Registration Dossier

Administrative data

Description of key information

The skin sensitizing potential of the test item was assessed by using the radioactive Murine Local Lymph Node Assay (OECD 429, GLP). The mean stimulation indices (expressed as multiples of the vehicle control) for3H thymidine incorporation, cell count, lymph node weight and ear weight are well below the threshold of regulation. No signs of systemic toxicity were noticed in all animals during general observation.

The test item does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay.

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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V.,lnc., Postbus 6174, 5960 AD Horst, The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-25g
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
propylene glycol
Concentration:
10, 25, 50%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: insoluble, max. 50% suspension in propylenglycol
- Irritation: no
- Systemic toxicity: no
- Ear weight and Lymphnode weight determined (S.I. well below 1)


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429, murine local lymph node assay
- Criteria used to consider a positive response: A test item is regarded as a sensitizer in the LLNA if exposure to at least one concentration of
the test item results in an incorporation of 3H thymidine at least 3-fold or greater than that recorded in control mice as indicated by the stimulation index. However, the biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed end points (i.e. cell counts, lymph node weights, ear weights).

TREATMENT PREPARATION AND ADMINISTRATION:
Randomization: Prior to first application, the animals will be distributed to the individual groups, will receive their animal numbers and will be allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 - 64".
Route of application: Epicutaneously to the dorsum of both ears
Application volume: 25 μL per ear
Site of application: Dorsal surface of both ears
Frequency of application: 3 consecutive applications (day O - day 2) on the same application site
Treatment of control group 1 analogously to the test groups, but only with the vehicle without test substance.

3H thymidine injection
On study day five (about 66 to 72 hours after the last application of test substance to the ears), 20 μCi 3H thymidine1 in 250 μL sterile saline will be injected intravenously (i.v.) into a tail vein of the mice.

Determination of ear weight: lmmediately after sacrifice, a circular piece of tissue (diameter 0.8 cm) will be punched out of the apical part of each ear of all animals. The weight of the pooled punches will be determined for each animal by using an analytical balance. These measurements serve for detecting a potential inflammatory ear swelling.

Removal and weight determination of the lymph nodes: Subsequently, the auricular lymph nodes (right and left) will be dissected. lmmediately after the removal, the total weight of both lymph nodes of each animal will be determined by using an analytical balance.

Preparation of cell suspension and determination of cell counts: Both lymph nodes (per animal) will be carefully passed through an iron mesh (mesh size 200 μm) into phosphate-buffered physiological saline. lmmediately afterwards, the cell suspension will be further diluted with isotone and measured by a cell counter straight away

Measurement of 3H thymidine incorporation in lymph node cells: The cell suspensions will be washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate will be transferred to scintillation fluid and incorporation of 3H thymidine will be measured by a ß-scintillation counter
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Cell count, 3H thymidine incorporation, lymph node weight and ear weight: WILCOXON - Test
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.69
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
50%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: see table below

EC3 CALCULATION

CLINICAL OBSERVATIONS: neither systemic toxicity nor skin irritation was not observed at any concentration

BODY WEIGHTS: see table below

   ear weight [mg]  body weight day 5 [g]  lymph node weight [mg]  cell counts [count / LN pair]
 vehicle propylene glycol  30.2  19.4  5.1  9,009,360
 10% in propylene glycol  31.8  19.3  4.7  7,643,520
 25% in propylene glycol  33.3  20.4  6.0  10,070,400
 50% in propylene glycol  33.8  19.3  5.2  9,534,000
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this murine local lymph node assay the test item is not considered to be a skin sensitizer
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitizing potential of the test item was assessed by using the radioactive Murine Local Lymph Node Assay. Groups of 5 female CBA/CaOlaHsd mice each were treated with 10%, 25% and 50% (w/w) preparations of the test substance in propylene glycol or with the vehicle alone. The study used 3 test groups and 1 control group. Each test animal was treated with 25 µL per ear of the appropriate test substance preparation applied to the dorsal surfaces of both ears on three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone. Three days after the last application, 20 µCi3H thymidine in 250 µL sterile saline were injected into the tail vein of the mice. About 5 hours after the3H thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring3H thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8cm diameter sample was punched out of the apical part of each ear and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

The mean stimulation indices (expressed as multiples of the vehicle control) for3H thymidine incorporation, cell count, lymph node weight and ear weight are well below the threshold of regulation. No signs of systemic toxicity were noticed in all animals during general observation.  When applied as 10%, 25% and 50% preparation in propylene glycol, the test substance did not induce a biologically relevant (no increase above the cut off SI of 3)or statistically significant increase of3H thymidine incorporation into the cells from the auricular lymph nodes. Concomitantly, the test substance did not induce a biologically relevant (no increase to 1.5-fold or above of control value = SI≥1.5) or statistically significant response in the auricular lymph node cell counts. No relevant or statistically significant increase in lymph node weights was noted at any concentration. The test-substance preparations did not cause relevant increases (SI > 1.25) in ear weights demonstrating the absence of ear skin irritation. The increases in ear weights are statistically significant at all concentrations.

Slight or moderate yellow discoloration of the ear skin wasnoted in all animals treated with the test substance during the observation period. Slight compound residues were observed on the ear skin of the animals treated with the 50% test substance preparation on study day 2 and 5.

Thus, it is concluded that the test item does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance does not need to be classified and labelled for skin sensitization under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EC) No 2016/1179.