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Description of key information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Vehicle:
other: Ethanol/water (3+7, v/v)
Concentration:
Low Dose: 5 %
Mid Dose: 10 %
High Dose: 25 %
No. of animals per dose:
5
Details on study design:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25 % in ethanol/water (3+7, v/v). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Parameter:
SI
Value:
1
Test group / Remarks:
Vehicle Control Group (ethanol/water (3+7, v/v))
Parameter:
SI
Value:
0.99
Test group / Remarks:
5 % Azo Initiator VA044
Parameter:
SI
Value:
0.96
Test group / Remarks:
10 % Azo Initiator VA044
Parameter:
SI
Value:
0.96
Test group / Remarks:
25 % Azo Initiator VA044
Interpretation of results:
GHS criteria not met
Conclusions:
The test item Azo Initiator VA044 was not a skin sensitiser under the test conditions of this study.
Executive summary:

In order to study a possible skin sensitising potential of Azo Initiator VA044, three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10, and 25 % in ethanol/water (3+7, v/v) by topical application to the dorsum of each ear for three consecutive days. The test item could be either suspended (25 %) or dissolved (10 + 5 %) in the vehicle. The appropriateness of the used concentrations was previously assessed by a pre-experiment.

A control group of five mice was treated with the vehicle ethanol/water (3+7, v/v) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised, pooled per animal and immediately weighed. Furthermore, after excision of the lymph nodes, both ears of the mice were punched at the apical area using a biopsy punch and were immediately weighed pooled per animal using an analytical balance. Afterwards single cell suspensions of lymph node cells were prepared from pooled lymph nodes per animal. An aliquot of each cell suspension was used for determination of lymph node cell count. Subsequently the suspensions were washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

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 increase in ear weights was observed in the mid and the high dose group in comparison to the vehicle control group (p < 0.05) 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 (see Ref. 9). None of the indices determined for the test item treated groups exceeded this threshold.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

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. None of the indices determined for the test item treated groups exceeded this threshold. A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 0.99, 0.96, and 0.96 were determined with the test item at concentrations of 5, 10, and 25% in ethanol/water (3+7, v/v), respectively. A dose response was not observed.

A statistically significant and biologically relevant increase in DPM values, lymph node weight or –cell count was not obtained in any group in comparison to the vehicle control group (p<0.05). Moreover, the cut-off value for a positive response regarding the lymph node cell count index reported for BALB/c mice was not exceeded in any of the test item groups. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

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

The test item Azo Initiator 2,2’-[azobis(1-methylethylidene)]bis[4,5-dihydro-1H-imidazole dihydrochloride was not a skin sensitiser under the test conditions of the available study.