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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
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 14 Jun 2021 - 21 Jun 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 021
- Report date:
- 2021
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- 14 June 2021
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- 26 June 2020
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Halohydrin dehalogenase
- Cas Number:
- 132421-41-1
- IUPAC Name:
- Halohydrin dehalogenase
- Test material form:
- solid: particulate/powder
- Details on test material:
- Identification: CDX-403
Physical Description: Light brown powder
Storage Conditions: In freezer (≤ -15°C)
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: SkinEthic Laboratories, Lyon, France.
- Details on animal used as source of test system:
- EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 21-EKIN-024 & 21-EKIN-022)
- Justification for test system used:
- In the interest of sound science and animal welfare, a sequential testing strategy is
recommended to minimize the need of in vivo testing. One of the validated in vitro skin
irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g.
OECD and EC). - Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Adult human donors
- Absence of HIV1 and 2 antibodies, hepatitis C antibodies, hepatitis B antigen HBs, bacteria, fungus and mycoplasma
- This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which will be seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
- Expiration date: 21June 2021 (21-EKIN-024) and 07 June 2021(21-EKIN-022)
KILLED TISSUES
Living epidermis (21-EKIN-022) was transferred to 12 well plates and incubated with 2 mL Milli-Q for 48 ±1 hours. After incubation, killed epidermis was stored in a freezer set to maintain -20°C.
Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates
on 2 mL maintenance medium. Further use of killed tissues was similar to living tissues.
TEMPERATURE USED FOR TEST SYSTEM
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, at 37.0 ± 1.0°C (actual range 36.1 - 39.7°C)
- Humid atmosphere of 80-100% (actual range 76 - 91%) containing 5.0 ± 0.5% CO2 in air in the dark
APPLICATION/TREATMENT OF THE TEST ITEM
The test was performed on a total of 3 tissues per test item together with negative and positive controls. The skin was moistened with 5 μL Milli-Q water to ensure close contact of the test item to the tissue and the solid test item (11.2 to 19.5 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. Since the test item reacted with the MTT medium, in addition three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT.
REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.
TEST FOR COLOR INTERFERENCE BY THE TEST ITEM
- To assess the color interference, 50 mg of the test item or 50 μL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water.
- The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark.
- 50 mg of the test item or 50 μL sterile Milli-Q water as a negative control was added to 2.0 mL
isopropanol.
- The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking.
-At the end of the exposure time, the mixtures were centrifuged for 30 seconds at 16000 g if
needed and the absorbance of the solutions was determined spectrophotometrically at 570 nm
in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement.
TEST FOR REDUCTION OF MTT BY THE TEST ITEM
- 50 mg of the test item was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline).
- The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark.
- A negative control, 50 μL sterile Milli-Q water was tested concurrently.
- If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 h at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 hours at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for approximately 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
ACCEPTABILITY CRITERIA
a) The absolute mean OD570 of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD439 (lower acceptance limit ≥0.6 and upper acceptance limit ≤1.5) and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.
d) The non-specific MTT reduction should be 30% relative to the negative control OD.
INTERPRETATION (See Table 1)
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- 11.2 to 19.5 mg applied directly on top of the skin tissue. The test item was spread to match the size of the tissue
- Duration of treatment / exposure:
- 15 ± 0.5 minutes at room temperature
- Duration of post-treatment incubation (if applicable):
- 42 ± 1 hours at 37°C
- Number of replicates:
- 3
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean of 3 replicates
- Value:
- 80
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 100
- Positive controls validity:
- valid
- Remarks:
- 19
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- See "attached full study report" for detailed results and historical control data.
- OTHER EFFECTS:
- Direct-MTT reduction:
A color change was observed in the presence of MTT therefore it was concluded that the test item interacted with the MTT endpoint. Three killed tissues treated with test item and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was 17% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.
- Colour interference with MTT:
The test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.0799 and 0.0040, respectively. Therefore it was concluded that the test item did not induce color interference.
ACCEPTABILITY CRITERIA
The positive control had a mean cell viability of 19% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 11%, indicating that the test system functioned properly.
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The substance does not cause skin irritation in the in vitro skin irritation test (OECD guideline 439).
- Executive summary:
In an in vitro skin irritation test, performed according to OECD guideline 439 and in accordance with GLP principles, the ability of the substance to induce skin irritation was tested using reconstructed human epidermis (EPISKIN-SM). The test substance was applied to 0.38 cm2 cultured skin. After 15 minutes exposure, the substance was removed and the tissues were incubated for 42 hours. Survival of unexposed tissue (negative control) was set at 100%. The positive control had a mean tissue viability of 19%. The test substance showed a mean tissue viability of 80% compared to the negative control. Since the mean relative tissue viability after exposure to the test substance was above 50%, no classification is required for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).
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