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EC number: 235-518-8 | CAS number: 12262-26-9 This substance is identified in the Colour Index by Colour Index Constitution Number, C.I. 53450.
- Life Cycle description
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- Ecotoxicological Summary
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- Short-term toxicity to fish
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- Long-term toxicity to aquatic invertebrates
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Endpoint summary
Administrative data
Description of key information
In an in vivo skin sensitisation assay (LLNA) in mouse according to OECD 429, the test item did not show skin sensitising properties.
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:
- January 10 - March 20, 2017
- 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:
- 22 July 2010
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
- Species:
- mouse
- Strain:
- CBA/Ca
- Remarks:
- Ola Hsd mice
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: TOXI-COOP ZRT., H-1103, Budapest, Cserkesz u. 90., Hungary
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: Young adult mice; 11-12 weeks old
- Weight at study initiation: 19.2 – 23.2 g, the weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
- Housing: in Type II. Polypropylene / polycarbonate cages
during acclimatization period: Grouped caging in small groups
during the test: Grouped caging (4 animals/cage),
- Diet: ssniff® Rat/Souris-Elevage E complete diet for rats and mice (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) ad libitum.
- Water: tap water from watering bottles ad libitum.
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12/12 from 6.00 a.m. to 6.00 p.m. - Vehicle:
- dimethyl sulphoxide
- Concentration:
- 1 %, 0.5 %, 0.25 % or 0.1 % (w/v)
- No. of animals per dose:
- 4 animals/treatment group
- Details on study design:
- Animals in the treatment groups were treated with the negative (vehicle) controls (DMSO or AOO), appropriate formulations of the test item or 25 % (w/v) concentration of the positive control substance. The test item was administered at four different concentrations according to the results of the dose range finding test.
Criteria for Erythema Scores:
Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to
eschar formation preventing grading of erythema 4
Test Concentrations in the Main Test:
Groups Test item Positive control No. of animals
Conc. (% w/v) conc.(% w/v)
1 Vehicle control for the positive control: AOO - - 4
2 Positive control: HCA in AOO - 25 4
3 Vehicle control for the test item: DMSO - - 4
4 Leuco Sulfur Blue 13 in DMSO 1 - 4
5 Leuco Sulfur Blue 13 in DMSO 0.5 - 4
6 Leuco Sulfur Blue 13 in DMSO 0.25 - 4
7 Leuco Sulfur Blue 13 in DMSO 0.1 - 4
In vivo Treatment
Each mouse was topically treated with 25 L of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette,
on the dorsal surface of each ear. After the treatments animals were 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 animals showed symptoms of systemic toxicity or excessive skin irritation, and no technical treatment failures were observed during the test: all animals treated were processed. Therefore no treatment group was excluded from the evaluation.
Injection of 3HTdR
On Day 6 each mouse was intravenously injected via the tail vein with 250 L of sterile PBS (1 x PBS, diluted from 10x concentrate) containing approximately 20 Ci# of
3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, 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 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 the mice from each test group were pooled and 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), pooled according to groups, 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). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 C. After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
Determination of Incorporated 3HTdR
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4C and decanting the supernatants, than the pellets were re-suspended 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 loaded into the -scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample. The -counter expressed the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots of 5 % TCA. - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- The randomization was checked by computer software [SPSS/PC+ (4.0.1)] according to the actual body weights verifying the homogeneity and deviations between the groups.
DPM (disintegration per minute) was measured for each treatment group. 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 the background DPM value. The results were expressed as DPM/mouse. The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Dose-response relationship was evaluated by linear regression using SI values. All calculations were made by Microsoft Excel Software. Based on the results an EC3 value (dose calculated to induce a stimulation index of 3) was not calculated for the test item. - Positive control results:
- The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. A significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 8.7). The results of the positive control item demonstrated an appropriate performance of the test in accordance with the relevant guidelines and confirmed the validity of the assay.
