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Description of key information

In the study, S.I. of 6.2, 10.1 and 8.1 were determined with the test item at concentrations of 5 %, 10 % and 25 % in DMF, respectively. The test item was therefore found to be a potential skin sensitizer in the LLNA test.

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:
Experimental start date - 15 Mar 2006;
Experiment completion date - 29 Mar 2006
Study completion date - 19 May 2006.
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Identity: FAT 40825/A
Batch number: CHU 297 / BOP 04/05
Purity: Organic part (Na-salt): approx. 83.7 %; All coloured components: approx. 80.54 %; Main component: approx. 59.1 %.
Appearance: Solid, black powder
Storage conditions: At room temperature at about 20 °C
Expiration date: December 31, 2010
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Test system: Mice, CBA/CaHsdRcc(SPF)
Number of animals for the pre-test (non-GLP): 3 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of negative control group: 1
Age: 8 - 12 weeks (beginning of acclimatization)
Body weight: 16 g - 24 g (ordered)
Identification: Each cage by unique cage card.
Randomization: Randomly selected by computer algorithm at time of delivery.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRY
Conditions: Standard Laboratory Conditions. Air-conditioned with ranges for room temperature 22 ±3 °C, relative humidity 30 - 70 % and 10 - 15 air changes per hour. There was a 12 hour fluorescent light /12 hour dark cycle with at least 8 hours music during the light period.
Accommodation: Individual in Makrolon type-2 cages with standard softwood bedding.
Diet: Pelleted standard Kliba 3433, batch no. 76/05 mouse maintenance diet available ad libitum.
Water: Community tap water from Itingen, available ad libitum.
Vehicle:
dimethylformamide
Concentration:
0 (Control group), 5 %, 10 % and 25 %
No. of animals per dose:
4
Details on study design:
Three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 % in N,N-dimethylformamide (DMF) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % was the highest technically achievable concentration in the chosen vehicle. A control group of four mice was treated with the vehicle DMF only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine, ³HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³HTdR measured in a ß-scintillation counter.
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.
Positive control results:
In the study STIMULATION INDICES of 1.8, 2.9 and 6.2 were determined with the test item at concentrations of 5 %, 10 % and 25 %, respectively, in acetone:olive oil, 4:1 (v/v). And Alpha-hexylcinnamaldehyde was therefore found to be a skin sensitizer in the LLNA tests and an EC3 value of 10.5 % was derived.
Parameter:
EC3
Remarks on result:
other: No dose-response relationship was observed. An EC3 value could not be determined because this calculation requires a S.I. value of less than 3.
Parameter:
SI
Value:
6.2
Test group / Remarks:
Group 2 : 5 % test item concentration
Parameter:
SI
Value:
10.1
Test group / Remarks:
Group 3 : 10 % test item concentration
Parameter:
SI
Value:
8.1
Test group / Remarks:
Group 4 : 25 % test item concentration

VIABILITY / MORTALITY
No deaths occurred during the study period.



CLINICAL SIGNS
No clinical signs of local toxicity at the ears of the animals and no systemic findings were observed during the study period.


 


BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

SIZE OF THE DRAINING LYMPH NODES
The size of the draining lymph nodes of Group 2 was 2-fold large compared to that of the control group. The size of the draining lymph nodes of Groups 3-4 was 3-fold large compared to that of the control group.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
S.I. of 6.2, 10.1 and 8.1 were determined with the test item at concentrations of 5 %, 10 % and 25 %, respectively, in DMF and the test item was therefore found to be a potential skin sensitizer in the LLNA tests.
Executive summary:

In order to study a possible contact allergenic potential of the test item, a LLNA study was conducted according to OECD test guideline 429 in a GLP-certified laboratory. Three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 % in N,N-dimethylformamide (DMF) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % was the highest technically achievable concentration in the chosen vehicle. A control group of four mice was treated with the vehicle DMF only. All treated animals survived the scheduled study period. No clinical / local signs were observed. After each topical application, the residual test item was found at all the local dosing sites in all mice of Group 3 (10 %) and Group 4 (25 %). After the second and the third topical application, the residual test item was found at all the local dosing sites in all mice of Group 2 (5 %). In the study, S.I. of 6.2, 10.1 and 8.1 were determined with the test item at concentrations of 5%, 10% and 25%, respectively, in DMF. The test item was therefore found to be a potential skin sensitizer in the LLNA test and it shall be included in the Skin sensitisers Category 1 according to the CLP Regulation (Regulation EC No. 1272/2008)

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In order to study a possible contact allergenic potential of the test item, a LLNA study was conducted according to OECD test guideline 429 in a GLP-certified laboratory. Three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 % in N,N-dimethylformamide (DMF) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % was the highest technically achievable concentration in the chosen vehicle. A control group of four mice was treated with the vehicle DMF only. All treated animals survived the scheduled study period. No clinical / local signs were observed. After each topical application, the residual test item was found at all the local dosing sites in all mice of Group 3 (10 %) and Group 4 (25 %). After the second and the third topical application, the residual test item was found at all the local dosing sites in all mice of Group 2 (5 %). In the study, S.I. of 6.2, 10.1 and 8.1 were determined with the test item at concentrations of 5 %, 10 % and 25 % in DMF, respectively. The test item was therefore found to be a potential skin sensitizer in the LLNA test and it shall be included in the Skin sensitisers Category 1 according to the CLP regulation (Regulation EC No. 1272/2008)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test article shall be classified as Category 1 skin sensitiser according to the CLP Regulation (Regulation EC No. 1272/2008). Data on respiratory sensitisation are lacking.