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EC number: 203-250-0 | CAS number: 104-90-5
- 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 sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Report date:
- 2012
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.2600 (Skin Sensitisation)
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Toxi-Coop ZRT.
- Type of study:
- mouse local lymph node assay (LLNA)
Test material
- Test material form:
- gas under pressure: refrigerated liquefied gas
- Details on test material:
- - Name of test material: 5-ethyl-2-methylpyridine
Constituent 1
In vivo test system
Test animals
- Species:
- mouse
- Strain:
- other: CRL: NMRI BR and CBA/Ca mice
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 10 - 12 weeks old
- Weight at study initiation: 16.9 – 20.7 g; The weight variation in animals involved in the study did not exceed +/- 20 % of the mean weight.
- Housing during acclimatization period: Grouped caging in small groups, during the test: Grouped caging (5 animals/cage), cage type: Type II. Polypropylene / polycarbonate
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 21 or 7 days
ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 30 - 70 %
- Photoperiod (hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
IN-LIFE DATES:
- From: May 16, 2012
- To: May 24, 2012
Study design: in vivo (LLNA)
- Vehicle:
- acetone/olive oil (4:1 v/v)
- Concentration:
- No., Groups , Test item concentration (% w/v ) , Positive control concentration (% w/v) , No. of animals
1, Vehicle control: AOO , - , - , 5
2, Positive control: HCA in AOO , - , 25, 5
3, LZ163 in AOO , 50, - , 5
4, LZ163 in AOO , 37.5, - , 5
5, LZ163 in AOO , 25, - , 5
6, LZ163 in AOO , 10, - , 5
7, LZ163 in AOO , 5, - , 5
AOO = Acetone:olive oil 4:1 (v/v) mixture
HCA = α-Hexylcinnamaldehyde - No. of animals per dose:
- Total of 5 animals per dose and control group.
- Details on study design:
- RANGE FINDING TESTS:
- Compound solubility: The test item was a liquid and based on preliminary test results formulated for the main test (apparently solutions) in Acetone: Olive oil 4:1 (v/v) mixture (AOO).
- Irritation: The preliminary irritation/toxicity screen (conducted in two steps at a concentration range from 100 % to 10 %) indicated systemic toxic effect of the test item at high test concentrations. Based on the results 50 % was selected as the maximum applicable concentration to be used in the main test.
- Lymph node proliferation response: Proliferation values obtained reflected the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
1. A minimum of four animals were used per dose group, with a minimum of three concentrations of the test substance, plus a negative control group treated only with the vehicle for the test substance and a positive control. In those cases in which individual animal data were collected and statistically analyzed, a minimum of five animals per dose group were used.
- Skin sensitizing effects were observed at the applied concentration in the positive control group (SI > 3).
TREATMENT PREPARATION AND ADMINISTRATION:
AOO was used as negative (vehicle) control both for the groups treated with the test item formulations and the positive control. The test item was administered in five different concentrations.
- In vivo Treatment: Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance (positive control group) or the vehicle (negative control group) using a pipette, on the dorsal surface of each ear. After the treatments animals returned to their cages. 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.
- Proliferation Assay: No technically failed treatment was observed during the test. Lymph nodes from all five animals per dose groups were processed (except the 50 % dose group where 4 animals were processed).
- Injection of 3H-methyl-thymidine (3HTdR): On Day 6, the animals were intravenously injected via the tail vein with 250 μL of sterile PBS containing 20 μCi of 3HTdR using a 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 humanely sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps. Once removed, the nodes of individual mice were collected separately in a Petri dish 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) of each individual animal was prepared and collected in disposable tubes by a gentle mechanical disaggregation of the lymph nodes through 200 μm-mesh stainless steel gauze using the plunger of a disposable syringe. The cell strainer (stainless steel gauze) was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4°C. After centrifugation, the majority of the supernatant was aspirated off, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated and the LNCs were resuspended in 10 mL of PBS. This washing procedure was repeated twice. This procedure was repeated for lymph nodes of each individual animal.
- Determination of Incorporated 3HTdR: After the final washing step, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before resuspending the LNCs in 3 mL of 5 % trichloracetic acid (TCA) to allow for the precipitation of the macromolecules. After incubation with 5 % TCA at 2-8 °C overnight (approx. 18 hrs), each precipitate was removed and the pellets were resuspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and stored at room temperature protected from light until measurement.
For the measurement of incorporated radioactivity the vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expressed the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % TCA.
Instrument used for the measurement:
Name: Packard Tri-Carb 2300 TR Liquid Scintillation Analyzer
Serial Number: 406 117 - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- The individual approach enabled statistical analysis of the data. Statistical analysis was performed by SPSS/PC+ (4.0.1) software package. The heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values. The normal distribution of data was examined by Kolmogorow-Smirnow test. Since no normal distribution was observed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. A positive result was detected and inter-group comparisons performed using the Mann-Whitney U-test. Dose-response was evaluated by linear regression using Microsoft Excel Software.