- Key result
- Parameter:
- EC3
- Remarks on result:
- not determinable
- Remarks:
- SI values were below 3 at all test concentrations
- Parameter:
- SI
- Value:
- 0.4
- Variability:
- p = 0.03, r = 0.97
- Test group / Remarks:
- test item concentration 1%
- Parameter:
- SI
- Value:
- 0.8
- Variability:
- p = 0.03, r = 0.97
- Test group / Remarks:
- test item concentration 0.5%
- Parameter:
- SI
- Value:
- 0.8
- Variability:
- p = 0.03, r = 0.97
- Test group / Remarks:
- test item concentration 0.25%
- Parameter:
- SI
- Value:
- 1
- Variability:
- p = 0.03, r = 0.97
- Test group / Remarks:
- test item concentration 0.1%
- Cellular proliferation data / Observations:
- CELLULAR PROLIFERATION DATA
No significant lymphoproliferative response (SI ≥ 3) compared to the relevant control (DMSO) was noted forthe test item at the applied test concentrations.
DETAILS ON STIMULATION INDEX CALCULATION
The observed stimulation index values were 0.4, 0.8, 0.8 and 1.0 at test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values. Statistical significance was observed (p = 0.03, r = 0.97).
EC3 CALCULATION
not determined
CLINICAL OBSERVATIONS:
No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group.
BODY WEIGHTS
A body weight decrease by > 5 % was observed in the positive control group (2 of the 4 animals, 6 % decrease both), in the DMSO group (2 of the 4 animals, 8 % decrease both), in the 1 % (w/v) dose group (1 of the 4 animals, 6 % decrease) and in the 0.1 % (w/v) dose group (1 of the 4 animals, 6 % decrease). No body weight decrease by > 5 % was observed in the other treatment groups (AOO group, 0.5 % or 0.25 % (w/v) dose groups). The mean body weights did not decrease significantly in any dose group. The observed effect was not dose-related and was considered not significant or toxicological relevant. - Interpretation of results:
- GHS criteria not met
- Conclusions:
- In an in vivo skin sensitisation assay (LLNA) in mouse according to OECD 429, the test item did not show skin sensitising potential.
- Executive summary:
In an in vivo skin sensitising assay (LLNA) in mouse according to OECD 429, the skin sensitization potential of the test item following dermal exposure was determined.
Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration. Solubility of the test item in vehicles preferred in the LLNA was evaluated. Based on these results the test item was formulated in Dimethyl sulfoxide (DMSO) in the LLNA. The maximum achievable concentration (based on solubility) was 1 % (w/v) using ultrasonic dispersion and stirring. According to the results of the DRF (where no adverse effect was observed up to this maximum concentration) the test item was examined in the main test as 1 %, 0.5 %, 0.25 % or 0.1 % (w/v) formulations in DMSO. All formulations were adequately homogeneous during the application (apparently solutions, observed by the naked eye) although dark blue colour of the formulations rendered observation of any insoluble particles more difficult.
An appropriate positive control (α-Hexylcinnamaldehyde, HCA), and furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.
The positive control item (25 % (w/v) HCA in Acetone:Olive oil 4:1 (v/v) mixture, AOO) induced significant stimulation over the relevant control (SI = 8.7) thus confirming the validity of the assay.
No mortality or obvious signs of systemic toxicity were observed during the test. No significant effects on body weights were observed although body weights decreased by > 5 % (but < 10 %) in some dose groups (including some control groups) but without a dose-related effect. No signs of irritation or any other local effects were observed at the treatment site (ears) in any treatment group.
No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (DMSO) was noted for the test item at the applied test concentrations. The observed stimulation index values were 0.4, 0.8, 0.8 and 1.0 at the test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v), respectively. No significant, biologically relevant dose-response relationship was considered, although statistical significance was observed (p = 0.03, r = 0.97; evaluated by linear regression using SI values).
According to the evaluation criteria of the relevant guidelines the lack of a significantly increased lymphoproliferation (indicated by an SI ≥ 3) up to the maximum attainable concentration of 1 % (w/v, based on solubility) and also the lack of a significant, biologically relevant dose-response relationship are considered as evidence that the test item is not a skin sensitizer.
In conclusion, under the conditions of the present assay, the test item tested at the maximum feasible concentration of 1 % (w/v, based on solubility) and also at concentrations of 0.5 %, 0.25 % or 0.1 % (w/v) as formulations (apparently solutions) in a suitable vehicle (DMSO) was shown to have no skin sensitization potential in the Local Lymph Node Assay.
