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EC number: 951-458-1 | 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 corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12 November 2019 - 28 January 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 guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
- Version / remarks:
- adopted 18 June 2019
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
- Version / remarks:
- 31 May 2008
- Deviations:
- no
- Principles of method if other than guideline:
- DEviation:
Cell Viability Measurement
• The start time for the MTT incubation was not noted correctly. The correct time cannot be
traced.
Evaluation: The responses for the negative and positive control indicated that an
appropriate incubation time was used. Therefore this deviation has no impact on the study
result.. - GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
- EC Number:
- 951-458-1
- Molecular formula:
- Not applicable - UVCB
- IUPAC Name:
- Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
- Test material form:
- liquid
- Details on test material:
- Substance name: Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
Test item handling: Use amber glassware or wrap container in aluminum foil
EC Number: 951-458-1
Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Constituent 1
- Specific details on test material used for the study:
- Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Purity/Composition correction factor: No correction factor required
EC Number: 951-458-1
Substance name: Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
Test item handling: Use amber glassware or wrap container in aluminum foil
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: Reconstructed human epidermis
- Source strain:
- not specified
- Details on animal used as source of test system:
- The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- Justification for test system used:
- Recommended test system in international guidelines (OECD and EC).
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Model used: EpiDerm Reconstucted Human Epidermmis
Tissue batch numbers: 30928 and 32126
Date of initiation of testing: 25 November 2019
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM.
TEST ITEM PREPARATION
No correction was made for the purity/composition of the test item. The liquid test item was applied undiluted (50 µL) directly on top of the tissue. To protect the test item from light glassware was wrapped in tin-foil.
TEMPERATURE USED FOR TEST SYSTEM
Temperature used during treatment / exposure: 36.5 - 37.2°C
ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 63 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data, these deviations are considered not to affect the study integrity.
APPLICATION/TREATMENT OF THE TEST ITEM
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before the test item was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 µL of the undiluted test item was added into the 6-well plates on top of the skin tissues. In addition, since the test item reacted with the MTT medium, two freeze-killed tissues were treated with test item and two freeze-killed non treated tissues were used per exposure time for the cytotoxicity evaluation with MTT.For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both the 3-minute and 1-hour time point. Negative and positive controls were shared with parallel studies. All information pertaining to shared tissues are archived in the raw data.
REMOVAL OF TEST MATERIAL AND CONTROLS
Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Incubation time: The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm
- Filter/Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported
The amount of extracted formazan was determined spectrophotometrically in triplicate.
Test for the Interference of the Test Item with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.
Test for Color Interference by the Test Item:
The test item was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the color interference, 50 µL of the test item or 50 µL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the
exposure time the mixture was shaken and it was checked if a blue / purple color change was observed.
Test for Reduction of MTT by the Test Item:
The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 µL of the test item or 50 µL Milli-Q water as a negative control were added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline.
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Fresh tissues / killed tissues: Killed tissues
N. of replicates : since the test item reacted with the MTT medium, two freeze-killed tissues were treated with test item and two freeze-killed non treated tissues were used per exposure time for the cytotoxicity evaluation with MTT.
Procedure used to prepare the killed tissues
Freeze-killed. Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to ≤-15°C. The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 mL DMEM. Further use of killed tissues was similar to living tissues. DMEM (Dulbecco’s Modified Eagle’s Medium) Supplemented DMEM, serum-free.
Cell Viability Measurement
The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature.
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.
ACCEPTABILITY CRITERIA
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8).
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
d) The non-specific MTT reduction should be ≤ 30% relative to the negative control OD. All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
INTERPRETATION
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Table 1 presents the data interpretation and optional sub-categorisation in case a test item will be corrosive
See Table 1: Data interpretation and sub-categorisation of test items in 'Any other information on materials and methods' incl. tables - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount applied: 50 µL liquid test item (undiluted)
NEGATIVE CONTROL
- Amount applied: 50 µL
POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8.0 normal solution - Duration of treatment / exposure:
- 3-minute and 1-hour treatment/exposures
- Number of replicates:
- Two (two tissues per exposure period and two tissues each for the positive and negative controls)
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3-minute treatment with the test item
- Value:
- 80
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1-hour treatment with the test item
- Value:
- 84
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.
Any other information on results incl. tables
The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint.
