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EC number: 222-908-8 | CAS number: 3658-77-3
- 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 corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05-07 April 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted according to OECD test Guideline No. 431 without any deviation.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- UK GLP Compliance Programme (inspected on 05 July 2016 / signed on 28 October 2016)
Test material
- Reference substance name:
- 4-hydroxy-2,5-dimethylfuran-2(3H)-one
- EC Number:
- 222-908-8
- EC Name:
- 4-hydroxy-2,5-dimethylfuran-2(3H)-one
- Cas Number:
- 3658-77-3
- Molecular formula:
- C6H8O3
- IUPAC Name:
- 4-hydroxy-2,5-dimethylfuran-3(2H)-one
- Test material form:
- solid
- Details on test material:
- - Physical state / Appearance: White solid
Constituent 1
- Specific details on test material used for the study:
- - Storage condition of test material: The test item was stored dry and in the dark in a closed aluminium bag at room temperature (20 ± 10 °C). On 23 February 2017 the aluminium bag was opened and the contents were split into single use aliquots. The test item aliquots were stored in amber jars flushed with nitrogen.
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Justification for test system used:
- Following the REACH up and down strategy, the EpiDerm™ Human Skin Model method was used to assess skin corrosion as recommended in the OECD test guideline No. 431.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: MatTek
- Tissue batch number(s): EpiDermTM Tissues (0.63cm2) lot number 25803
- Production Date: 03 April 2017
- Shipping date: 03 April 2017
- Delivery date: 04 April 2017
- Date received: 04 April 2017
- Date used: 06 April 2017
- Assay Medium lot number: 033017TME
- Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C, in a humidified atmosphere of 5% CO2
- Temperature of post-treatment incubation: 37 °C, in a humidified atmosphere of 5% CO2
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsing was performed using a wash bottle and was achieved by filling and emptying each tissue under a constant soft stream of DPBS for approximately 40 seconds to gently remove any residual test item
- Observable damage in the tissue due to washing: No visible damage to the rinsed tissues was noted following the rinsing step..
- Modifications to validated SOP: none reported
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT solution
- Incubation time: 3 hours at 37 °C, humidified atmosphere of 5% CO2.
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570 nm
- Filter: without reference filter
- Filter bandwidth: filter band pass ± 10
- Linear OD range of spectrophotometer: 0.0 to 2.6 nm.
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
In order to confirm the integrity of the test system a comparison was made with historical data for negative and positive controls obtained by the laboratory in the previous six months. All values for negative and positive controls were within the historical range and this was taken to indicate the adequate functioning of the test system.
- Viability: OD (540-570 nm) = 1.7 ± 0.105 and 1.739 ± 0.134 for viable and non-viable tissues, respectively (acceptance criteria: between 1.0-3.0)
- Barrier function: ET-50 = 5.36 hours and 6.71 hours for viable and no-viable tissues, respectively (acceptance criteria: between 4.77-8.72 hours)
- Morphology: Tissue viability and the barrier function test are within acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: sterile (Long term antibiotic and antimycotic free culture)
- Reproducibility: For the previous 43 experiments conducted between August 2016 and February 2017 using this test method, the mean OD of the positive control was 0.079 ± 0.025 after 3 minutes of exposure and 0.068 ± 0.025 after 60 minutes of exposure. The mean percentage viability was 4.3 ± 1.0 after 3 minutes of exposure and 3.8 ± 1.0 after 60 minutes of exposure (The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤15% relative to the negative control treated tissues after 1 hour exposure). In this same period the mean OD of the negative control was 1.845 ± 0.239 after 3 minutes of exposure and 1.810 ± 0.235 after 60 minutes of exposure (The assay establishes the acceptance criterion for an acceptable test if the mean OD for the negative control treated tissues was ≥0.8 and ≤2.8 for every exposure time).
The historical control values given were obtained on a different microplate reader to the machine that was used in this study. They were also measured at 562nm whereas in this study the values were measured at a wavelength of 570nm. Even though the values were obtained on a different machine the values are still representative of the historical range for the laboratory and the minor difference of 8nm between 562nm and 570nm is not considered to impact upon the applicability of the historical comparison for this study.
NUMBER OF REPLICATE TISSUES: Duplicate tissues for test item, negative and positive controls
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues : Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12 well plate and storing in a freezer (-14 to -30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
- N. of replicates : 2
- Method of calculation used: True viability = mean OD (treated viable tissues) - [OD (treated killed tissues)-OD (untreated killed tissues)]
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PR
EDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- If the test item is corrosive with the viability less than 25% after 3 min exposure: H314; Sub-category 1A
- If the test item is corrosive with the viability greater than or equal to 25% after 3 min exposure: H314; Sub-category 1A - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of the unformulated test item (undiluted) wetted with 25 μL of sterile water to increase tissue surface contact.