Reference
Table 1: Individual Body Weights of the Animals with Group Means, the Associated Error Termsand Body Weight Changes in the Main Test
Animal |
Dose Group |
Initial |
Terminal |
Body Weight |
Number |
Body Weight |
Body Weight |
Change |
|
(g) |
(g) |
(%) |
||
1 |
Vehicle control for the positive control: |
21.4 |
21.7 |
1 |
2 |
AOO |
19.9 |
19.8 |
-1 |
3 |
|
23.2 |
22.9 |
-1 |
48 |
|
20.8 |
21.0 |
1 |
|
Mean |
21.3 |
21.4 |
0 |
|
SD |
1.4 |
1.3 |
|
4 |
Positive control: |
19.3 |
19.3 |
0 |
5 |
25 % HCA |
21.8 |
20.6 |
-6 |
49 |
in AOO |
22.0 |
20.7 |
-6 |
50 |
|
20.8 |
20.4 |
-2 |
|
Mean |
21.0 |
20.3 |
-3 |
|
SD |
1.2 |
0.6 |
|
6 |
Vehicle control for the test item: |
19.9 |
18.4 |
-8 |
7 |
DMSO |
22.9 |
22.4 |
-2 |
8 |
|
21.2 |
19.6 |
-8 |
51 |
|
20.9 |
20.6 |
-1 |
|
Mean |
21.2 |
20.3 |
-5 |
|
SD |
1.2 |
1.7 |
|
9 |
Test Item |
19.2 |
18.2 |
-5 |
10 |
1 % |
21.5 |
20.7 |
-4 |
11 |
in DMSO |
22.8 |
21.5 |
-6 |
52 |
|
20.7 |
20.5 |
-1 |
|
Mean |
21.1 |
20.2 |
-4 |
|
SD |
1.5 |
1.4 |
|
12 |
Test Item |
20.4 |
20.3 |
0 |
13 |
0.5 % |
21.4 |
21.3 |
0 |
14 |
in DMSO |
22.4 |
21.8 |
-3 |
53 |
|
19.9 |
19.3 |
-3 |
|
Mean |
21.0 |
20.7 |
-2 |
|
SD |
1.1 |
1.1 |
|
15 |
Test Item |
20.4 |
20.4 |
0 |
16 |
0.25 % |
22.7 |
21.9 |
-4 |
54 |
in DMSO |
21.2 |
22.2 |
5 |
55 |
|
19.8 |
19.6 |
-1 |
|
Mean |
21.0 |
21.0 |
0 |
|
SD |
1.3 |
1.2 |
|
17 |
Test Item |
20.0 |
19.8 |
-1 |
18 |
0.1 % |
22.4 |
21.1 |
-6 |
56 |
in DMSO |
19.8 |
19.6 |
-1 |
57 |
|
21.4 |
21.4 |
0 |
|
Mean |
20.9 |
20.5 |
-2 |
|
SD |
1.2 |
0.9 |
|
HCA = a-Hexylcinnamaldehyde AOO = Acetone: Olive oil 4:1 (v/v) mixture
DMSO = Dimethyl sulfoxide SD = Standard Deviation
Table 2: DPM and Stimulation Index Values for all Groups in the Main Test
Dose Group |
Measured |
Group* |
DPM/Mouse# |
Stimulation |
DPM/group |
DPM |
Index Values |
||
Vehicle control for the positive control: |
2998 |
2972.0 |
743.0 |
1.0 |
AOO |
|
|
|
|
Positive control: |
25961 |
25935.0 |
6483.8 |
8.7 |
25 % HCA in AOO |
|
|
|
|
Vehicle control for the test item: |
7728 |
7702.0 |
1925.5 |
1.0 |
DMSO |
|
|
|
|
Test Item |
3438 |
3412.0 |
853.0 |
0.4 |
1 % in DMSO |
|
|
|
|
Test Item |
6161 |
6135.0 |
1533.8 |
0.8 |
0.5 % in DMSO |
|
|
|
|
Test Item |
6187 |
6161.0 |
1540.3 |
0.8 |
0.25 % in DMSO |
|
|
|
|
Test Item |
7915 |
7889.0 |
1972.3 |
1.0 |
0.1 % in DMSO |
|
|
|
|
HCA = a-Hexylcinnamaldehyde
AOO = Acetone: Olive oil 4:1 (v/v) mixture
DMSO = Dimethyl sulfoxide
*Group DPM = measured DPMgroup- average DPMbackground
Average DPMbackground= 26
# Number of animals/group = 4
Table 3: Individual Ear Thickness Values and the Deviations from the Initial Values in the Dose Range Finding Test
Dose Group |
Animal |
Ears |
Day 1* |
Day 3$ |
Day 3 |
Day 6# |
Day 6 |
|
Number |
value (mm) |
value (mm) |
% deviation |
value (mm) |
% deviation |
|
|
978 |
L |
0.19 |
0.20 |
5.3 |
0.20 |
5.3 |
Test Item |
R |
0.19 |
0.20 |
5.3 |
0.20 |
5.3 |
|
1 % in DMSO |
31 |
L |
0.20 |
0.21 |
5.0 |
0.20 |
0.0 |
|
R |
0.20 |
0.21 |
5.0 |
0.20 |
0.0 |
|
|
979 |
L |
0.21 |
0.21 |
0.0 |
0.21 |
0.0 |
Test Item |
R |
0.21 |
0.21 |
0.0 |
0.21 |
0.0 |
|
0.5 % in DMSO |
32 |
L |
0.20 |
0.20 |
0.0 |
0.20 |
0.0 |
|
R |
0.20 |
0.20 |
0.0 |
0.20 |
0.0 |
|
|
980 |
L |
0.20 |
0.21 |
5.0 |
0.21 |
5.0 |
Test Item |
R |
0.20 |
0.21 |
5.0 |
0.21 |
5.0 |
|
0.25 % in DMSO |
33 |
L |
0.20 |
0.20 |
0.0 |
0.21 |
5.0 |
|
R |
0.20 |
0.20 |
0.0 |
0.21 |
5.0 |
L = Left
R = Right
DMSO = Dimethyl sulfoxide
* Ear thickness was measured prior to the first treatment.
