Registration Dossier

Administrative data

Description of key information

The test substance was determined to be not 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:
2003-08-28 to 2003-09-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
yes
Remarks:
non-radioactive, measurement of proliferation, comparable to OECD 442A
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
yes
Remarks:
Please refer to Principles of method
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 442A (Skin Sensitization: Local Lymph Node Assay: DA)
Version / remarks:
2010
Principles of method if other than guideline:
The deviations refer to the measurement of cell proliferation by cell counting instead of radioactive labeling. In addition, the acute inflammatory skin reaction was determined to discriminate specific from non-specific activation of immune competent cells in the draining lymph nodes. The principle was comparable to OECD Guideline 442A (2010-07-22).
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 27 - 36 g
- Housing: Acclimation: MakrolonTM type III cages, up to 8 animals Study period: MakrolonTM type II cages, single animals
- Diet: ad libitum, PROVIMI KLIBA SA 3883 (Provimi Kliba SA, CH-4303 Kaiseraugst)
- Water: ad libitum, tap water
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
3, 10 and 30 %
No. of animals per dose:
6 per group
Details on study design:
- Compound solubility: was soluble in Aceton/Olive oil (4:1)

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: cell count index; ear swelling
- Criteria used to consider a positive response: cell count index: 1.35; ear swelling: 2x10-2 mm increase

TREATMENT PREPARATION AND ADMINISTRATION:
- formulation immediately before treatment in Aceton/Olive oil (4:1), visual: solution
- Application: epicutaneously onto the dorsal part of both ears, three consecutive days (d1, d2, d3)
- Volume: 25 µL/ear
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
when reasonable the Mann-Whitney or Wilcoxon significance test was performed
Positive control results:
Alpha hexyl cinnamic aldehyde was formulated in Aceton/Olive oil (4:1) at the concentrations of 3 %, 10 % and 30 %. The results showed that the positive control substance had a clear sensitizing potential.
Parameter:
other: Cell count index [%]
Value:
1
Variability:
+/- 40.39
Test group / Remarks:
Control group
Key result
Parameter:
other: Cell count index [%]
Value:
0.88
Variability:
+/- 29.27
Test group / Remarks:
3 % treatment group
Key result
Parameter:
other: Cell count index [%]
Value:
1.13
Variability:
+/- 11.94
Test group / Remarks:
10 % treatment group
Key result
Parameter:
other: Cell count index [%]
Value:
1.06
Variability:
+/- 15.83
Test group / Remarks:
30 % treatment group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
The mice did not show an increase in the stimulation indices for cell counts or for weights of the draining lymph nodes after application of the test item.

CLINICAL OBSERVATIONS:
Both in ear swelling and ear weight the values of the treatment group were in the same range as the values of the control group.

BODY WEIGHTS
The body weights of the animals were not affected by any treatment.
Direct LLNA
Dose [%] Weight index (mean ± SD in %) Cell count index (mean ± SD in %)
Vehicle control 1.00± 30.36 1.00 ± 40.39
3 0.87 ± 28.92 0.88 ± 29.27
10 1.04 ± 13.95 1.13 ± 11.94
30 0.97 ± 15.75 1.06 ± 15.83

The positive level was 1.35. No treatment group reached this level. All values were in the same range as the control group. Therefore the cell count index showed no increase in proliferating lymphocytes and the test substance is not sensitizing.

Ear swelling 
Dose [%] day 1 (mean ± SD in %) day 4 (mean ± SD in %) Index day 4
Vehicle control 18.25 ± 4.13 19.08 ± 7.23 1
3 18.00 ± 3.35 18.25 ± 6.66 0.96
10 18.83 ± 3.07 19.25 ± 7.39 1.01
30 17.83 ± 4.68 19.00 ± 5.50 1

Ear weight
Dose [%] day 4 (mean ± SD in %) Index day 4
Vehicle control 13.28 ± 5.78 1
3 12.77 ± 6.71 0.96
10 13.35 ± 8.06 1.01
30 12.39 ± 8.99 0.93

Both in ear swelling and ear weight the values of the treatment group were in the same range as the values of the control group. The index was never raised significantly. Therefore the ear swelling and ear weight showed no increase in proliferating lymphocytes and the test substance is not sensitizing.

Interpretation of results:
GHS criteria not met
Conclusions:
A modified Local Lymph Node Assay in mice was performed. Analysis of LLN weight and cell count index, ear swelling and weight showed no results that reached or exceeded the positive levels. Therefore the test substance was not sensitising.
Executive summary:

The modified Local Lymph Node Assay (IMDS) was performed on 24 female NMRI mice of the strain Hsd Win:NMRI (6 animals/test item group and 6 control animals) to determine if there was any specific (sensitizing) or non-specific (irritating) stimulating potential of the test substance. The study was conducted according to OECD Guideline No. 429, EC Guideline 96/54/EC (22th Adaptation of Guideline 67/548/EEC)/Health Effects Test Guideline, OPPTS 870.2600 (EPA) with the concentrations of 0 %; 3 %; 10 % and 30 %. The test substance was formulated in Aceton/Olive Oil (A/OO, 4:1) to yield a solution. No increase in the stimulation indices for cell counts or for weights of the draining lymph nodes after application of the test substance was observed. The "positive level" which was 1.35 for the cell count index was never reached or exceeded in any dose group. The "positive level" of ear swelling which was 2x10^-2 mm increase, i.e. about 10% of the control values, had also not been exceeded in any dose group and no increases of the ear weights could be determined compared to control animals. The results showed that the test substance had neither an irritating nor a sensitizing potential in mice after dermal application.

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

Skin sensitisation

A modified Local Lymph Node Assay in mice was performed comparable to OECD Guideline 442A. The measurement of cell proliferation was done by cell counting instead of radioactive labeling. In addition, the acute inflammatory skin reaction was determined to discriminate specific from non-specific activation of immune competent cells in the draining lymph nodes. The test substance was applied in concentrations of 3, 10 and 30 % to ears of 6 mice each. The LLN weight and cell count index, ear swelling and weight were analyzed. No increase in the stimulation indices for weights of the draining lymph nodes after application of the test substance was observed compared to the vehicle control. The maximal weight index was found with a 10 % test substance at 1.04 (± 13.95 %) (control: 1.00 (± 30.36 %)). The "positive level" which was 1.35 for the cell count index was never reached or exceeded in any dose group. The maximal index was found for cell count index with a concentration of 10 % at 1.13 (± 11.94 %) (control: 1.00 (± 40.39%)). The "positive level" of ear swelling which was 2x10^-2 mm increase, i.e. about 10% of the control values, had also not been exceeded in any dose group and no increases of the ear weights could be determined compared to control animals. The maximal values were found with a 10 % concentration of the test substance at an Index of 1.01 while the control index was 1.00. In conclusion in can be said that the results of the different analysis did not reach or exceed the positive levels. Therefore the test substance was concluded to be not skin sensitising.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

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

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the nineth time in Regulation (EU) No 2016/1179.