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Description of key information

A Local Lymph Node Assay (LLNA) is available, conducted according to OEC guideline 429 and GLP principles (Klimisch 1 study performed in 2015). The results do not indicate skin sensitising properties.

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:
2015
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
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst, The Netherlands
- Age at study initiation: pretest: 10-11 weeks, main study: 9-10 weeks
- Housing: group-housed
- Diet: 2018C Teklad Global 18 % protein rodent diet, ad libitum
- Water: tap water ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:
dimethyl sulphoxide
Concentration:
5, 10 and 20% in DMSO
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
The highest test item concentration, which could be technically used was a 20% solution in DMSO. At the tested concentrations the animals showed neither any signs of local skin irritation, such as swelling, nor systemic toxicity. Due to the inherent color of the test item an erythema of the ear skin could not be determined.

MAIN STUDY
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
- the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as a statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05).
Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice.
Parameter:
SI
Value:
1.45
Variability:
1.1 - 2.1
Test group / Remarks:
5%
Parameter:
SI
Value:
1.51
Variability:
1.2 - 2.0
Test group / Remarks:
10%
Parameter:
SI
Value:
1.7
Variability:
0.9 - 2.2
Test group / Remarks:
20%
Cellular proliferation data / Observations:
The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. However, a possible erythema of the ear skin could not be determined due to the inherent color of the test item. On day 6, all animals treated with 10% and two animals treated with 20% test item concentration showed scabby ear skin. A statistically significant increase in ear weights was observed in the mid and the high dose group in comparison to the vehicle control group (p<0.05).Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. The high dose group slightly exceeded this threshold (index of 1.16). However, this was considered to be not biologically relevant, as the observed increase did not exceed the threshold value of 25% for excessive local skin irritation mentioned in OECD guideline 429. Nevertheless, the increased ear weights in the high dose group indicated a slight irritant property of the test item.

DPM values per animal (2 lymph nodes): control group: 1510, 1996, 1904, 2422, 1935 (mean: 1934.9) test group 1 (5% test item concentration): 3109, 2375, 2173, 4149, 2331 (mean: 2808.9) test group 2 (10% test item concentration): 2306, 2600, 3212, 3900, 2731 (mean: 2931.3) test group 3 (20% test item concentration):1686, 4108, 2789, 4276, 3692 (mean: 3393.5).
Stimulation Indices relative to the mean of the control group: test group 1 (5% test item concentration): 1.6, 1.2, 1.1, 2.1, 1.2 (mean: 1.45) test group 2 (10% test item concentration): 1.2,1.3, 1.7, 2.0, 1.4 (mean: 1.51) test group 3 (20% test item concentration): 0.9, 2.1, 1.4, 2.2, 1.9 (mean: 1.70) .

A statistically significant or biologically relevant increase in DPM values, lymph node weights and lymph node cell counts was not observed in any treated group in comparison to the vehicle control group.

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance did not show a skin sensitising potential in the Local Lymph Node Assay (OECD 429, GLP).
Executive summary:

To assess the skin sensitizing potential of the test substance, a Local Lymph Node Assay (LLNA) was conducted in mice (CBA/CaOlaHsd) according to OEC guideline 429 and GLP principles. Test item suspensions at different concentrations (5, 10 and 20 % w/w) were prepared using DMSO as a vehicle.

In this study Stimulation Indices (S.I.) of 1.45, 1.51 and 1.70 were determined with the test item at concentrations of 5, 10, and 20% (w/w) in DMSO, respectively. A statistically significant or biologically relevant increase in DPM values, lymph node weights and lymph node cell counts was not observed in any treated 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 not exceeded in any dose group. Thus, the test item was not a skin sensitiser under the test conditions of this study.

 

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

To assess the skin sensitizing potential of the test substance, a Local Lymph Node Assay (LLNA) was conducted in mice (CBA/CaOlaHsd) according to OEC guideline 429 and GLP principles. Test item suspensions at different concentrations (5, 10 and 20 % w/w) were prepared using DMSO as a vehicle.

In this study Stimulation Indices (S.I.) of 1.45, 1.51 and 1.70 were determined with the test item at concentrations of 5, 10, and 20% (w/w) in DMSO, respectively. A statistically significant or biologically relevant increase in DPM values, lymph node weights and lymph node cell counts was not observed in any treated 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 not exceeded in any dose group. Thus, the test item was not a skin sensitiser under the test conditions of this study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available experimental test data, PR63:1 is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008.