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EC number: 951-275-7 | 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:
- key study
- Study period:
- The study was conducted between 14 March 2018 and 02 April 2018.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Deviations:
- yes
- Remarks:
- This deviation was considered to have not affected the integrity or validity of the study.
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Reaction mass of Rel-(3aR,4S,7R,7aS)-1-(methoxymethylene)octahydro-1H-4,7-methanoindene and Rel-(3aR,4S,7R,7aS)-2-(methoxymethylene)octahydro-1H-4,7-methanoindene
- EC Number:
- 951-275-7
- Molecular formula:
- C12H18O
- IUPAC Name:
- Reaction mass of Rel-(3aR,4S,7R,7aS)-1-(methoxymethylene)octahydro-1H-4,7-methanoindene and Rel-(3aR,4S,7R,7aS)-2-(methoxymethylene)octahydro-1H-4,7-methanoindene
Constituent 1
- Specific details on test material used for the study:
- Identification: FRET 14-0383
Envigo Description: Colorless liquid
Sponsor Description: Clear yellow liquid
Storage Conditions: Approximately 4 °C, in the dark
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- other: reconstructed human epidermis
- Cell source:
- other: not specified
- Source strain:
- not specified
- Details on animal used as source of test system:
- EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : EpiSkin Laboratories, Lyon, France
Date received : 27 March 2018
EpiSkinTM Tissues (0.38cm^2) lot number : 18-EKIN-013
Maintenance Medium lot number : 18-MAIN3-015
Assay Medium lot number : 18-ESSC-012 - Justification for test system used:
- The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 Hour post exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end point can be used to either confirm a non-irritant result or will be used to override the non irritant result.
The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13 Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items. - Vehicle:
- unchanged (no vehicle)
- Details on test system:
- Reference Items
Negative Control
Information as provided by the Supplier.
Identification: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
Batch: 1754643
Purity: Not applicable
Expiry Date: 01 February 2019
Storage Conditions: Approximately 4 °C in the dark
Supplier: Gibco
Positive Control
Information as provided by the Supplier.
Identification: Sodium dodecyl sulphate
Batch: SLBT3991
Purity: 99.5%
Expiry Date: 01 March 2020
Storage Conditions: Room temperature
Supplier: Sigma-Aldrich
Preparation of Negative and Positive Control Items and MTT
The negative control item, Dulbecco’s Phosphate Buffered Saline (DPBS), was used as supplied.
The positive control item, Sodium dodecyl sulphate (SDS), was prepared as a 5% w/v aqueous solution. The positive control was formulated within 2 hours of being applied to the test system.
A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.
Study Design
Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.
Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 µL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.
Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 µL of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.
Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre labeled 12 well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.
Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12 well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL/cm^2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7 Minute contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
MTT Loading/Formazan Extraction (Day 3)
Following the 42 Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT 4500 microplate reader. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- 10 µL of the test item
10 µL of DPBS
10 µL of SDS 5% w/v - Duration of treatment / exposure:
- 15 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours
- Number of replicates:
- Triplicate
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- other: relative mean viability (%)
- Run / experiment:
- Mean
- Value:
- 57.1
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- The test was repeated due to a failure to meet the assay acceptance criteria. The relative mean tissue viability for the positive control treated tissues was 9.9% relative to the negative control treated tissues and the standard deviation value of the viability was 2.8%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.685 and the standard deviation value of the viability was 3.5%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 2.7%. The test item acceptance criterion was therefore satisfied.
Any other information on results incl. tables
Direct MTT Reduction
The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
Assessment of Color Interference with the MTT endpoint
The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.
Test Item, Positive Control Item and Negative Control Item
The relative mean viability of the test item treated tissues was 57.1% after a 15‑Minute exposure period and 42‑Hour post‑exposure incubation period.
It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.
Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item
Item |
OD570 of tissues |
Mean OD570 of triplicate tissues |
±SD of OD570 |
Relative individual tissue viability (%) |
Relative mean viability (%) |
± SD of Relative mean viability (%) |
Negative Control Item |
0.671 |
0.685 |
0.024 |
98.0 |
100* |
3.5 |
0.671 |
98.0 |
|||||
0.712 |
104.0 |
|||||
Positive Control Item |
0.061 |
0.068 |
0.019 |
8.9 |
9.9 |
2.8 |
0.053 |
7.7 |
|||||
0.090 |
13.1 |
|||||
Test Item |
0.412 |
0.391 |
0.019 |
60.1 |
57.1 |
2.7 |
0.385 |
56.2 |
|||||
0.376 |
54.9 |
OD = Optical Density
SD = Standard deviation
* = The mean viability of the negative control tissues is set at 100%
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Remarks:
- Criteria used for interpretation of results: EU
- Conclusions:
- The relative mean viability of FRET 14-0383 treated tissues was 57.1% after the 15 Minute exposure period and 42 Hours post exposure incubation period. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment, the substance is considered to be non-irritant.
- Executive summary:
The possible skin irritation potential of FRET 14 -0383 was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD TG 439. Skin tissue was treated by topical application of 10 µL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.
Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean viability of FRET 14-0383 treated tissues was 57.1% after the 15 Minute exposure period and 42 Hours post exposure incubation period.
Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment, the substance is considered to be non-irritant. The positive control had a mean cell viability of ≤40% relative to the negative control treated tissues after 15 minutes treatment. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.
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