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EC number: 219-283-9 | CAS number: 2402-79-1
- 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:
- 14 July 2009 to 16 July 2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 009
- Report date:
- 2009
Materials and methods
Test guideline
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes (1, 2).
The SkinEthic RHC model consists of transformed human keratinocytes of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. Test materials are applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium.
The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHC model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.
Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the test material treated tissues (quantitative measurement of tissue viability) relative to the negative control. - GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- 2,3,5,6-tetrachloropyridine
- EC Number:
- 219-283-9
- EC Name:
- 2,3,5,6-tetrachloropyridine
- Cas Number:
- 2402-79-1
- Molecular formula:
- C5HCl4N
- IUPAC Name:
- 2,3,5,6-tetrachloropyridine
- Details on test material:
- Batch number XF09160201 of purity = 99.4%
Constituent 1
Test animals / tissue source
- Details on test animals or tissues and environmental conditions:
- Not applicable
Test system
- Amount / concentration applied:
- Not applicable
- Duration of treatment / exposure:
- Not applicable
- Observation period (in vivo):
- Not applicable
- Number of animals or in vitro replicates:
- Not applicable
- Details on study design:
- Pre-Test
Assessment of Direct Test Material Reduction of MTT
One limitation of the assay is possible interference of the test material with MTT. A test material may directly reduce MTT to formazan, thus mimicking dehydrogenase activity of the cellular mitochondria of viable cells. This property of the test substance is only a problem, if at the time of the MTT test (after the chemical has been rinsed off) there are still sufficient amounts of the test material on or in the tissues. To identify this possible interference, the test material was checked for its ability to reduce MTT directly.
30 mg of test material was added to a 0.5 mg/ml MTT solution and incubated at room temperature in the dark for 60 minutes. Untreated MTT solution was used as a control. If the MTT solution turned black/purple, the test material was presumed to have reduced the MTT.
Receipt of Tissues
On arrival, the SkinEthic HCE tissues (Day 6 cultures), were stored at room temperature prior to transferring into 24-well plates designated ‘arrival plates’ containing 300 µl of maintenance medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37°C, 5% CO2 in air.
Preparation of Tissues
Using sterile techniques, 1 ml of maintenance medium at room temperature, was dispensed into the appropriate number of wells of 6-well plates designated ‘treatment plates’. Each well was labelled with details of the treatment and the appropriate exposure time. Separate treatment plates were used for the test material and negative and positive controls to avoid the possibility of cross contamination occurring. Before treatment, the 7 day old tissues were transferred from the ‘arrival plates’ into the wells of the ‘treatment plates’ containing the maintenance medium.
Main Test
Triplicate tissues were treated with 30 mg of the test material for 10 minutes. The tissues were dosed at regular timed intervals to allow for the periodtaken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 30 µl of olution A to serve as negative controls and triplicate tissues were treated with 30 µl of 1 % w/v SDS to serve as positive controls. The plates were incubated at 37°C, 5% CO2 in air during the exposure time.
At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (DPBS). Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24 well plate designated ‘holding plate’ containing 300 µl of maintenance medium (at room temperature) until all the tissues were rinsed. Following rinsing, the tissues (two per group) were transferred to a pre labelled 24 well plate designated ‘MTT Loading plate’ containing 300 µl of a 0.5 mg/ml MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37°C, 5% CO2 in air.
At the end of the incubation period, the tissues were visually examined and the degree of MTT staining evaluated (qualitative evaluation of tissue viability). The inserts were blotted on absorbent paper to remove residual MTT solution and transferred to a pre labelled 24 well plate designated ‘MTT extraction plate’ containing 0.75 ml of Isopropanol in each of a sufficient number of wells. An extra 0.75 ml of Isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminium foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.
At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 µl tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue triplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µl of Isopropanol alone was added to three wells designated as ‘blanks’. The optical density was measured (quantitative measurement of tissue viability) at 540nm (OD540) using the Anthos 2001 microplate reader.
Tissue Histology
One tissue for each treatment group was retained for possible tissue histopathology.
The tissues were carefully cut out of the polycarbonate inserts with a sharp scalpel. The tissues were carefully cut in half. Both halves were placed into a pre-labelled 1.5 ml Eppendorf tube containing 1 ml of 10% Formalin and stored at room temperature.
Results and discussion
In vivo
Results
- Irritation parameter:
- other: Reduction of MTT
- Time point:
- other: 10 minutes
- Score:
- 128.8
- Remarks on result:
- other: The score given is the relative mean viability of the test material treated tissues in % terms.
- Irritant / corrosive response data:
- Assessment of Direct Test Material Reduction of MTT
The test material was not able to directly reduce MTT.
Assessment of Eye Irritation Potential
The mean OD540 values and mean viabilities for each treatment group are given in Table 1.
The relative mean viability of the test material treated tissues after a 10 minute exposure was 128.8%.
It was considered unnecessary to proceed with tissue histopathology.
Qualitative Evaluation of Tissue Viability (MTT Uptake Visual Assessment)
The qualitative evaluation of tissue viability is presented in Table 2.
The test material and negative control material treated tissues appeared blue which was considered to be indicative of viable tissue. The positive control material treated tissues appeared blue/white which was considered to be indicative of semi viable tissue.
Assay Acceptance Criterion
The quality criterion required for the acceptance of results in the test was satisfied.
CONCLUSION
According to the protocol followed the test material was considered to be a Non-Irritant (NI).
Any other information on results incl. tables
Table1 Assessment of Eye Irritation Potential – Viability of RHC Tissues
Material |
Mean Tissue Viability |
Mean OD540 |
Viability (%) |
Negative Control |
0.977 |
0.961 |
100* |
0.944 |
|||
Positive Control |
0.704 |
0.652 |
67.8 |
0.599 |
|||
Test Material |
1.164 |
1.238 |
128.8 |
1.312 |
*= The mean viability of the negative control tissues is set at 100%
Table2 Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)
Material |
Score |
|
Tissue 1 |
Tissue 2 |
|
Negative Control |
- |
- |
Positive Control |
+ |
+ |
Test Material |
- |
- |
MTT Visual Scoring Scheme of SkinEthic Tissues
- = Blue tissue (viable)
+ = Blue/White tissue (semi viable)
++ = Tissue completely white (dead)
*= The mean viability of the negative control tissues is set at 100%
Applicant's summary and conclusion
- Interpretation of results:
- not irritating
- Remarks:
- Migrated information Criteria used for interpretation of results: other: In accordance with the principles of the test method
- Conclusions:
- According to the protocol followed the test material was considered to be a Non-Irritant.
- Executive summary:
The eye irritation potential of the test material was evaluated using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories) after a treatment period of 10 minutes. The substance was considered to be Non-Irritant under the conditions of the test.
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