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EC number: 251-718-8 | CAS number: 33885-52-8
- 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:
- 2015-09-02 till 2015-09-07
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 015
- Report date:
- 2015
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- TNO Triskelion, Utrechtseweg 48, 3700 AV, Zeist
Test material
- Reference substance name:
- α,α,6,6-tetramethylbicyclo[3.1.1]hept-2-ene-2-propionaldehyde
- EC Number:
- 251-718-8
- EC Name:
- α,α,6,6-tetramethylbicyclo[3.1.1]hept-2-ene-2-propionaldehyde
- Cas Number:
- 33885-52-8
- Molecular formula:
- C14H22O
- IUPAC Name:
- 3-{6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl}-2,2-dimethylpropanal
- Test material form:
- liquid
1
In vitro test system
- Test system:
- human skin model
- Remarks:
- EpiDerm™ (EPI-200) skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- foreskin from a single donor
- Source strain:
- other: Not relevant
- Details on test system:
- The EpiDerm™ (EPI-200) skin model consisted of normal human epidermal keratinocytes from one single donor, derived from neonatal-foreskin tissue. The keratinocytes were plated on chemically modified, collagen-coated, 9 mm ID cell culture inserts (surface area 0.64 cm2). The skin models are commercially available and were obtained from MatTek In Vitro Life Science Laboratories (IVLSL), Slovakia.
Preliminary tests:
- Some chemicals are known to change into a coloured substance in aqueous conditions and consequently stain tissues during the exposure. To determine this possibility, 30 μL of the test substance was incubated in 300 μL MilliQ water for 60 min at ca. 37 ºC and 5% CO2 and a colour change was assessed visually. Some chemicals are known to non-specifically reduce MTT, resulting in a blue/purple precipitate and/or blue/purple staining of a MTT solution. To test the MTT reducing capacity of the test substance, 30 μL of the test substance was incubated in 1 mL of a MTT solution (1 mg/mL) for 185 min at ca. 37ºC and 5% CO2 and the formation of a blue/purple formazan product was assessed visually. Based on the results, the negative control and test substance were applied to frozen controls (n=2 per test group) in the in vitro skin irritation test. To test if the test substance had the potential to damage a nylon mesh, a mesh compatibility test was performed in which 30 μL of the test substance was applied to a nylon mesh. After 60 min incubation at ambient temperature, the possible interaction with the mesh was visually checked using a microscope.
Exposure to study substances:
- The skin models were topically exposed to 30 μL of the test substance, negative or positive control, at the end of the acclimatization period. Immediately after application a nylon mesh was placed on the skin model surface to facilitate an equal distribution of the study substances. Exposure was initiated at ambient temperature. After dosing the last skin model, all skin models were transferred to a humidified incubator (ca. 37ºC and 5% CO2). Frozen controls were included as required. After 36 min, the plates were removed from the incubator and kept at ambient temperature until the exposure period of 60 min was completed. Subsequently, the skin models were removed from the well, washed using an excess of PBS to remove the study substances and mesh. The skin models were transferred to a clean 6-well plate containing fresh NMM (900 μL/well) and incubated in a humidified incubator (ca. 37ºC and ca. 5% CO2). Medium was refreshed after 18 h post-exposure. Following an additional 24 h incubation period (i.e. the total post-exposure period was 42 h), viability was determined using the MTT test
MTT test:
- The MTT solution of 1 mg/mL was freshly prepared by diluting MTT concentrate five times in MTT diluent. The inserts were transferred to a 24-well plate containing 300 μL of MTT solution per well. After 178 min incubation in a humidified incubator at ca. 37 ºC and 5% CO2, the skin models were rinsed three times with PBS. The formazan product was extracted from the skin model using 2 mL MTT extractant (provided with the MTT-100 assay kit). Extraction was performed at 2-10 ºC for three days. Following extraction, the optical density was measured in triplicate in 200 μL sub fractions per well in a 96-well plate using a spectrophotometer set at 570 nm. MTT extractant was used as blank. The mean optical density (OD) was calculated and expressed as the percentage viability compared to the negative control (mean tissue viability).
