,487 |
2,435 |
2,508 |
2,479 |
2,459 |
Positive control (PC) |
0,087 |
0,086 |
0,088 |
0,075 |
0,075 |
0,074 |
Test sample |
2,127 |
2,114 |
2,089 |
2,222 |
2,232 |
2,224 |
ASSAY VALIDITY CRITERIA |
Value |
Acceptability |
Result |
|
Negative |
Mean OD value |
2.431 |
≥0.8 and≤2.5 |
Comply |
Difference of viability % |
0.822 |
<20% |
Comply |
|
Positive |
Mean % Viability |
1.65 |
<50% |
Comply |
Difference of viability % |
0.493 |
<20% |
Comply |
|
Sample |
Difference of viability % |
4.772 |
<20% |
Comply |
SAMPLE |
% VIABILITY |
TRISODIUM (2S)-2,6-BIS(3- |
87.5 |
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- On the basis of the results, interpreted according to OECD 492:2017, can be stated that the test item “TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE” belong to No Category for eye.
- Executive summary:
On the test item “TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE” anin vitro toxicological study aimed to evaluate ocular irritation potential has been carried out.
The following test has been performed:
-in vitro ocular irritation – Reconstructed EpiocularTMtissue model test method.
To perform thein vitro Ocular Irritation on EpiOcularTM, a Tissue Model from MatTek Corporation, Cornea-Like 3-D Tissue Structure, has been used.
MatTek’s EpiOcular system consists of normal, primary human-derived epidermal keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. Cultured on specially prepared cell culture inserts using serum-free culture medium, the cells differentiate to form a multi-layered structure which closely parallels the corneal epithelium.
Histological examination of the RhCE tissue construct on one tissue not treated that should demonstrate appropriated human cornea-like epithelium structure (including at least 3 layers of viable epithelial cells and a non-keratinized surface) has been performed.
Pre-tests to identify identification potential direct MTT reducers and/or interfering and define, if additional controls need to be used to detect and correct for potential interference from such test item, have been performed.
On the basis of obtained results in pre-tests, no additional controls have been performed.
To performin vitro Ocular Irritation test, two replicates of three tissues series, one for test sample, one for negative control and one for positive control have been used.
The test sample has been topically applied on tissues replicates for 30 minutes at 37±1°C, 5±1% CO2. At the end of the treatment tissues have been rinsed with 100 ml for 3 times of DPBS without Ca2+and Mg2+.then transferred in a 12 well plate with 5 ml of Assay Medium and incubated for 12±2 minutes at room temperature in order to remove any test article absorbed by the tissues, then tissues have been transferred in 6-well plate containing 1 ml of Assay Medium and incubated for 120±15 minutes at 37±1°C, 5±1% CO2.
Cell viability determination is based on cellular dehydrogenase activity, measured by MTT reduction and conversion into blue formazan salt that is quantified after extraction from tissues.
The aim of this assay has been to assess quantitatively the effects of the tested product on cell survival through the MTT assay.
After incubation, tissues have been treated with MTT SOLUTION 1 mg/ml for 180±10 minutes at 37±1°C, 5±1%CO2,then tissues have been treated with Isopropanol for 2 hours at room temperature with gentle agitation (about 120 rpm) for formazan extraction. The percentage reduction in viability is used to predict the irritation potential.
On the basis of the results, interpreted according to OECD 492:2017, can be stated that the test item TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE belong to No Category for eye.
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