Registration Dossier
Registration Dossier
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EC number: 310-132-3 | CAS number: 61902-31-6 This substance is identified in the Colour Index by Colour Index Constitution Number, C.I. 53235
Endpoint summary
Administrative data
Description of key information
In an in vivo local lymphnode assay (LLNA) 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:
- August 18 - October 10, 2016
- 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 JULY 22
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- 2008 May 31
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
- Specific details on test material used for the study:
- Date of production: 29.04.2015
Expiration date: 29.04.2020
Storage: Room temperature - Species:
- mouse
- Strain:
- CBA/Ca
- Remarks:
- Ola Hsd Mice (SPF)
- 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: Hygienic level at arrival was SPF; Hygienic level during the study was good conventional.
- Age at study initiation: Young adult mice; 9-11 weeks old (at start of the main test)
- Weight at study initiation: 17.8 – 23.0 g (the weight variation in animals involved in the study did not exceed ± 20 % of the mean weight)
- Housing during the test: Grouped caging (4 animals/cage) in Type II cages
- Diet: ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum
- Water: tap water from watering bottles ad libitum
- Acclimation period: 7 days
- Indication of any skin lesions: None
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:
- dimethylformamide
- Concentration:
- 0.25 %, 0.1 %, 0.05 %, and 0.025 % (w/v)
- No. of animals per dose:
- 4 animals / treatment groups
- Details on study design:
- MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
The test item is considered as a skin sensitizer, if:
Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.
TREATMENT PREPARATION AND ADMINISTRATION:
Each mouse was topically treated with 25 µL 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. Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6. During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test. Individual records were maintained.
Injection of 3HTdR
On Day 6 each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (1 x PBS, diluted from 10x concentrate) containing approximately 20 µCi 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 2-8 °C overnight (ca. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4 °C 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 beta-scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample. The beta-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. Instrument used for the measurement: Tri-Carb 3100TR Liquid Scintillation Analyzer, Serial Number: 072971
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 no EC3 value (dose calculated to induce a stimulation index of 3) was calculated for the test item. - 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)]
- Positive control results:
- 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 = 13.8) thus confirming the validity of the assay.
- Key result
- Parameter:
- EC3
- Remarks on result:
- not determinable
- Remarks:
- SI values were below 3 for all tested concentrations.
- Parameter:
- SI
- Value:
- 1
- Test group / Remarks:
- 0.25%
- Parameter:
- SI
- Value:
- 2
- Test group / Remarks:
- 0.1%
- Parameter:
- SI
- Value:
- 0.7
- Test group / Remarks:
- 0.05%
- Parameter:
- SI
- Value:
- 1
- Test group / Remarks:
- 0.025%
- Cellular proliferation data / Observations:
- CELLULAR PROLIFERATION DATA
No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (DMF) was noted for the test item at the applied test concentrations.
DETAILS ON STIMULATION INDEX CALCULATION
The observed stimulation index values were 1.0, 2.0, 0.7 and 1.0 at test item concentrations of 0.25 %, 0.1 %, 0.05 % and 0.025 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.94, r = 0.06; evaluated by linear regression using SI values).
EC3 CALCULATION
not determinable
CLINICAL OBSERVATIONS AND BODY WEIGHTS
No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group. - Interpretation of results:
- GHS criteria not met
- Conclusions:
- In an in vivo local lymphnode assay (LLNA) according to OECD 429, the test item did not show skin sensitising properties.
- Executive summary:
The aim of this study was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay according to OECD guideline 429. Preliminary tests were performed 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 the results the test item was formulated in N,N-Dimethylformamide (DMF) in the main test. The maximum achievable concentration (based on solubility) was 0.25 % (w/v) using ultrasonic dispersion and stirring. According to the results of the DRF (no adverse effects were observed up to this maximum concentration) the test item was examined in the main test in concentrations of 0.25 %, 0.1 %, 0.05 % or 0.025 % (w/v) in DMF. All formulations were adequately homogeneous during the application. Appropriate positive control (a-Hexylcinnamaldehyde, HCA), furthermore two negative control groups (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 = 13.8) thus confirming the validity of the assay.
No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI of above 3) compared to the relevant control (DMF) was noted for the test item at the applied test concentrations. The observed stimulation index values were 1.0, 2.0, 0.7 and 1.0 at test item concentrations of 0.25 %, 0.1 %, 0.05 % and 0.025 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.94, r = 0.06; evaluated by linear regression using SI values). According to the evaluation, the lack of a significantly increased lymphoproliferation (indicated by an SI of above 3) up to the maximum attainable concentration of 0.25 % (w/v, based on solubility) and also the lack of a significant dose-response relationship are considered evidence that the test item is not a skin-sensitizer.
