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EC number: 632-619-2 | CAS number: 881685-58-1
- 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
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- Endpoint summary
- Stability
- Biodegradation
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- 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 sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05 Feb 2014 to 11 Feb 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- 2010
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- 2012
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
Test material
- Reference substance name:
- 632-619-2
- EC Number:
- 632-619-2
- Cas Number:
- 881685-58-1
- Molecular formula:
- C20 H23 F2 N3 O
- IUPAC Name:
- 632-619-2
Constituent 1
In vivo test system
Test animals
- Species:
- mouse
- Strain:
- CBA:J
- Remarks:
- Rj (SPF)
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Sex: Females
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old (age-matched, within one week)
- Weight at study initiation: 19.0-19.9 grams (the weight variation in animals in the study did not exceed ± 20 % of the mean weight)
- Housing: Group caging / mice were provided with glass tunnel-tubes
- Diet: autoclavable Complete diet for rats/mice – Breeding and maintenance. Ad libitum
- Water: tap water. Ad libitum
- Acclimatisation period: 20 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 - 25.3
- Humidity (%): 28 - 70
- Air changes (per hr): 15-20
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- IN-LIFE DATES: From: 05 Feb 2014 To: 11 Feb 2014
Study design: in vivo (LLNA)
- Vehicle:
- dimethyl sulphoxide
- Concentration:
- 50, 25 and 10 % w/v
- No. of animals per dose:
- 4
- Details on study design:
- PRE-SCREEN TESTS:
A Preliminary Irritation/ Toxicity Test was performed on CBA/J Rj mice using two doses, at test material concentrations of 100 and 50 % (w/v) in DMSO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on day 6 with a body weight measurement and the radioactive proliferation assay was not performed. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
MAIN STUDY
Substance Administration:
- Each mouse was topically dosed on the dorsal surface of each ear with 25 μL of the test material applied using a pipette. Each animal was dosed once a day for 3 consecutive days (days 1, 2 and 3). There was no treatment on days 4, 5 and 6.
Proliferation Assay:
- Thymidine (3HTdR) Administration: on day 6, each mouse was intravenously injected via the tail vein with 250 μL of sterile phosphate buffered saline (PBS) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" 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: 5 hours (± 30 minutes) after intravenous injection, the mice were euthanised. 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 nodes of mice from each test group was pooled and 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 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 by centrifuging. Pellets were resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice.
- Determination of incorporated 3HTdR: pellets were gently agitated, re-suspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight incubation at 2-8 °C, precipitates were centrifuged and supernatants were removed. Pellets were re-suspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured.
Observations:
- Clinical observations: all animals were observed at least once daily for any clinical signs, including local irritation and systemic toxicity.
- Body weight: Individual body weights were recorded on day 1 (beginning of the assay) and day 6 (prior to 3HTdR injection) with a precision of +/- 0.1 g.
- Ear thickness: measured by ear punch weight determinations, which was performed on day 6 after the animals were humanely killed.
Terminal Procedures:
- Five hours after intravenous injection, the mice were euthanised by asphyxiation by a rising concentration of carbon dioxide, followed by confirmation of death. - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- Disintegrations per minute (DPM) was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value. The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as 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.
Results and discussion
In vivo (LLNA)
Resultsopen allclose all
- Key result
- Parameter:
- SI
- Value:
- 1.5
- Test group / Remarks:
- 10 % test material
- Key result
- Parameter:
- SI
- Value:
- 2.6
- Test group / Remarks:
- 25 % test material
- Key result
- Parameter:
- SI
- Value:
- 8.5
- Test group / Remarks:
- 50 % test material
- Cellular proliferation data / Observations:
- CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the study. Rigid ears were observed in the 50 % (w/v) dose group on Days 2-5 and in the 25 % (w/v) group on Days 2-3. Slight alopecia was observed in the 50 % (w/v) dose group on Days 4-6. Test material precipitate was detected on the ears of the animals in the 50 % (w/v) dose group on Days 1-6, in the 25 % (w/v) group on Days 1-4 and in the 10 % (w/v) group on Days 2-3.
BODY WEIGHT MEASUREMENT
No treatment related effects were observed on body weight, however marked body weight loss (>5 %) was detected for one animal in the 10 % (w/v) dose group.
EAR THICKNESS MEASUREMENT
The ear punch weights were out of the historical control range for two animals in the 50 % (w/v) dose group, however, they did not exceed the limit of positive response (upper limit of historical control range + 25%).
PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative (vehicle) control group and in the 10 % (w/v) dose group. Larger than normal lymph nodes were observed for 1 animal in the 50 % (w/v) dose group. Slightly enlarged lymph nodes were detected for three animals in the 50 % (w/v) dose group and for all animals in 25 % (w/v) dose group.The stimulation index values were 8.5, 2.6 and 1.5 at concentrations of 50, 25 and 10 % (w/v), respectively.
INTERPRETATION OF OBSERVATIONS
The test item was a solid, which was formulated in DMSO. 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 item to cause lymphoproliferation in the Local Lymph Node Assay. The resulted stimulation index observed above the threshold limit of 3 at the concentration of 50 % (w/v) (SI value is 8.5) under these exaggerated test conditions were considered to be good evidence that the test material is a sensitiser. No EC3 value was derived, however it can be calculated from the results to be >2%.
Any other information on results incl. tables
Preliminary Screening Test
No mortality or signs of systemic toxicity were detected. Rigid ears were observed in both dose groups on Day 3 and alopecia was detected for both animals in the 100 % (w/v) group on Days 5-6. Alopecia on the crown was observed for one animal in the 50 % (w/v) group on Days 2-6. No marked body weight loss was detected for the animals. Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6. Increased ear thickness values (>25 % increase) were detected in the 100 % (w/v) dose group indicating excessive local skin irritation. The appearance of the lymph nodes was normal in both dose groups. Based on these results, 100 % (w/v) dose was considered too high due to the increased ear thickness values indicating excessive local skin irritation. Therefore, 50, 25 and 10 % (w/v) doses were examined in the main test.
Table 1. Individual Body Weights for all Animals with Group Means (Preliminary Irritation/Toxicity Test).
Animal Number |
Test Group |
Initial Body Weight (g) |
Terminal Body Weight* (g) |
Change# % |
1 |
100 % (w/v) |
23.5 |
24.6 |
4.7 |
2 |
100 % (w/v) |
23.9 |
24.3 |
1.7 |
|
Mean |
23.7 |
24.5 |
3.2 |
3 |
50 % (w/v) |
23.6 |
24.1 |
2.1 |
4 |
50 % (w/v) |
23.1 |
23.1 |
0.0 |
|
Mean |
23.4 |
23.6 |
1.1 |
Notes:
1. *: Terminal body weights were measured on Day 6.
2. #: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100
Table 2. Individual ear thickness for all animals (Preliminary Irritation/Toxicity Test).
Animal Number |
Test Group |
Ear Thickness on Day 1 (mm) |
Ear Thickness on Day 3 (mm) |
Ear Thickness on Day 6 (mm) |
Biopsy weight* on Day 6 (mg) |
|||
|
|
Right |
Left |
Right |
Left |
Right |
Left |
|
1 |
100 % (w/v) |
0.20 |
0.21 |
0.37 |
0.35 |
0.25 |
0.23 |
19.77 |
2 |
100 % (w/v) |
0.21 |
0.22 |
0.38 |
0.32 |
0.24 |
0.21 |
21.73 |
3 |
50 % (w/v) |
0.22 |
0.21 |
0.23 |
0.23 |
0.24 |
0.23 |
23.21 |
4 |
50 % (w/v) |
0.21 |
0.22 |
0.23 |
0.24 |
0.23 |
0.23 |
22.69 |
Note:
1. *: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (≥25%).
Table 3. Summarised Clinical Observations (Preliminary Irritation/Toxicity Test).