Results and discussion
- Positive control results:
- Statistically significant increase of the proliferation values compared to the negative control was observed in the positive control group (p < 0.01, Mann-Whitney U-test).
In vivo (LLNA)
Resultsopen allclose all
- Parameter:
- SI
- Remarks on result:
- other: see Remark
- Remarks:
- Visually larger lymph nodes than the control was observed in the positive control group and in the 50 % dose group. Visual appearance of the lymph nodes was normal in the negative (vehicle) control group and in the other test item treated groups. Significant (SI ≥ 3) lymphoproliferation was noted for LZ163 at concentrations of 50 %, 37.5 % and 25 %. No significant (SI ≥ 3) lymphoproliferation was noted for LZ163 at concentrations of 10 % and 5 %. The observed stimulation index values were 4.74, 5.45, 3.27, 1.55 and 2.26 at test item concentrations of 50 %, 37.5 %, 25 %, 10 % and 5 %, respectively. Dose-related response was observed, but not statistically significant (see Statistical Analysis).
- Parameter:
- other: disintegrations per minute (DPM)
- Remarks on result:
- other: see Remark
- Remarks:
- Individual DPM and DPN Values, the Mean DPM and DPN Values and the SI values for all Groups in the Main Test are shown in table 1 below. Individual DPM values (corrected with the mean background value) were statistically evaluated in the 37.5 %, 25 %, 10 % and 5 % test item treated groups and in the control groups (both positive and negative control). Statistically significant increase of the proliferation values compared to the negative control was observed in the positive control group and in the 37.5 % test item treated group (p < 0.01, Mann-Whitney U-test) and in the 25 % dose group (p < 0.05, Mann-Whitney U-test). The increase of the lymphoproliferation over the control at test item concentrations of 10 % or 5 % was not statistically significant. Results of the statistical analysis are given in Table 9 of Appendix I. Significance of the dose-response was evaluated by linear regression using the SI values calculated for evaluated test concentrations (37.5 %, 25 %, 10 % and 5 %). Although the effect was considered as dose-related, no statistical significance was observed: the r value was 0.93, the p value was 0.068.
Any other information on results incl. tables
Individual DPM and DPN Values, the Mean DPM and DPN Values and the SI values for all Groups in the Main Test are shown in table 1 below:
Table 1: Individual DPM and DPN Values, the Mean DPM and DPN Values and the SI values for all Groups in the Main TestTest Group | Animal | Individual DPM | Individual DPM | Individual DPN | Group DPM | Group DPN | SI |
Name | (Measured) | (Corrected)$ | (DPM/Node)# | (Mean) | (Mean) | Values | |
684 | 369 | 326.5 | 163.3 | 724.3 | 362 | 1.00 | |
Negative (vehicle) | 685 | 1211 | 1168.5 | 584.3 | |||
control: | 700 | 575 | 532.5 | 266.3 | |||
AOO | 705 | 393 | 350.5 | 175.3 | |||
716 | 1286 | 1243.5 | 621.8 | ||||
683 | 7223 | 7180.5 | 3590.3 | 8824.5 | 4412 | 12.18 | |
Positive control: | 686 | 13383 | 13340.5 | 6670.3 | ** | ||
25 % HCA | 701 | 8891 | 8848.5 | 4424.3 | |||
in AOO | 704 | 8301 | 8258.5 | 4129.3 | |||
714 | 6537 | 6494.5 | 3247.3 | ||||
690 | 2824 | 2781.5 | 1390.8 | 3432.3 | 1716 | 4.74 | |
LZ163 | 691 | 4648 | 4605.5 | 2302.8 | NE | ||
50 % in AOO | 702 | 3821 | 3778.5 | 1889.3 | |||
711 | 2606 | 2563.5 | 1281.8 | ||||
689 | 5730 | 5687.5 | 2843.8 | 3944.3 | 1972 | 5.45 | |
LZ163 | 693 | 1526 | 1483.5 | 741.8 | ** | ||
37.5 % in AOO | 703 | 2508 | 2465.5 | 1232.8 | |||
706 | 6666 | 6623.5 | 3311.8 | ||||
717 | 3504 | 3461.5 | 1730.8 | ||||
687 | 1892 | 1849.5 | 924.8 | 2371.3 | 1186 | 3.27 | |
LZ163 | 694 | 1271 | 1228.5 | 614.3 | * | ||
25 % in AOO | 718 | 3112 | 3069.5 | 1534.8 | |||
719 | 3801 | 3758.5 | 1879.3 | ||||
720 | 1993 | 1950.5 | 975.3 | ||||
688 | 1483 | 1440.5 | 720.3 | 1123.5 | 562 | 1.55 | |
LZ163 | 692 | 1681 | 1638.5 | 819.3 | NS | ||
10 % in AOO | 707 | 829 | 786.5 | 393.3 | |||
710 | 554 | 511.5 | 255.8 | ||||
712 | 1283 | 1240.5 | 620.3 | ||||
682 | 2399 | 2356.5 | 1178.3 | 1637.7 | 819 | 2.26 | |
LZ163 | 708 | 804 | 761.5 | 380.8 | NS | ||
5 % in AOO | 709 | 1109 | 1066.5 | 533.3 | |||
713 | 2191 | 2148.5 | 1074.3 | ||||
715 | 1898 | 1855.5 | 927.8 |
AOO = Acetone: Olive oil 4:1 (v/v) mixture
HCA =a-Hexylcinnamaldehyde
$ = Measured DPM value – Mean background value
# = Corrected DPM value / No. of lymph nodes (per animal)
Mean background value = 42.5
No. of lymph nodes (per animal) = 2
NE= Not evaluated. The 50 % dose group was not statistically evaluated, because mortality and significant clinical symptoms were observed in this dose group during the main test.
