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EC number: 441-520-1 | 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
Endpoint summary
Administrative data
Description of key information
LLNA
Under the conditions of the study, the test material was shown to have no sensitisation potential.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 15 December 2010 to 21 December 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- 2010
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
- Species:
- mouse
- Strain:
- CBA:J
- Remarks:
- CBA/J Rj mice
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 8 – 9 weeks old
- Weight at study initiation: 19.0 – 21.8 grams (the weight variation in animals involved in the study did not exceed ± 20% of the mean weight)
- Housing: Group caging in Type II polypropylene/ polycarbonate cages. Bedding was available to animals during the study and they were provided with glass tunnel-tubes.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30 - 70%
- Air changes: 15- 2 0 air exchange/hour
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m. - Vehicle:
- acetone/olive oil (4:1 v/v)
- Concentration:
- 10, 5 and 2.5 (w/v) %
- No. of animals per dose:
- 4 animals per dose
- Details on study design:
- PRE-SCREEN TESTS:
- Compound solubility: -During the Preliminary Compatibility Test, based on the solubility of the test material acetone/olive oil 4:1 (v/v) mixture (AOO) was chosen as vehicle for the experiment. The maximum attainable concentration was 10 (w/v) %. The test material was weighed and formulations prepared daily on a weight: volume basis.
- A Preliminary Irritation/Toxicity Test was performed on CBA/J Rj mice using two doses, at test material concentrations of 10 and 5 (w/v) %, respectively. This preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6. An assessment of ear swelling was made by measuring thickness on Days 1, 3 and 6, and the weight of an ear punch on Day 6.
- During the Preliminary Irritation / Toxicity Test no mortality, systemic toxicity or local irritation were observed (score of erythema was 0 during the observation time). There was no local swelling on Day 3 and on Day 6 in either group. The ear punch weights were in the normal range. No treatment related effect on body weights was observed. The observations recorded in this preliminary test suggested that the application of the material at 10 and 5% were acceptable for the LLNA main study.
MAIN STUDY
- Topical application: During the assay each mouse was topically dosed on the dorsal surface of each ear with 25 μL of the appropriate formulation applied using a pipette. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
- Injection of Tritiated Thymidine (3HTdR): On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
- Removal and Preparation of Draining Auricular Lymph Nodes: Five hours (± 30 minutes) after intravenous injection the mice were euthanised by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses). The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice were collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
- Preparation of Single Cell Suspension of Lymph Node Cells: A single cell suspension (SCS) of lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4°C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice.
- Determination of Incorporated 3HTdR: After the final wash, supernatants were removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. Each pellet was gently agitated before suspending the LNCs in 3 mL of 5% TCA (trichloroacetic acid) for precipitation of macromolecules. After incubation with 5% TCA at 2 - 8°C overnight (approximately 18 hours) precipitate was recovered by centrifugation at 190 x g for 10 minutes, supernatants were removed and pellets were suspended in 1 mL of 5% TCA and dispersed using ultrasonic water bath. Each precipitate was transferred to a suitable sized scintillation vial with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded to a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM) for each animal, and the results were pooled. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5% TCA.
OBSERVATIONS
- Clinical Observations: During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
- Measurement of Body Weight: Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.
EVALUATION OF THE RESULTS
- DPM was measured for each group. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 (w/v) % TCA solutions was used as the background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation. Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
- Interpretation of Results: The test material is regarded as a sensitiser if both of the following criteria are fulfilled:
(1) That exposure to at least one concentration of the test material resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
(2) The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression. - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Positive control results:
- - The positive control group animals were treated with 25 (w/v) % HCA solution in a relevant vehicle (AOO) concurrent to the test material groups. The positive control substance was chosen according to the OECD guideline. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. A significant lymphoproliferative response (stimulation index value of 4.2) was noted for α-hexylcinnamaldehyde in this experiment. The results of the positive control group demonstrated the appropriate performance of the assay.
- Key result
- Parameter:
- SI
- Value:
- 0.5
- Test group / Remarks:
- 10 (w/v) %
- Key result
- Parameter:
- SI
- Value:
- 0.7
- Test group / Remarks:
- 5 (w/v) %
- Key result
- Parameter:
- SI
- Value:
- 0.8
- Test group / Remarks:
- 2.5 (w/v) %
- Cellular proliferation data / Observations:
- CLINICAL OBSERVATION
- No mortality or signs of systemic toxicity were observed during the study.