In addition to the normal 3-minute and 1-hour procedure, two tissues were treated with test item. Instead of MTT solution these tissues were incubated with DMEM. The non-specific reduction of MTT by the test item was 0.63% and 1.62% of the negative control tissues after 3 minutes and 1 hour respectively.
The mean absorption at 570 nm measured after treatment with the test item and controls are presented in Appendix 1, Table 1. The individual OD570 measurements are presented in Appendix 2.
3-minute application | 1-hour application | |||||||||
A (OD570) | B (OD570) | Mean (OD570) | SD | A (OD570) | B (OD570) | Mean (OD570) | SD | |||
Negative Control | 1.787 | 2.106 | 1.946 | ± | 0.226 | 1.837 | 1.986 | 1.946 | ± | 0.226 |
Test Item(1) | 1.565 | 1.559 | 1.562 | ± | 0.004 | 1.741 | 1.480 | 1.562 | ± | 0.004 |
Positive Control | 0.187 | 0.178 | 0.183 | ± | 0.007 | 0.129 | 0.187 | 0.183 | ± | 0.007 |
SD = Standard deviation | ||||||||||
Duplicate exposures are indicated by A and B. | ||||||||||
(1)The test item values are corrected for the non-specific MTT reaction (0.63 and 1.62 at the 3 minute and 1 hour treatment, respectively). |
The individual OD570 measurements are presented in the following table (attached in Appendix 2):
3-minute application (OD570) | 1-hour application (OD570) | |||
A | B | A | B | |
Negative control | ||||
OD570measurement 1 | 1.8243 | 2.1438 | 1.9075 | 2.0896 |
OD570measurement 2 | 1.8348 | 2.1432 | 1.8636 | 1.9975 |
OD570measurement 3 | 1.8304 | 2.1618 | 1.8709 | 2.0008 |
Test item | ||||
OD570measurement 1 | 1.6544 | 1.6057 | 1.8401 | 1.5761 |
OD570measurement 2 | 1.6024 | 1.6116 | 1.7981 | 1.5495 |
OD570measurement 3 | 1.6047 | 1.6261 | 1.8085 | 1.5357 |
Test item on freeze killed tissue | ||||
OD570measurement 1 | 0.1645 | 0.1743 | 0.1560 | 0.1557 |
OD570measurement 2 | 0.1623 | 0.1689 | 0.1554 | 0.1519 |
OD570measurement 3 | 0.1642 | 0.1859 | 0.1552 | 0.1544 |
Non - treated freeze killed tissue | ||||
OD570measurement 1 | 0.1214 | 0.2338 | 0.1231 | 0.1284 |
OD570measurement 2 | 0.1207 | 0.2157 | 0.1203 | 0.1251 |
OD570measurement 3 | 0.1228 | 0.1324 | 0.1212 | 0.1249 |
Positive control | ||||
OD570measurement 1 | 0.2307 | 0.2255 | 0.1762 | 0.2416 |
OD570measurement 2 | 0.2301 | 0.2177 | 0.1702 | 0.2284 |
OD570measurement 3 | 0.2312 | 0.2204 | 0.1701 | 0.2209 |
OD = Optical density | ||||
Duplicate exposures are indicated by A and B. |
The following table (Table 2, Appendix 1) shows the mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues:
3-minute application viability (percentage of control) | 1-hour application viability (percentage of control) |
|
Negative control | 100 | 100 |
Test item | 80 | 84 |
Positive control | 9.4 | 8.3 |
Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 80% and 84% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit≤2.8) and the laboratory historical control data range (See Appendix 3). The mean relative tissue viability following the 1-hour exposure to the positive control was 8.3%.
In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was≤15%, indicating that the test system functioned properly (Appendix 1, Table 3).
3 minute | 1 hour | |
Negative control | 15 | 7.5 |
Test item | 0.4 | 15 |
Positive control | 5.1 | 31 |
CV (%) = 100 - [(lowest OD570/highest OD570) x 100%] |
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
- Executive summary:
The objective of this study was to evaluate the test item for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour.
The study procedures described in this report were based on the most recent OECD and EC guidelines.
The test item was a light orange liquid. The test item was applied undiluted (50 µL) directly on top of the skin tissue.
The positive control had a mean relative tissue viability of 8.3% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤15%, indicating that the test system functioned properly.
Because a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint.
In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each time point. The non-specific reduction of MTT by the test item was 0.63% and 1.62% of the negative control tissues after 3 minutes and 1 hour respectively.
Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 80% and 84%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.
In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
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