Interpretation of results:
- The OD of the viable skin models was corrected for non-specific MTT reduction of the test substance according to the following formula: True OD = mean OD viable skin models treated with the test substance – mean OD frozen controls, where mean OD frozencontrols = mean OD of the frozen controls treated with the test substance – the mean OD of the frozen controls treated with the negative control.
- If the non-specific reduction of MTT by the test substance was comparable to the negative control, a correction is not required. If the non-specific reduction of MTT by the test substance was > 30% compared to the negative control, the test substance will be considered incompatible with the test (expert judgement).
- The in vitro skin corrosion test was considered valid if the OD of the negative control was ≥ 0.8 and ≤ 2.8, the OD of the blank was < 0.1, and skin models treated with the positive control showed mean tissue viability ≤ 20% compared to the negative control
- The test was considered invalid if the test did not meet these acceptance criteria.
- The test group was considered valid if the SD calculated from individual tissue viability percentages of the three replicates was ≤ 18%. Test substances showing tissue viabilities in a range of 30-70% may show standard deviation (SD) > 18%. If this was typical for the test substance, and consistent in a repeat experiment, the results were accepted, although the acceptance criterion was not met.
- The test group was considered invalid (inconclusive) if the SD calculated from individual tissue viability percentages of the three replicates was >18% and if the test was not repeated.
- The in vitro irritation potential of the test substance was determined from the relative mean tissue viabilities compared to the negative control tissues, using the following prediction model:
Mean tissue viability (% of negative control) Prediction
Mean tissue viability ≤ 50 % Irritant
Mean tissue viability > 50 % Non-irritant (No Category) - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 uL
- Concentration: Undiluted - Duration of treatment / exposure:
- 60 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours
- Number of replicates:
- 3
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Remarks:
- % of negative control
- Run / experiment:
- mean
- Value:
- 105
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Remarks:
- Time point: 60 min. Max. score: 100.0. Remarks: (SD = 11)
- Other effects / acceptance of results:
- Preliminary tests
- At the end of the incubation period of the test substance in MilliQ, the solution did not significantly changed colour to blue/purple, indicating that there would be no colour interference in the test. Therefore, no additional controls were required in the in vitro skin irritation test.
- At the end of the incubation period of the test substance with a MTT solution, the MTT solution showed a purple top layer, indicating that the test substance had the potential to reduce MTT. Therefore, frozen controls were included in the in vitro skin irritation test.
- During the mesh compatibility test it was observed that the test substances did not damage the nylon mesh and therefore the nylon mesh was used in the in vitro skin irritation test to facilitate equal distribution of the test substance.
In vitro skin irritation test:
- The OD of the blank, the negative control (PBS) and the positive control (5% SDS) demonstrated the expected response. The SD calculated from individual tissue viability percentages of the three replicates was <18%. All acceptance criteria were met and therefore the study was considered valid.
- The OD of the frozen control membranes exposed to the test substance was comparable to the OD of the frozen control membranes exposed to the negative control. Therefore no data correction was applied.
Applicant's summary and conclusion
- Interpretation of results:
- other: Not a skin irritant
- Remarks:
- in accordance with EU CLP (EC 1272/2008 and its updates)
- Conclusions:
- Under the test conditions (OECD 439 and GLP) the test substance is not considered to be a skin irritant.
- Executive summary:
In accordance to OECD guideline 439 and GLP the test substance was examined for its in vitro skin irritation potential using EpiDerm™ reconstructed skin membranes. In the in vitro skin irritation test, the skin membranes were topically exposed to the undiluted test substance for 60 min. The test was performed in triplicate. Viability of the epidermal cells was assessed using the MTT test after 42 h of culture. Negative and positive controls were run in parallel. All acceptance criteria were met and therefore the study was considered valid. After exposure to the test substance the mean tissue viability was 105 ± 11 % compared to the concurrent negative control group. Based on the results obtained in the present study the test substance is not considered to be a skin irritant.
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