BODY WEIGHT MEASUREMENT
- No treatment related effects were observed on animal body weights.
PROLIFERATION ASSAY
- Appearance of the lymph nodes was normal in the negative control group and in the test material treated groups. Larger than normal lymph nodes was observed in the positive control group.
INTERPRETATION OF OBSERVATIONS
- The test material was a light yellow solid, which was dissolved in acetone/olive oil 4:1 (v/v) mixture (AOO). Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test material to cause lymphoproliferation in the Local Lymph Node Assay. The lack of any positive result under these exaggerated test conditions is considered to be good evidence that the test material is not a sensitiser.
RELIABILITY OF THE TEST
- The results of the positive control group demonstrated the appropriate performance of the assay. - Interpretation of results:
- other: Not classified in accordance with EU Criteria.
- Conclusions:
- Under the conditions of this study, the test material was shown to have no sensitisation potential.
- Executive summary:
The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 429, under GLP conditions.
Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline, the test material was dissolved in acetone/olive oil 4:1 (v/v) mixture (AOO). The maximum attainable concentration was 10 (w/v) %.
The Preliminary Irritation/Toxicity Test was performed in CBA/J Rj mice using two doses (test material concentrations of 10 and 5 (w/v) %) in the selected vehicle. The applicability and biocompatibility of the test material on the ears of animals at the maximum concentration of test material of 10 (w/v) % was acceptable.
In the main assay, twenty female CBA/J Rj mice were allocated to five groups of four animals each: three groups received the appropriate formulation of the test material at concentrations of 10, 5 and 2.5 (w/v) %, the negative control group received AOO and the positive control group received 25% α-hexylcinnamaldehyde (HCA) in AOO. The solutions of the test material were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).
No mortality, systemic toxicity or local irritation was observed during the study. No treatment related effects were observed on animal body weights in any treated groups. Stimulation index values of the test material were 0.5, 0.7 and 0.8 at treatment concentrations of 10, 5 and 2.5 (w/v) %, respectively. α-hexylcinnamaldehyde (25 (w/v) % dissolved in AOO) was used as a positive control to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 4.2) was noted for the positive control chemical and this result confirmed the validity of the assay.
Under the conditions of this study, the test material was shown to have no sensitisation potential.
Reference
Table 1: DPM, DPN and Stimulation Index Values for all Groups
Test Group |
Measured DPM/group |
Group DPM |
No. of Nodes |
DPN |
Stimulation Index Values |
Background (5 (w/v) % TCA ) |
34 |
- |
- |
- |
- |
Negative control AOO |
1245 |
1211 |
8 |
151.4 |
1.0 |
Test material 10 (w/v) % in AOO |
689 |
655 |
8 |
81.9 |
0.5 |
Test material 5 (w/v) % in AOO |
867 |
833 |
8 |
104.1 |
0.7 |
Test material 2.5 (w/v) % in AOO |
974 |
940 |
8 |
117.5 |
0.8 |
Positive control 25 % HCA in AOO |
5160 |
5126 |
8 |
640.8 |
4.2 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
LLNA
The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 429, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline, the test material was dissolved in acetone/olive oil 4:1 (v/v) mixture (AOO). The maximum attainable concentration was 10 (w/v) %.
The Preliminary Irritation/Toxicity Test was performed in CBA/J Rj mice using two doses (test material concentrations of 10 and 5 (w/v) %) in the selected vehicle. The applicability and biocompatibility of the test material on the ears of animals at the maximum concentration of test material of 10 (w/v) % was acceptable.
In the main assay, twenty female CBA/J Rj mice were allocated to five groups of four animals each: three groups received the appropriate formulation of the test material at concentrations of 10, 5 and 2.5 (w/v) %, the negative control group received AOO and the positive control group received 25% α-hexylcinnamaldehyde (HCA) in AOO. The solutions of the test material were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).
No mortality, systemic toxicity or local irritation was observed during the study. No treatment related effects were observed on animal body weights in any treated groups. Stimulation index values of the test material were 0.5, 0.7 and 0.8 at treatment concentrations of 10, 5 and 2.5 (w/v) %, respectively. α-hexylcinnamaldehyde (25 (w/v) % dissolved in AOO) was used as a positive control to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 4.2) was noted for the positive control chemical and this result confirmed the validity of the assay.
Under the conditions of this study, the test material was shown to have no sensitisation potential.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin sensitisation.
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