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EC number: 941-434-9 | 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 20 May 2014 and 23 May 2014.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 439 using a Reconstructed Human Epidermis Test Method and in compliance with GLP.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- ethyl (1R,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1R,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate
- Molecular formula:
- C10H14O2
- IUPAC Name:
- ethyl (1R,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1R,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate
- Test material form:
- other: Liquid
- Details on test material:
- Identity: IFF TM 09-218
Chemical name: Ethyl bicyclo[2.2.1]hept-5-ene-2 carboxylate
CAS number: 10138-32-6
Intended use: Fragrance ingredient in household products
Appearance: Clear liquid
Storage conditions: Room temperature (ca. 20˚C, in the dark)
Constituent 1
Test animals
- Species:
- other: EpiSkin™ human epidermis skin constructs
- Strain:
- other: Not applicable
Test system
- Type of coverage:
- other: Not applicable
- Preparation of test site:
- other: Not applicable
- Vehicle:
- unchanged (no vehicle)
- Controls:
- other: Not applicable
- Amount / concentration applied:
- 10mg/tissue.
- Duration of treatment / exposure:
- 15 minutes
- Observation period:
- 42 hours post-exposure incubation period
- Number of animals:
- Not applicable
- Details on study design:
- Reduction of MTT by test substance
It is possible that a test substance may reduce MTT, resulting in the production of a blue colour without any involvement of cellular mitochondrial dehydrogenase. Since the test substance is rinsed off the tissue before the MTT assay, this is usually avoided. However, it is possible that small amounts of test substance may be present after washing or be released through the tissue into the MTT medium. If the mixture with the test substance turns
blue/purple after approximately 3 hours incubation at 37 ±2ºC in 5% CO2 in air, MTT reduction may have occurred.
The MTT reducing capability of the test substance, IFF TM 09-218, was investigated by mixing 10 μL of the test substance with 2 mL of 0.3 mg/mL MTT solution in duplicate. A control of 10 μL of purified water, mixed with 2 mL of 0.3 mg/mL MTT solution was also included in duplicate.
Check for colouring potential of test substance
The test substance, IFF TM 09-218, was evaluated for its colour or ability to become coloured in contact with water (simulating a tissue humid environment). Evaluation was achieved by mixing 10 μL of the test substance, with 90 μL of purified water in a transparent container. 100 μL of purified water was included as a control. The solution was mixed for 15 minutes on a shaker. At the end of the shaking period the colour of the solution was
assessed by eye. If a coloured solution was detected, the tissue staining ability was tested by following the test procedure, however, MTT was replaced with the assay medium and only one tissue for the test substance and the Dulbecco’s phosphate buffered saline (DPBS) control was used.
Receipt of tissues
On receipt, the kit contents were checked and the inserts with tissues on agarose were stored at room temperature until use. The kit was used within
the expiry date indicated by the supplier (expiry date: 26 May 2014). The maintenance medium was pre-warmed to 37ºC. The tissues were removed from the agar and placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 24 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. The inserts used for killed tissues were received on a previous occasion (4 February 2014). The tissues had been previously killed by transferring the inserts to a 12 well plate containing 2 mL of water per well and then incubated at 37 ± 2ºC in humidified 5 % CO2 in air for 48 ± 1 hours. The killed tissues were transferred to a 12 well plate after blotting on absorbent
paper and then stored at ca. -20°C until use.
Controls
The negative control was sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium.
The positive control was 5% Sodium Dodecyl Sulphate (SDS) in purified water.
Preparation/application of samples
The test substance, IFF TM 09-218, a clear liquid, positive and negative controls were in liquid form and were applied by dispensing a volume of 10 µLover each tissue using a positive displacement pipette.
Test procedure
After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 15 ± 0.5 minutes with the test substance, negative or
positive control at room temperature. A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10 μL, the positive control was spread over the tissue for approximately 30 seconds and then respread with a curved flat spatula after 8 minutes application time. At least one hour before dosing, thawed water killed tissues were transferred into wells of 12
well plates containing 2 mL pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 1 hour at room temperature. Triplicate tissues were dosed for 15 ± 0.5 minutes with the test substance (10 ± 2 mg per tissue) at room temperature. As killed tissues may retain somereducing enzymes (which may reduce MTT), triplicate killed tissues were left untreated as a control.
After 15 ± 0.5 minutes, each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 ± 1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
After 42 ± 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
At the end of 3 hours ± 5 minutes, the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro-tube. When all tissues had been punched, the tissues were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration). The tissues were extracted by storing at room temperature, protected from light, for 4 hours, vortexing after approximately 2 hours.
After formazan extraction, duplicate 200 µL aliquots of the extractant from each tube were pipetted into the wells of flat-bottomed 96-well plates. Theextractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol
(0.04 N HCl final concentration) as a blank.
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- other: other: Mean
- Value:
- 23.9
- Remarks on result:
- other:
- Remarks:
- Basis: mean. Time point: 15 minutes exposure. Remarks: Irritant. (migrated information)
In vivo
- Irritant / corrosive response data:
- The test substance was predicted to be an irritant. The mean tissue vaibility as a percentage of mean OD negative control for the test substance was 23.9 ± 7.7%.