Period |
Animal Number |
Test Group |
Clinical observations before treatment |
Clinical observations after treatment |
Day 1 |
1 |
100 % (w/v) |
Symptom free, ES: 0 |
Symptom free*, ES i.s.: 0 |
2 |
100 % (w/v) |
Symptom free, ES: 0 |
Symptom free*, ES i.s.: 0 |
|
3 |
50 % (w/v) |
Symptom free, ES: 0 |
Symptom free, ES: 0 |
|
4 |
50 % (w/v) |
Symptom free, ES: 0 |
Symptom free, ES: 0 |
|
Day 2 |
1 |
100 % (w/v) |
Symptom free*, ES: 0 |
Symptom free*, ES i.s.: 0 |
2 |
100 % (w/v) |
Symptom free*, ES: 0 |
Symptom free*, ES i.s.: 0 |
|
3 |
50 % (w/v) |
Alopecia on the crown**, ES: 0 |
Alopecia on the crown*, ES: 0 |
|
4 |
50 % (w/v) |
Symptom free**, ES: 0 |
Symptom free*, ES: 0 |
|
Day 3 |
1 |
100 % (w/v) |
Symptom free*, ES: 0 |
Rigid ears*, ES: i. s.: 0 |
2 |
100 % (w/v) |
Symptom free*, ES: 0 |
Rigid ears*, ES: i. s.: 0 |
|
3 |
50 % (w/v) |
Alopecia on the crown*, ES: 0 |
Alopecia on the crown, rigid ears*, ES: 0 |
|
4 |
50 % (w/v) |
Symptom free*, ES: 0 |
Rigid ears*, ES: 0 |
|
Day 4 |
1 |
100 % (w/v) |
Symptom free*, ES: 0 |
|
2 |
100 % (w/v) |
Symptom free*, ES: 0 |
|
|
3 |
50 % (w/v) |
Alopecia on the crown*, ES: 0 |
|
|
4 |
50 % (w/v) |
Symptom free*, ES: 0 |
|
|
Day 5 |
1 |
100 % (w/v) |
Slight alopecia*, ES: 0 |
|
2 |
100 % (w/v) |
Slight alopecia*, ES: 0 |
|
|
3 |
50 % (w/v) |
Alopecia on the crown**, ES: 0 |
|
|
4 |
50 % (w/v) |
Symptom free**, ES: 0 |
|
|
Day 6 |
1 |
100 % (w/v) |
Alopecia**, ES: 0 |
|
2 |
100 % (w/v) |
Alopecia**, ES: 0 |
|
|
3 |
50 % (w/v) |
Alopecia on the crown, ES: 0 |
|
|
4 |
50 % (w/v) |
Symptom-free, ES: 0 |
|
*: test item precipitate; **: minimal amount of test item precipitate, i. s.: Score made on inner surface of ear
Main Test Estimation of the Proliferation Response of Lymph Node Cells
Table 1. The radioactive disintegrations per minute per animal and the stimulation index. The stimulation index is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.
Test Group Name |
Measured DPM / group |
DPM |
Number of Lymph Nodes |
DPN |
Stimulation Index |
Background/ (5 % (w/v) TCA) |
34/ 36 |
- |
- |
- |
- |
Negative control DMSO |
1912 |
1877.0 |
8 |
234.6 |
1.0 |
Isopyrazam 50 % (w/v) in DMSO |
15923 |
15888.0 |
8 |
1986.0 |
8.5 |
Isopyrazam 25 % (w/v) in DMSO |
4832 |
4797.0 |
8 |
599.6 |
2.6 |
Isopyrazam 10 % (w/v) in DMSO |
2764 |
2729.0 |
8 |
341.1 |
1.5 |
Applicant's summary and conclusion
- Interpretation of results:
- Category 1B (indication of skin sensitising potential) based on GHS criteria
- Conclusions:
- The test material tested in a suitable vehicle, was shown to have skin sensitisation potential (sensitiser) in the Local Lymph Node Assay. No EC3 value was derived, however it can be calculated from the results to be >2%.
- Executive summary:
The skin sensitisation potential of the test material following dermal exposure to female CBA/J Rj mice was determined using the LLNA method according to OECD TG 429 following GLP principles . The test material solutions (50, 25, 10% test material formulated in DMSO) were applied to 4 females per group on the dorsal surface of ears of experimental animals (25 μL/ear) for 3 consecutive days (days 1, 2 and 3). The negative control group received the vehicle (DMSO). There was no treatment on days 4, 5 and 6. On day 6, 5 hours prior to termination, animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR). Cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the values obtained were used to calculate stimulation indices (SI).
No mortality or signs of systemic toxicity were observed during the study. Rigid ears were observed in the 50 % dose group on days 2-5 and in the 25 % group on days 2-3. Slight alopecia was observed in the 50 % dose group on days 4-6. Test material precipitate was detected on the ears of the animals in the 50 % dose group on days 1-6, in the 25 % group on days 1-4 and in the 10 % group on days 2-3. The appearance of the lymph nodes was normal in the negative control group and in the 10 % dose group. Larger than normal lymph nodes were observed for 1 animal in the 50 % dose group. Slightly enlarged lymph nodes were detected for 3 animals in the 50 % dose group and for all animals in 25 % dose group. The observed stimulation index values were 8.5, 2.6 and 1.5 at concentrations of 50, 25 and 10 %, respectively.
The test material tested in a suitable vehicle, was shown to have skin sensitisation potential (sensitiser) in the Local Lymph Node Assay. No EC3 value was derived, however it can be calculated from the results to be >2%.
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