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Description of key information

The aim of this study was to determine the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay.

Under the conditions of the present Local Lymph Node Assay, The test item tested at the maximum applicable (non-toxic, non-irritant) concentration of 10 % and at concentrations of 5 %, 2.5 % and 1 % (w/v) as formulations in an appropriate vehicle (DMF) was shown to have no skin sensitization potential.

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:
September - November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
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)
Specific details on test material used for the study:
TEST MATERIAL
- Substance identification/name in the report: LZ 596
- Molecular formula: C21H30N2
- Batch no.: 1229
- Analysis date: August 12, 2014
- Date of production: August 11, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25ºC, <70 RH%), protected from light and humidity.
- Stability under test conditions: stable for min. 3 years from manufacturing date
- Expiry date: August 10, 2017
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT., H-1103, Budapest, Cserkesz u. 90, Hungary
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 12 weeks old
- Weight at study initiation: 20.0 - 24.1 g
- Housing: grouped caging (5 animals/cage)
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water (e.g. ad libitum): tap water
- Acclimation period: 8 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Vehicle:
dimethylformamide
Concentration:
concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v)
No. of animals per dose:
5 animals/treatment group
Details on study design:
Selection of test item concentrations was based on the results of a formulation evaluation and also a preliminary irritation/toxicity test to find an appropriate solvent and the maximum applicable concentration according to the relevant guidelines. Based on the preliminary test results the test item was examined in the main test at 10 %, 5 %, 2.5 % and 1 % (w/v) concentrations as formulations in the selected vehicle of N,N-Dimethylformamide (DMF). Appropriate positive control furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis was performed by SPSS/PC+ (4.0.1) software package. The measured DPM values corrected with the mean background (TCA) value were used for statistical analysis of the proliferation data. The heterogeneity of variance between the groups treated with the test item and the relevant vehicle controls was checked by Bartlett's test. Since significant heterogeneity was detected, the normal distribution of data was examined by Kolmogorow-Smirnow test followed by the non-parametric method of Kruskal-Wallis One-Way analysis of variance. As a results of this analysis the inter-group comparisons was performed using Mann-Whitney U-test to assess the significance of inter-group differences.
Significance of the positive control response was evaluated by T-test versus the relevant vehicle control (AOO). All other calculations and evaluation of dose-response relationship by linear regression were made by Excel.
Positive control results:
The positive control item (25 % (w/v) α-Hexylcinnamaldehyde (HCA) in Acetone: Olive oil 4:1 (v/v) mixture (AOO)) induced the appropriate, statistically significant stimulation compared to the relevant control (SI = 16.4; p < 0.01; T-test versus the AOO control), thus confirming the validity of the assay.
No significant, treatment related effect on the body weights was observed in any treatment group. No significant irritation (indicated by erythema ≥ 3 on the ears) or any other local effect was observed in any treatment group. Ear thickness was also monitored in the test item treated groups: no significant ear thickness changes compared to the initial values were observed.
Key result
Parameter:
SI
Value:
2.5
Test group / Remarks:
Test item concentration 10%
Key result
Parameter:
SI
Value:
2.1
Test group / Remarks:
Test item concentration 5%
Key result
Parameter:
SI
Value:
3.4
Test group / Remarks:
Test item concentration 2.5%
Remarks on result:
other: SI value of 3.4 observed at concentration of 2.5 % (w/v) was borderline but considered not biologically relevant.
Key result
Parameter:
SI
Value:
2.1
Test group / Remarks:
Test item concentration 1%
Cellular proliferation data / Observations:
No significant, biologically relevant increased lymphoproliferation

The SI value of 3.4 observed at concentration of 2.5 % (w/v) was on the borderline but considered not biologically relevant. Although statistical significance (p < 0.05) was observed at all test concentrations it was considered also not biologically significant since no dose-related response was observed.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present Local Lymph Node Assay, The test item tested at the maximum applicable (non-toxic, non-irritant) concentration of 10 % and at concentrations of 5 %, 2.5 % and 1 % (w/v) as formulations in an appropriate vehicle (DMF) was shown to have no skin sensitization potential.
Executive summary:

The aim of this study was to determine the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. Selection of test item concentrations was based on the results of a formulation evaluation and also a preliminary irritation/toxicity test to find an appropriate solvent and the maximum applicable concentration according to the relevant guidelines. Based on the preliminary test results the test item was examined in the main test at 10 %, 5 %, 2.5 % and 1 % (w/v) concentrations as formulations in the selected vehicle of N,N-Dimethylformamide (DMF). Appropriate positive control 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) α-Hexylcinnamaldehyde (HCA) in Acetone: Olive oil 4:1 (v/v) mixture (AOO)) induced the appropriate, statistically significant stimulation compared to the relevant control, thus confirming the validity of the assay. No significant, treatment related effect on the body weights was observed in any treatment group. No significant irritation (indicated by erythema ≥ 3 on the ears) or any other local effect was observed in any treatment group. Ear thickness was also monitored in the test item treated groups: no significant ear thickness changes compared to the initial values were observed. Since the test was valid and no sign of systemic toxicity or irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. No Stimulation Index (SI) equal to or above 3 was observed for the test item at the maximum applicable concentration of 10 % and at concentrations of 5 % and 1 % (w/v). The observed stimulation index values were 2.5, 2.1, 3.4 and 2.1 at test item concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. The SI value of 3.4 observed at concentration of 2.5 % (w/v) was on the borderline but considered not biologically relevant.

Under the conditions of the present Local Lymph Node Assay, The test item tested at the maximum applicable (non-toxic, non-irritant) concentration of 10 % and at concentrations of 5 %, 2.5 % and 1 % (w/v) as formulations in an appropriate vehicle (DMF) was shown to have no skin sensitization potential.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of this study, the test item does not need to be classified as skin sensitizer according to the UN GHS system.