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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15-MAR-2006 TO 28-MAR-2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
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)

Test material

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: Black powder
Stability of test item: Stable under storage conditions
Storage conditions: At room temperature (range of 20 ±5 °C), light and moisture protected. The test item is very hygroscopic! Stored in a desiccator.

In vivo test system

Test animals

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.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
N,N-dimethylformamide
Concentration:
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.

Results and discussion

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.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 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.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM of 18568, 30345 and 24532 were determined with the test item at concentrations of 5 %, 10 % and 25 %, respectively, in DMF.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
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 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.

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)