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EC number: 944-610-3 | CAS number: 286472-48-8
- 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
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Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- This study was conducted between 05 July 2016 and 28 July 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- 22 July 2010
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- EC No. 440/2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
Test material
- Reference substance name:
- 2-ethoxy-1,3-dimethylcyclohexane
- EC Number:
- 944-610-3
- Cas Number:
- 286472-48-8
- Molecular formula:
- C10H20O
- IUPAC Name:
- 2-ethoxy-1,3-dimethylcyclohexane
- Test material form:
- liquid
- Details on test material:
- - Substance name as cited in test report: FRET 05-0293
- Phystical state: clear, colorless liquid
- Storage conditions: ambient temperature (15-25 °C), protected from light
1
In vivo test system
Test animals
- Species:
- mouse
- Strain:
- CBA/Ca
- Remarks:
- CBA/CaOlaHsd strain
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands.
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: not reported
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23g
- Housing: in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: >5 days
- Indication of any skin lesions: not reported
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25”C
- Humidity (%): 30 - 70%
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 05 July 2016 To: 28 July 2016
Study design: in vivo (LLNA)
- Vehicle:
- acetone/olive oil (4:1 v/v)
- Concentration:
- undiluted (as supplied) or 50% or 25% in acetone/olive oil 4:1
- No. of animals per dose:
- Preliminary Screening Test = 1
Main Test = 5 - Details on study design:
- Study Design
Preliminary Screening Test
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item or the test item at a concentration of 50% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Table 1. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.
Main Test
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 50%vor 25% in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.
The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item, α Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.
3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control or positive control item (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.
Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.
Preparation of Single Cell Suspension:
A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re suspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was re suspended in 3 mL of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation:
After approximately 18 hours incubation at approximately 4”C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA). - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)
Results and discussion
- Positive control results:
- The positive control α Hexylcinnamaldehyde gave a Stimulation Index of greater than 3 (5.29) when tested at a concentration of 25% v/v in acetone/olive oil 4:1
In vivo (LLNA)
Resultsopen allclose all
- Key result
- Parameter:
- SI
- Value:
- ca. 2.28
- Test group / Remarks:
- 25% v/v in acetone/olive oil 4:1
- Remarks on result:
- other: Negative
- Key result
- Parameter:
- SI
- Value:
- ca. 4.43
- Test group / Remarks:
- 50% v/v in acetone/olive oil 4:1
- Remarks on result:
- other: Positive
- Key result
- Parameter:
- SI
- Value:
- ca. 4.47
- Test group / Remarks:
- 100%
- Remarks on result:
- other: Positive
- Cellular proliferation data / Observations:
- Preliminary Screening Test
No signs of systemic toxicity were noted.
No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
Based on this information the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.
Main Test
Estimation of the Proliferative Response of Lymph Node Cells
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are summarised in Table 2 (below):
Clinical Observations and Mortality Data
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.
Body Weight
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.
Any other information on results incl. tables
Table 2: Stimulation Indices
Teatment Group | Concentration | Stimulation Index | Result |
Test Item | 25% v/v in acetone/olive oil 4:1 |
2.28 | Negative |
50% v/v in acetone/olive oil 4:1 |
4.43 |
Positive |
|
|
100% |
4.47 |
Positive |
Positive Control Item |
25% v/v in acetone/olive oil 4:1 |
5.29 |
Positive |
Calculation of EC3 Value
EC3 = c + [[(3-d)/(b-d)] x (a-c)]
a = 50
b = 4.43
c = 25
d = 2.28
EC3 = 25 + [[(3-2.28)/(4.43-2.28)] x (50-25)] = 33
The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 33%.
Applicant's summary and conclusion
- Interpretation of results:
- Category 1B (indication of skin sensitising potential) based on GHS criteria
- Conclusions:
- The test item was considered to be a sensitizer under the conditions of the test.
The test item was also classified as a contact sensitizer (Category 1B) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H317: May cause an allergic skin reaction” are therefore required.
The test item was classified as a contact sensitizer (Category 1B) according to the Globally Harmonized System of Classification and Labelling of Chemicals.
The positive control α Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (5.29) when tested at a concentration of 25% v/v in acetone/olive oil 4:1 thus demonstrating the sensitivity and reliability of the test system - Executive summary:
A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.
Methods
Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1
Results
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:
Teatment Group Concentration Stimulation Index Result Test Item 25% v/v in acetone/olive oil 4:1
2.28 Negative 50% v/v in acetone/olive oil 4:1
4.43
Positive
100%
4.47
Positive
Positive Control Item
25% v/v in acetone/olive oil 4:1
5.29
Positive
The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 33%.
Conclusion
The test item was considered to be a sensitizer under the conditions of the test.
The test item was also classified as a contact sensitizer (Category 1B) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H317: May cause an allergic skin reaction” are therefore required.
The test item was classified as a contact sensitizer (Category 1B) according to the Globally Harmonized System of Classification and Labelling of Chemicals.
The positive control α Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (5.29) when tested at a concentration of 25% v/v in acetone/olive oil 4:1 thus demonstrating the sensitivity and reliability of the test system.
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