Reference
Table 2: Clinical Observations in the Main Test
Dose Group |
Animal |
Days |
||||||||
1 |
2 |
3 |
4 |
5 |
6 |
|||||
PT |
AT |
PT |
AT |
PT |
AT |
|||||
Vehicle control for the positive control: |
461 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
473 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
474 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
522 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Positive control: 25 % HCA in AOO |
462 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
475 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
476 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
523 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Vehicle control for the test item: DMF |
463 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
477 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
524 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
525 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Leuco Sulfur Blue 11 |
478 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
479 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
526 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
527 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Leuco Sulfur Blue 11 |
480 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
481 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
528 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
529 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Leuco Sulfur Blue 11 |
482 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
483 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
530 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
531 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Leuco Sulfur Blue 11 |
484 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
485 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
486 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
532 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
PT = Prior to the treatment
AT = After the treatment
HCA =a-Hexylcinnamaldehyde
AOO = Acetone: Olive oil 4:1 mixture (v/v)
DMF =N,N-Dimethylformamide
N = Normal (no symptoms observed)
Table: 3 Erythema Scores in the Main Test
Dose Group |
Animal Number |
Ears |
Days |
||||||||
1 |
2 |
3 |
4 |
5 |
6 |
||||||
PT |
AT |
PT |
AT |
PT |
AT |
||||||
Vehicle control for the positive control: |
461 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
473 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
474 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
522 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Positive control: |
462 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
475 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
476 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
523 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Vehicle control for the test item: |
463 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
477 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
524 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
525 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Dose Group |
Animal Number |
Ears |
Days |
||||||||
1 |
2 |
3 |
4 |
5 |
6 |
||||||
PT |
AT |
PT |
AT |
PT |
AT |
||||||
Leuco Sulfur Blue 11 |
478 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
479 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
526 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
527 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Leuco Sulfur Blue 11 |
480 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
481 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
528 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
529 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Leuco Sulfur Blue 11 |
482 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
483 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
530 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
531 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Leuco Sulfur Blue 11 |
484 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
485 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
486 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
532 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
L = Left R = Right
PT = Prior to treatment AT = After the treatment
AOO = Acetone: Olive oil 4:1 (v/v) mixture
DMF =N,N-Dimethylformamide
HCA =a-Hexylcinnamaldehyde
Table 3: Visual Observations of the Lymph Nodes in the Main Test
Dose Group |
Animal |
Appearance of Lymph Nodes |
Vehicle control for the positive control: |
461 |
N |
473 |
N |
|
474 |
N |
|
522 |
N |
|
Positive control: |
462 |
Larger than the relevant control (AOO) |
475 |
Larger than the relevant control (AOO) |
|
476 |
Larger than the relevant control (AOO) |
|
523 |
Larger than the relevant control (AOO) |
|
Vehicle control for the test item: |
463 |
N |
477 |
N |
|
524 |
N |
|
525 |
N |
|
Leuco Sulfur Blue 11 |
478 |
N |
479 |
N |
|
526 |
N |
|
527 |
N |
|
Leuco Sulfur Blue 11 |
480 |
N |
481 |
N |
|
528 |
N |
|
529 |
N |
|
Leuco Sulfur Blue 11 |
482 |
N |
483 |
N |
|
530 |
N |
|
531 |
N |
|
Leuco Sulfur Blue 11 |
484 |
N |
485 |
N |
|
486 |
N |
|
532 |
N |
AOO = Acetone: Olive oil 4:1 (v/v) mixture
HCA =a-Hexylcinnamaldehyde
DMF =N,N-Dimethylformamide N = Normal
Table 5: 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: |
1745 |
1703.5 |
425.9 |
1.0 |
AOO |
|
|
|
|
Positive control: |
23627 |
23585.5 |
5896.4 |
13.8 |
25 % HCA in AOO |
|
|
|
|
Vehicle control for the test item: |
1571 |
1529.5 |
382.4 |
1.0 |
DMF |
|
|
|
|
Leuco Sulfur Blue 11 |
1571 |
1529.5 |
382.4 |
1.0 |
0.25 % in DMF |
|
|
|
|
Leuco Sulfur Blue 11 |
3048 |
3006.5 |
751.6 |
2.0 |
0.1 % in DMF |
|
|
|
|
Leuco Sulfur Blue 11 |
1079 |
1037.5 |
259.4 |
0.7 |
0.05 % in DMF |
|
|
|
|
Leuco Sulfur Blue 11 |
1570 |
1528.5 |
382.1 |
1.0 |
0.025 % in DMF |
|
|
|
|
HCA =a-Hexylcinnamaldehyde
AOO = Acetone: Olive oil 4:1 (v/v) mixture
DMF =N,N-Dimethylformamide
*Group DPM = measured DPMgroup- average DPMbackground
Average DPMbackground= 41.5
# Number of animals/group = 4
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
The aim of this study was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay according to OECD guideline 429. Preliminary tests were performed 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 the results the test item was formulated in N,N-Dimethylformamide (DMF) in the main test. The maximum achievable concentration (based on solubility) was 0.25 % (w/v) using ultrasonic dispersion and stirring. According to the results of the DRF (no adverse effects were observed up to this maximum concentration) the test item was examined in the main test in concentrations of 0.25 %, 0.1 %, 0.05 % or 0.025 % (w/v) in DMF. All formulations were adequately homogeneous during the application.Appropriate positive control (a-Hexylcinnamaldehyde, HCA), furthermore two negative control groups (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 = 13.8) thus confirming the validity of the assay.
No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group.No significantly increased lymphoproliferation (indicated by an SI of above 3) compared to the relevant control (DMF) was noted for the test item at the applied test concentrations. The observed stimulation index values were 1.0, 2.0, 0.7 and 1.0 at test item concentrations of 0.25 %, 0.1 %, 0.05 % and 0.025 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.94, r = 0.06; evaluated by linear regression using SI values). According to the evaluation, the lack of a significantly increased lymphoproliferation (indicated by an SI of above 3) up to the maximum attainable concentration of 0.25 % (w/v, based on solubility) and also the lack of a significant dose-response relationship are considered evidence that the test item is not a skin-sensitizer.
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 1272/2008 (CLP). As a result
the test item is not considered to be classified for skin sensitisation
under Regulation (EC) No 1272/2008, as amended for the twelfth
time in Regulation (EU) No 2019/52.
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