Registration Dossier

Administrative data

Description of key information

Based on a local lymph node assay according to OECD guidline 429, the test item was detemined not to be skin sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 January 2017 - 20 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/Ca Ola Hsd mice
Sex:
female
Details on test animals and environmental conditions:
Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Justification of strain: On the basis of comparative investigations in other laboratories, mice of the CBA/Ca strain were found to exhibit a more marked response than other strains. Females are used because the existing database is predominantly based on females.
Number of animals: 28 animals/main test (4 animals/treatment group, 12 shared control animals)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice; 11-12 weeks old (at start of the main test)
Body weight range at starting: 19.0 – 23.2 g, The weight variation in animals involved in the study did not exceed 20 % of the mean weight.
Acclimatization time: 7 days

Husbandry
Animal health: Only healthy animals (and not showing any sign of skin lesion) were used
Housing duringacclimatization period: Grouped caging in small groups
Housing during the test: Grouped caging (4 animals/cage)
Cage type: Type II. Polypropylene / polycarbonate
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 – 70 %
The same conditions were used for the dose range finding and main test animals. There were no deviations from these specifications during the experimental phase. The temperature and relative humidity were recorded daily during the acclimatization and experimental phases. Before housing the animals, the microbiological status of the room was checked.

Food and Water Supply
Animals received ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
The food is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Animals received tap water from watering bottles ad libitum. The drinking water is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Vehicle:
dimethyl sulphoxide
Concentration:
The test item was examined in the main test at concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v).
No. of animals per dose:
4 animals / group
Details on study design:
Based on the preliminary test results the maximum attainable concentration (based on solubility) of 1 % (w/v) was used in the main test with the aim of testing the highest possible concentration. The test item was tested also at three additional, lower concentrations (0.5 %, 0.25 % and 0.1 %, w/v) to evaluate a dose-response relationship and ensure validity of the test in accordance with the relevant guidelines.
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.

In vivo Treatment
Each mouse was topically treated with 25 µL of the appropriate formulations of the test item, the positive control substance or the vehicles (see Table 3) 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 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 (approx. 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:
Name: Tri-Carb 3100TR Liquid Scintillation Analyzer
Serial Number: 072971
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Randomization based on body weigt was checked by SPSS PC+.
Calculation of stimulation indices were made by EXCEL software.
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 = 8.7) thus confirming the validity of the assay.
Key result
Parameter:
SI
Value:
0.5
Variability:
p=0.43; r= 0.57
Test group / Remarks:
test item concentrations of 1 % v/w
Parameter:
SI
Value:
1
Variability:
p = 0.43, r = 0.57
Test group / Remarks:
test item concentrations of 0.5 % v/w
Parameter:
SI
Value:
1.7
Variability:
p = 0.43, r = 0.57
Test group / Remarks:
the test item concentrations of, 0.25 %
Parameter:
SI
Value:
0.8
Variability:
p = 0.43, r = 0.57
Test group / Remarks:
test item concentrations of 0.1 % (w/v),
Key result
Parameter:
EC3
Remarks on result:
not determinable
Remarks:
the SI values were below 3 at all test concentrations
Cellular proliferation data / Observations:
No significantly increased lymphoproliferation (indicated by an SI >= 3) compared to the relevant control (DMSO) was noted for Blue Sema at the applied test concentrations. The observed stimulation index values were 0.5, 1.0, 1.7 and 0.8 at the test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.43, r = 0.57; evaluated by linear regression using SI values).

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 effect were observed at the treatment site (ears) in any treatment group.

According to the evaluation criteria of the relevant guidelinesthe 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 dose-related response are considered as evidence that Blue Semais not a skin sensitizer.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was determined not to have a skin sensitization potential.
Executive summary:

The aim of this study according to OECD guideline 429 was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. 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 colour of the formulations rendered observation of any insoluble particles more difficult. An appropriate positive control (a-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 effect 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 0.5, 1.0, 1.7 and 0.8 at the test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.43, r = 0.57; 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 dose-related response are considered as evidence that the test item is not a skin sensitizer. 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The aim of the study according to OECD guideline 429 was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. 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 colour of the formulations rendered observation of any insoluble particles more difficult. An appropriate positive control (a-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 effect 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 0.5, 1.0, 1.7 and 0.8 at the test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.43, r = 0.57; 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 dose-related response are considered as evidence that the test item is not a skin sensitiser. 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on this data, the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EC) No 2019/521.