Any other information on results incl. tables
Possible reduction of MTT by test substance
There was no change in the water control/MTT solution after three hours incubation in the dark at 37 ± 2 °C in a humidified atmosphere of 5% CO2 in air. There was a change in the colour of the test substance, IFF TM 09-218/MTT solution from yellow/green to yellow/green with purple suspension after three hours incubation in the dark at 37 ± 2 °C in a humidified atmosphere of 5% CO2 in air. The test substance had interacted with the MTT, additional controls were used.
Check for colouring potential of test substance
The test substance, IFF TM 09-218/water solution and water control were colourless after the 15 minute shaking period. The test substance had not shown any potential for colouring water.
Assay validity
Negative control
The mean absorbance of the triplicate negative control values was 0.853 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 7.5 which was below the maximum value of 18.
Positive control
The percentage mean viability of the positive control was 28.1 ± 10.5 of the negative control.These were below the maximum acceptance values of 40% viability and SD of 18%.
EpiSkin™ results
The test substance OD values were corrected for nonspecific MTT reduction by the test substance. The results of the assay are summarised in the table below.
Sample |
Tissue viability as percentage of mean OD negative control |
Prediction MTT endpoint |
|||
Replicate tissues |
Mean±SD |
||||
A |
B |
c |
|||
Negative control |
108.1 |
93.4 |
98.5 |
100.0±7.5 |
Not applicable |
Positive control |
40.1 |
23.4 |
20.7 |
28.1± 10.5 |
Irritant |
Test substance |
18.2 |
32.6 |
20.9 |
23.9± 7.7 |
Irritant |
Nonspecific MTT reduction data
Sample |
Tissue replicate |
Optical density (OD) |
Corrected OD |
Corrected OD – Untreated corrected mean OD |
(OD – Blank) |
||||
Untreated |
A |
0.329 |
0.186 |
N/A |
0.369 |
0.226 |
|||
B |
0.354 |
0.211 |
||
0.376 |
0.232 |
|||
C |
0.361 |
0.217 |
||
0.365 |
0.213 |
|||
Replicates a, b, c |
Mean |
0.214 |
||
SD |
0.016 |
|||
Test substance |
A |
0.289 |
0.145 |
-0.042 |
0.308 |
0.165 |
|||
B |
0.304 |
0.161 |
||
0.252 |
0.108 |
|||
C |
0.371 |
0.227 |
||
0.368 |
0.225 |
|||
Replicates a, b, c |
Mean |
0.172 |
||
SD |
0.046 |
|||
Blank |
|
0.138 |
N/A |
|
|
0.148 |
|||
|
0.145 |
|||
|
0.139 |
|||
|
0.146 |
|||
|
0.143 |
|||
Mean |
0.143 |
|||
SD |
0.004 |
sd = Standard Deviation
Note. Rounded values only are displayed; unrounded numbers are used for calculations by Excel spreadsheet.
EpiSkin™ study data
Sample |
Tissue replicate |
Optical density (OD) |
Corrected OD |
Correction for MTT reduction |
% negative control |
(OD – Blank) |
|||||
Negative control |
A |
1.053 |
0.910 |
N/A |
108.1 |
1.079 |
0.935 |
||||
B |
0.925 |
0.782 |
93.4 |
||
0.955 |
0.812 |
||||
C |
0.974 |
0.831 |
98.5 |
||
0.994 |
0.850 |
||||
Replicates a, b, c |
Average |
0.853 |
100 |
||
SD |
0.059 |
7.5 |
|||
Positive control |
A |
0.509 |
0.365 |
N/A |
40.1 |
0.462 |
0.318 |
||||
B |
0.345 |
0.202 |
23.4 |
||
0.341 |
0.197 |
||||
C |
0.332 |
0.189 |
20.7 |
||
0.308 |
0.165 |
||||
Replicates a, b, c |
Average |
0.239 |
28.1 |
||
SD |
0.082 |
10.5 |
|||
Test substance |
A |
0.259 |
0.115 |
0.157 |
18.2 |
0.254 |
0.111 |
0.153 |
|||
B |
0.383 |
0.240 |
0.282 |
32.6 |
|
0.376 |
0.233 |
0.275 |
|||
C |
0.274 |
0.131 |
0.173 |
20.9 |
|
0.286 |
0.143 |
0.185 |
|||
Replicates a, b, c |
Average |
0.162 |
0.204 |
23.9 |
|
SD |
0.059 |
0.059 |
7.7 |
||
Blank |
|
0.138 |
|
|
|
0.148 |
|||||
0.145 |
|||||
0.139 |
|||||
0.146 |
|||||
0.143 |
|||||
Average |
0.143 |
||||
SD |
0.004 |
Correction for MTT reduction = corrected OD – MTT nonspecific reduction value (-0.042)
sd = Standard Deviation
Note. Rounded values only are displayed; unrounded numbers are used for calculations by Excel spreadsheet.
Applicant's summary and conclusion
- Interpretation of results:
- irritating
- Remarks:
- Migrated information Criteria used for interpretation of results: EU
- Conclusions:
- It was concluded that the test substance, IFF TM 09-218, with a mean tissue viability of 23.9 ± 7.7%, was predicted as irritant to the skin.
- Executive summary:
The skin irritation potential of the test substance, TM 09-218, was positive according to OECD Test Guideline 439 using a Reconstructed Human Epidermis Test Method. A mean tissue viability of 23.9 ± 7.7%, was predicted and is therefore considered as irritant to the skin.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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