NS= Not significant; Mann-Whitney U-test versus control (AOO)
* Significant; p < 0.05 Mann-Whitney U-test versus control (AOO)
** Significant; p < 0.01 Mann-Whitney U-test versus control (AOO)
The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and also results of the preliminary irritation/toxicity tests.
The test item was a liquid and based on preliminary test results formulated for the main test (apparently solutions) in Acetone: Olive oil 4:1 (v/v) mixture (AOO). The preliminary irritation/toxicity screen (conducted in two steps at a concentration range from 100 % to 10 %) indicated systemic toxic effect of the test item at high test concentrations. Based on the results 50 % was selected as the maximum applicable concentration to be used in the main test. To ensure validity of the test should unexpected adverse effects occur, a total of five concentrations were examined in the main test. Concentrations tested were 50 %, 37.5 %, 25 %, 10 % and 5 % in vehicle (AOO).
The mortality and intensity of the clinical symptoms observed in the 50 % dose group indicated significant systemic toxic effects. Therefore even though the SI value was calculated, results were not statistically evaluated. Taking into account both the preliminary test results and main test data (body weights, clinical observations, ear thickness values and erythema scores) clinical symptoms observed in the 37.5 % and 25 % dose groups were not considered toxicologically significant. Proliferation values obtained reflected the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.
Although no strong dose-response relationship was observed (as indicated by the linear regression performed using data points of the statistically evaluated test concentrations of 37.5 %, 25 %, 10 % and 5 %), the statistically significant (SI ≥ 3) proliferation observed at 37.5 % and 25 % concentrations was considered evidence that LZ163 has sensitization potential, according to evaluation criteria of the relevant guidelines.
EC3calculation was conducted by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve based on published method. The calculated EC3value of LZ163 was 22.4 % in this LLNA. Using this EC3value according to the published data for classification of contact allergens LZ163 can be ranked among weak sensitizers (10 ≤ EC3≤ 100) in this LLNA.
Additionally EC3value based on the equation of the regression curve was also calculated: the relevant value was 18.1 in this LLNA. Using the EC3value based on the regression curve LZ163 can be ranked among the same category (weak sensitizer) as based on the actual dose-response curve.
Applicant's summary and conclusion
- Interpretation of results:
- sensitising
- Remarks:
- Migrated information Criteria used for interpretation of results: EU
- Conclusions:
- Under the conditions of the present Local Lymph Node Assay, the test item at concentrations of 50%, 37.5%, 25%, 10% and 5 % as homogenous formulations (solutions) in a suitable vehicle (AOO) was shown to have sensitization potential (sensitizer). Based on the EC3 values calculated using dose-response and regression curve analysis the test item was considered a weak sensitizer in this LLNA.
- Executive summary:
A study according to EU Method B.42, OECD Guideline 429 and EPA OPPTS 870.2600 (Skin Sensitisation: Local Lymph Node Assay) was carried out. Under the conditions of the assay the test item was tested at concentrations of 50 %, 37.5 %, 25 %, 10 % and 5 % as homogenous formulations (solutions) in a suitable vehicle (AOO). The test item was shown to shown to have sensitization potential (sensitizer).The calculated EC3-value in this LLNA was 22.4 % based on linear interpolation and 18.1 % based on regression curve analysis. Based on the EC3 values calculated using dose-response and regression curve analysis the test item was considered a weak sensitizer.
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