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Description of key information

The test substance was investigated for its skin sensitizing potential within an LLNA. The test item was not a sensitizer to skin.

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:
2007-01-07 to 2007-03-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2002)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan Netherlands B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 7 - 8 weeks
- Housing: Single caging. The animals were distributed into the test groups at random and identified by cage number. Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen), granulated soft wood bedding
- Diet: Pelleted standard diet, ad libitum, (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water: Tap water

ENVIRONMENTAL CONDITIONS
- Temperature: 22 + 3°C
- Humidity: 30- 70 %
- Photoperiod: Artificial light 6.00 a.m. - 6.00 p.m.
Vehicle:
dimethylformamide
Concentration:
Test substance concentration: 25, 50 and 100 %.
No. of animals per dose:
2 females for pre-testing
16 females for main trial (4/ females/group)
Details on study design:
RANGE FINDING TESTS:
To determine the highest non-irritant test concentration or the highest technically applicable concentration, a non-GLP pretest was performed in two mice (pretest excluded from Statement of Compliance). The data showed that the highest test item concentration, which can be used is 100 %. At this concentration the treated mouse did not show any signs of irritation. The test item in the main study was therefore assayed at 25, 50, and 100 %.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay
- Criteria used to consider a positive response: Disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that 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.
- Second, that 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.


TREATMENT PREPARATION AND ADMINISTRATION:
Topical Application: Each test group of mice was treated by topically to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 25, 50, and 100 % (w/v) in Dimethylformamide. The application volume of 25 µL was spread over the entire dorsal surface of 8 mm2 of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). Five days after the first topical application, all mice were administered with 250 µL of 79.6 µCi/mL 3HTdR (corresponds to 19.9 µCi 3HTdR per mouse) by intravenous injection via the tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanised. The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately + 4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of ‘Ultima Gold’ scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL- aliquots of 5 % trichloroacetic acid. The scintillation counter expresses 3HTdR.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated for the body weight.
Further calculations were made for DPM and SI values.
Positive control results:
Test item concentration % (w/v); Stimulation Index (S.I.)
5%; 2.04
10%; 6.31
25% ; 12.45
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
In this study Stimulation Indices of 1.6, 1.8, and 2.4 were determined with the test item at concentrations of 25, 50, and 100 % in Dimethylformamide.The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: For the control, DPM- value of 294 DPM/node was obtained. For the three test item concentrations of 25, 50 and 100 % DPM- values of 480, 516 and 693 DPM/node were obtained respectively.
Interpretation of results:
not sensitising
Remarks:
Migrated information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitization

In the study the test substance was dissolved in Dimethylformamide (DMF) and was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed according to OECD 429, adopted in 2002. The test substance was applied to the dorsum of both mouse ears in concentrations of 25, 50, and 100 %. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 1.6, 1.8, and 2.4 were determined with the test item at concentrations of 25, 50, and 100 % in DMF, respectively. The test item is considered to be "not sensitizing" to skin.


Migrated from Short description of key information:
The test substance was investigated for its skin sensitizing potential within an LLNA. The test item was not a sensitizer to skin.

Justification for selection of skin sensitisation endpoint:
GLP and guideline compliant study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, there is no sensitisation potential of the test substance. Therefore there is no need for classification and labeling acorrding to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).

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