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Diss Factsheets

Administrative data

Description of key information

The test item was assessed for its skin sensitising potential at concentrations of 5, 10 and 25% using the Local Lymph Node Assay (LLNA) in mice (OECD 429, GLP). The test material was found to be a skin sensitizer and an EC3 value of 13.7% was derived. There are hints that the skin irritation potential of the test item may have an influence on the outcome of the local lymph node assay. Therefore, a Bühler assay (OECD 406, GLP) in guinea pig was proposed. The Bühler assay was performed at concentrations of 100 % (induction and challenge) and 75% (re-challenge). Skin irritation was observed in control and test groups. The test item is not considered to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
Buehler test
Justification for non-LLNA method:
There are hints that the skin irritation potential of the test item may have an influence on the outcome of the local lymph node assay. Therefore, a Bühler assay (OECD 406, GLP) in guinea pig was performed.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HARLAN (Kreuzelweg 53, 5961 NM HORST The Netherlands)
- Age at study initiation: 5-6 weeks
- Housing: in groups of two
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
pre-test: 100% and diluted at 75%, 50% and 25% in liquid paraffin
induction phase: 100 %
challenge: 100 %
re-challenge: 75%
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
pre-test: 100% and diluted at 75%, 50% and 25% in liquid paraffin
induction phase: 100 %
challenge: 100 %
re-challenge: 75%
No. of animals per dose:
control: 10
test group: 20
Details on study design:
RANGE FINDING TESTS:

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6h
- Test groups: 20 female animals
- Control group: 10 female animals
- Site: inter-scapular zone
- Frequency of applications: D0, D7, D14
- Concentrations: 100 %

Rest phase
The animals of both groups were left untreated for 13 days.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 1
- Exposure period: 6h
- Test groups: 10 animals
- Control group: 20 animals
- Site: dorso-lumbar zone
- Concentrations: 100%, re-challenge: 75%
- Evaluation (hr after challenge): 24h
Challenge controls:
vehicle control
Positive control substance(s):
yes
Remarks:
a-Hexylcinnamaldehyde
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100%. No with. + reactions: 2.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
11
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 11.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 72.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
75%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 75%. No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
75%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 75%. No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
75%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 72.0. Group: negative control. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
rechallenge
Hours after challenge:
72
Group:
test chemical
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 72.0. Group: test group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
75%
No. with + reactions:
4
Total no. in group:
20
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 75%. No with. + reactions: 4.0. Total no. in groups: 20.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
75%
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 75%. No with. + reactions: 1.0. Total no. in groups: 20.0.
Reading:
rechallenge
Hours after challenge:
72
Group:
test chemical
Dose level:
75%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 72.0. Group: test group. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Skin irritation was observed in control and test animals. Effects recovered within 48h.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In this study the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solutionat different concentrations was prepared in the vehicle DMF. For this purpose a local lymph node assay was performed using test item concentrations of 5, 10 and 25% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by two pre-experiments). The animals did neither show any signs of systemic toxicity nor local skin effects during the course of the study and no cases of mortality were observed. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. In this study Stimulation Indices (S.I.) of 2.14, 2.71 and 3.89 were determined with the test item at concentrations of 5, 10 and 25% (w/w) in DMF, respectively. A clear dose response was observed. A statistically significant and biologically relevant increase in DPM value and also in lymph node cell count was observed in all test item treated groups and a statistically significant and biologically relevant increase in lymph node weight was observed in the highest dose group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was exceeded in all test item treated groups (indices of 1.6, 1.6 and 2.2, respectively). Thus, the test item was found to be a skin sensitizer and an EC3 value of 13.7% was derived.


After the second pre-test the animal treated with 10% test item concentration showed an erythema of the ear skin (score 1) between day 3 and 5. The animal treated with 25% test item concentration showed an erythema of the ear skin (score 1 or 2) between day 1 and 5. Furthermore, the increase in DMP (appr. 2-fold) does not correlate with the increase in lymph node cell count (appr. 4-fold). In an acute dermal irritation study the substance was found to be irritating to the skin. In conclusion, there are hints that the skin irritation potential of the test item may have an influence on the outcome of the local lymph node assay. Therefore, a Bühler assay in guinea pig was proposed.


The aim of the Buehler test was to evaluate the possible allergenic activity of the test item after topical administration in guinea pigs. The induction phase was conducted with three topical applications of the test item to 20 guinea-pigs with occlusive dressing and a 13-day rest phase. The challenge phase, conducted under an occlusive dressing for 6 hours, consisted of a single topical application of the test item at 100% or an application of a negative control (liquid paraffin).


In the treatment group (treatment dose of 100%), a discrete erythema was noted in eleven animals (11/20) at the reading time 24 hours. No macroscopic cutaneous reactions attributable to allergy were observed 48 hours after the removal of the occlusive dressing. In the control group (associated with the treatment dose of 100%), a discrete erythema was noted in two animals (2/10) at the reading time 24 hours. No cutaneous intolerance reaction was observed 48 hours after the removal of the occlusive dressing. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (vehicle). As irritation was observed in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization. Therefore, no sensitization reaction was noted in animals from the treated group 24 and 48 hours after the challenge phase, on the area challenged with the test item at 100%.


In view to confirm or infirm these results, a new challenge phase was performed with the test item diluted at 75% (w/w) in liquid paraffin and with liquid paraffin as control (vehicle), after a rest phase of 5 days. In the treatment group (treatment dose of 75%), a discrete to moderate erythema was noted in 20% (4/20), 5% (1/20) and 5% (1/20) at the reading time 24, 48, 72 hours, respectively. In the control group (associated with the treatment dose of 75%), a discrete erythema was noted in one animal (1/10) at the reading time 24 and 48 hours. No cutaneous intolerance reaction was observed 72 hours after the removal of the occlusive dressing. No cutaneous reaction was recorded in animals from the treated and control groups after the rechallenge phase, on the treated area with liquid paraffin (vehicle). As irritation was observed in the control group animals, only reactions in the test group animals that exceed the most severe reactions seen in the control group animals were attributed to skin sensitization. So, a sensitization reaction was noted in 10%, 5% and 5% of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, on the area challenged with the test item at 75%. In conclusion, in view of these results, under these experimental conditions, 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. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008.