$ Ear thickness was measured approximately 48 hours after the first treatment (prior to the third treatment).
# Ear thickness was measured at the end of the test.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
In an in vivo skin sensitising assay (LLNA) in mouse according to OECD 429, the skin sensitization potential of the test item following dermal exposure was determined.
Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration. Solubility of the test item in vehicles preferred in the LLNA was evaluated. Based on these results the test item was formulated in Dimethyl sulfoxide (DMSO) in the LLNA. The maximum achievable concentration (based on solubility) was 1 % (w/v) using ultrasonic dispersion and stirring. According to the results of the DRF (where no adverse effect was observed up to this maximum concentration) the test item was examined in the main test as 1 %, 0.5 %, 0.25 % or 0.1 % (w/v) formulations in DMSO. All formulations were adequately homogeneous during the application (apparently solutions, observed by the naked eye) although dark blue colour of the formulations rendered observation of any insoluble particles more difficult.
An appropriate positive control (α-Hexylcinnamaldehyde, HCA), and furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.
The positive control item (25 % (w/v) HCA in Acetone:Olive oil 4:1 (v/v) mixture, AOO) induced significant stimulation over the relevant control (SI = 8.7) thus confirming the validity of the assay.
No mortality or obvious signs of systemic toxicity were observed during the test. No significant effects on body weights were observed although body weights decreased by > 5 % (but < 10 %) in some dose groups (including some control groups) but without a dose-related effect. No signs of irritation or any other local effects were observed at the treatment site (ears) in any treatment group.
No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (DMSO) was noted for the test item at the applied test concentrations. The observed stimulation index values were 0.4, 0.8, 0.8 and 1.0 at the test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v), respectively. No significant, biologically relevant dose-response relationship was considered, although statistical significance was observed (p = 0.03, r = 0.97; evaluated by linear regression using SI values).
According to the evaluation criteria of the relevant guidelines the lack of a significantly increased lymphoproliferation (indicated by an SI ≥ 3) up to the maximum attainable concentration of 1 % (w/v, based on solubility) and also the lack of a significant, biologically relevant dose-response relationship are considered as evidence that the test item is not a skin sensitizer.
In conclusion, under the conditions of the present assay, the test item tested at the maximum feasible concentration of 1 % (w/v, based on solubility) and also at concentrations of 0.5 %, 0.25 % or 0.1 % (w/v) as formulations (apparently solutions) in a suitable vehicle (DMSO) was shown to have no skin sensitization potential in the Local Lymph Node Assay.
Justification for classification or non-classification
Classification, Labelling, and Packaging
Regulation (EC) No 1272/2008
The available experimental test
data are reliable and suitable for classification purposes under
Regulation (EC) No 1272/2008. Based on available in vivo data on skin
sensitisation, the test item is not classified according to Regulation
(EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation
(EU) No 2019/521.
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