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of sterile distilled water was used as supplied.
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of 8.0 N Potassium Hydroxide was used as supplied. - Duration of treatment / exposure:
- 3 and 60 minutes
- Duration of post-treatment incubation (if applicable):
- Not applicable
- Number of replicates:
- Duplicate tissues for test item, negative and positive controls
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 minutes exposure
- Value:
- 73.1
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 100%
- Positive controls validity:
- valid
- Remarks:
- 4.6%
- Remarks on result:
- other: corrosive to the skin
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 60 minutes exposure
- Value:
- 5
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 100%
- Positive controls validity:
- valid
- Remarks:
- 4.2%
- Remarks on result:
- other: corrosive to the skin
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: The MTT solution containing the test item turned blue which indicated that the test item directly reduced MTT. Direct reduction was <30% relative to the negative control and therefore acceptable.
- Colour interference with MTT: No
DEMONSTRATION OF TECHNICAL PROFICIENCY: yes
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.542 for the 3 Minute exposure period and 1.589 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 4.2% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.
- Historical data: Furthermore, in order to confirm the integrity of the test system a comparison was made with historical control data for negative and positive controls obtained by the laboratory in the previous six months.
The historical control values were obtained on a different microplate reader to the machine that was used in this study. They were also measured at 562nm whereas in this study the values were measured at a wavelength of 570nm. Even though the values were obtained on a different machine the values are still representative of the historical range for the laboratory and the minor difference of 8nm between 562 nm and 570 nm is not considered to impact upon the applicability of the historical comparison for this study. All values obtained were within the historical range of the testing laboratory.
Any other information on results incl. tables
Table 7.3.1/1: Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item
Tissue |
Exposure Period |
Mean OD570 of individual tissues (tvt) |
Mean OD570 of duplicate tissues |
Corrected OD570 of tissues |
Standard Deviation |
Coefficient of Variation (%) |
Relative Mean Viability (%) |
Negative Control |
3 Minutes |
1.566 |
1.542 |
- |
0.035 |
2.2 |
100* |
1.517 |
|||||||
60 Minutes |
1.631 |
1.589 |
0.059 |
3.7 |
|||
1.547 |
|||||||
Positive Control |
3 Minutes |
0.081 |
0.071 |
- |
0.014 |
N/A |
4.6 |
0.061 |
|||||||
60 Minutes |
0.075 |
0.068 |
0.011 |
N/A |
4.2 |
||
0.060 |
|||||||
Test Item |
3 Minutes |
1.281 |
1.367 |
1.128 |
0.121 |
8.8 |
73.1 |
1.452 |
|||||||
60 Minutes |
0.507 |
0.475 |
0.080 |
0.046 |
9.7 |
5.0 |
|
0.442 |
3 minute exposure corrected mean OD570= 0.344 (tkt) – 0.105 (ukt) = 0.239
60 minute exposure corrected mean OD570= 0.521 (tkt) – 0.126 (ukt) = 0.395
3
minute exposure direct MTT reduction relative to the negative control =
15.5%
60 minute exposure direct MTT reduction relative to the negative control
= 24.9%
Relative mean viability (%) = (Mean OD570 of test item / Mean OD570 of negative control) x 100
Coefficient of variation = Standard deviation / Mean OD570 of duplicate tissues
* = The mean % viability of the negative control tissue is set at 100%
OD = optical density
N/A = not applicable
tvt = treated viable tissues
tkt = treated killed tissues
ukt = untreated killed tissues
Applicant's summary and conclusion
- Interpretation of results:
- Category 1B (corrosive) based on GHS criteria
- Remarks:
- combination of category 1B-and-1C
- Conclusions:
- Under the experimental conditions of this study, the test substance is classified as H314 “Causes severe skin burns and eye damage”, combination of category 1B-and-1C, according to the Regulation (EC) No 1272/2008 (CLP) and to the GHS.
- Executive summary:
An in vitro skin corrosion study was performed according to the OECD Guideline 431 and in compliance with GLP, using the EpiDerm™ Human Skin Model.
Duplicate tissues were treated with 25 mg of the undiluted test item wetted with 25 µL of sterile water to increase tissue surface contact for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).
The relative mean viability of the test item treated tissues was 73.1 and 5.0 % after 3 and 60 minutes exposure, respectively. The relative mean viability of the negative control treated tissues was 100 % after 3 and 60 minutes exposure. The relative mean viability of the positive control treated tissues was 4.6 and 4.2 % after 3 and 60 minutes exposure, respectively. The quality criteria required for acceptance of results in the test were satisfied.
Under the experimental conditions of this study, the test substance is classified as H314 “Causes severe skin burns and eye damage”, combination of category 1B-and-1C, according to Regulation (EC) No 1272/2008 (CLP) and to the GHS.
This study is considered as acceptable and satisfies the requirement for skin icorrosion endpoint.
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