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EC number: 915-634-1 | 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:
- 06 to 09 December 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study performed according to OECD Guideline No.431 and under GLP compliance.
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: Human Skin Model Test)
- Version / remarks:
- Dated to 29 July 2016.
- Deviations:
- no
- GLP compliance:
- yes
Test material
- Reference substance name:
- Ethyl 2,6,6-trimethylcyclohexa-1,3-ene-1-carboxylate
- EC Number:
- 252-335-9
- EC Name:
- Ethyl 2,6,6-trimethylcyclohexa-1,3-ene-1-carboxylate
- Cas Number:
- 35044-59-8
- Molecular formula:
- C12H18O2
- IUPAC Name:
- ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate
- Test material form:
- liquid
- Details on test material:
- SOURCE OF TEST MATERIAL
- Physical appearance: colourless to pale yellow liquid
- Stored in a dry, well ventilated and dark location at room
temperature (i.e. 10–30°C)
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- other: reconstituted epidermis (EpiDerm Skin Model)
- Cell type:
- other: EpiDerm Skin Model
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- HUMAN SKIN MODEL
- The EpiDerm Skin Model (EPI-200, Lot no.: 24940 kits J and K) 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.
- Source: MatTek Corporation, Ashland MA, U.S.A.
- Rationale: Recommended test system in international guidelines (OECD and EC).
CELL CULTURE
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 medium (Supplemented DMEM medium, serum-free supplied by MatTek Corporation). The plates were incubated for approximately 1.5 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test item was applied.
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 47 - 82%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 36.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
EVALUATION OF DIRECT INTERACTION WITH MTT
- Colour interference by the test item in aqueous conditions: To assess the colour 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 colour change was observed.
- Reduction of MTT by the test item: To assess the ability of the test item to reduce MTT, 50 μl of the test item was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently. At the end of the exposure time it was checked if a blue / purple colour change or a blue / purple precipitate was observed.
> Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.
TREATMENT
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. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively.
REMOVAL OF TEST MATERIAL
After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM), both
supplied by MatTek Corporation, was prepared.
The DMEM medium 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 (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.
PREDICTION MODEL / DECISION CRITERIA:
- Acceptability of the assay:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the acceptance limits of OECD 431 (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%.
In addition to the quality criteria a comparison of laboratory historical data on negative and positive controls was made to verify the functioning of the test system.
- Data evaluation and statistical procedures:
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%.
The following information presents the data interpretation and optional sub-categorisation in case a test item will be corrosive.
Viability measured after 3-minutes and 1 hour
STEP 1 (corrosive or not corrosive)
< 50% after 3 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND < 15% after 60 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure ==> Non-corrosive
STEP 2 (subcategories for items identified as corrosive in step 1)
< 25% after 3 min exposure ==> Optional Sub-category 1A
≥ 25% after 3 min exposure ==> A combination of optional Sub-categories 1B-and-1C - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted - Duration of treatment / exposure:
- During 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.
- Duration of post-treatment incubation (if applicable):
- The skin sample was placed in MTT solution of 1 mg/mL concentration for 3 hours at 37°C± 1°C, 5% CO2.
- Number of replicates:
- 2 living human skin models for each time
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Value:
- 95
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: at 3 minutes
- Irritation / corrosion parameter:
- % tissue viability
- Value:
- 112
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: at 60 minutes
- Other effects / acceptance of results:
- MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues, after 3 minutes exposure, was 95%, versus 7% in the positive control.
- The mean percent viability of the treated tissues, after 1 hour exposure, was 112 %, versus 14% in the positive control.
Any other information on results incl. tables
Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls
INDIVIDUAL AND AVERAGE VALUES AFTER 3 MINUTES EXPOSURE
|
Skin |
OD |
Mean OD / disc (#) |
Mean OD / product |
± SD |
Mean viability % |
Viability difference between replicates % |
Negative control |
A |
1.7327 1.7449 1.7441 |
1.698 |
1.658 |
± 0.056 |
100.00 |
4.7 |
B |
1.6579 1.6502 1.6745 |
1.6118 |
|||||
Positive control |
A |
0.1701 0.1674 0.1657 |
0.125 |
0.120 |
± 0.008 |
7 |
9.0 |
B |
0.1577 0.1562 0.1553 |
0.114 |
|||||
Test item |
A |
1.6523 1.7420 1.6830 |
1.650 |
1.567 |
± 0.117 |
95 |
10 |
B |
1.5378 1.5326 1.5101 |
1.484 |
INDIVIDUAL AND AVERAGE VALUES AFTER 1 HOUR EXPOSURE
|
Skin |
OD |
Mean OD / disc (#) |
Mean OD / product |
± SD |
Mean viability % |
Viability difference between replicates % |
Negative control |
A |
1.4840 1.4800 1.4840 |
1.440 |
1.406 |
± 0.049 |
100.00 |
4.8 |
B |
1.4386 1.3726 1.4303 |
1.371 |
|||||
Positive control |
A |
0.2751 0.2888 0.2664 |
0.234 |
0.202 |
± 0.046 |
14 |
28 |
B |
0.2121 0.2125 0.2126 |
0.170 |
|||||
Test item |
A |
1.6631 1.6308 1.5944 |
1.587 |
1.578 |
± 0.013 |
112 |
1.2 |
B |
1.6585 1.5719 1.6022 |
1.568 |
SD: standard deviation
In these tables the values are corrected for background absorption (0.0424). Isopropanol was used to measure the background absorption.
Acceptability criteria:
- 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. - The mean
relative tissue viability following the 1-hour exposure to the
positive control was 14% ( < 15%).
- In the range of 20 - 100% viability the Coefficient of Variation
between tissue replicates was ≤ 10%, indicating that the test system
functioned properly (≤ 30%).
Applicant's summary and conclusion
- Interpretation of results:
- Category 2 (irritant) based on GHS criteria
- Conclusions:
- 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 95% and 112% respectively. Because the mean relative tissue viability for ETHYL SAFRANATE was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered non corrosive in the in vitro skin corrosion test.
Therefore, in accordance with the CLP Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”, and based on the results from the previous EpiSkin study OECD 439 as irritant to the skin, the test item can be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required. - Executive summary:
An in vitro skin corrosion test according to the OECD Guideline OECD 431 and in compliance with GLP was performed.
The test item Ethyl Safranate was applied as supplied, at the dose of 50 μL to 2 living Human skin model surfaces for each time (EpiDerm (EPI-200)) for 3 minutes and 1 hour. The application was followed by a rinse with PBS to remove residual test item. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Additionally, the remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control).
3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item and treated with the positive control item (potassium hydroxide 8N) were respectively 95% and 112% versus 7% and 14% respectively, with the positive control.
Because the mean relative tissue viability for ETHYL SAFRANATE was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered non corrosive in the in vitro skin corrosion test.
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 95% and 112% respectively. Because the mean relative tissue viability for ETHYL SAFRANATE was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered non corrosive in the in vitro skin corrosion test.
Therefore, in accordance with the CLP Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”, and based on the results from the previous EpiSkin study OECD 439 as irritant to the skin, the test item can be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required.
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