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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation, other
Remarks:
in vivo study already available
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant OECD guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical purity: 98%

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbB (Borchen, Germany)
- Age at study initiation: six to ten week old
- Housing: individually in Makrolon type I cages
- Diet : ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): fully airconditioned rooms
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
other: acetone
Concentration:
0.3, 1 and 3%
No. of animals per dose:
6
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: in addition to the endpoints described in OECD guideline 429, lymph node cell counts as well as ear weight and thickness were determined. These latter parameters served as indicators for acute ear reactions which could lead to non-specific activation of draining lymph node cells

TREATMENT PREPARATION AND ADMINISTRATION: For each study, groups of 6 female CBA/Ca mice were treated with different concentrations of the test substances in acetone or with acetone alone (vehicle control). Because single animal evaluation was performed, six animals per test group were used, in order to ensure at least 5 valid results per test group to achieve sufficient statistical power. The studies used 3 test groups and 1 control group, each. Twenty five microliter per ear of the respective test substance preparation was applied to the dorsum of both ears for three consecutive days. The control group was treated solely with 25 µL per ear of the vehicle. The correctness of the concentrations and the stability of the test substance preparations were routinely checked by GC analyses in acetonitrile/deionized water mixtures (1:1 v/v).
Three days after the last application, the mice were injected intravenously (i.v.) with 20 µCi of [3H]-thymidine in 250 µL of sterile saline into a tail vein. About 5 h after the [3H]-thymidine injection, the mice were sacrificed and the auricular lymph nodes on both sides were removed and the weight of each animal’s pooled lymph nodes was determined. Lymph node response was evaluated by measuring the cellular content and [3H]-thymidine incorporation into lymph node cells (indicators of cell proliferation). To this end, single cell suspensions were prepared from the pooled lymph nodes of each animal as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 µm) into 6 mL of phosphate-buffered physiological saline.
For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy®-Counter. The remaining cell suspensions were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of [3H]-thymidine into the cells was measured in a β-scintillation counter.
Moreover, the ear thickness was measured in each animal and thereafter a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear. For each animal the weight of the pooled punches was determined. These measurements were performed to obtain an indication of possible acute skin reaction due to irritation.
Statistics:
Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of cell counts, [3H]-thymidine incorporation, lymph node weights, ear weights and ear thickness measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the concurrent vehicle control group. For cell counts, [3H]-thymidine incorporation, lymph node weights, ear weights and ear thickness, the WILCOXON test was applied.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
SI values based on incorporated [3H]Thymidine: 0.3%: 2.16** 1%: 3.17** 3%: 12.4** ** Wilcoxon test of the absolute values significant p < 0.01%. The EC 3 value was 0.9%. SI values based on lymph node cell count: 0.3%: 1.16* 1%: 1.50* 3%: 2.5** * Wilcoxon test of the absolute values significant p < 0.05%. ** Wilcoxon test of the absolute values significant p < 0.01%. The EC 1.5 value was 1.0%.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM values were not provided in the publication.

Any other information on results incl. tables

Ear thickness was statistically singnificantly increased at 1 % and 3 %.

Ear weight was statistically significantly increased at 3 %.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Based on the results of the LLNA the test substance was considered as skin sensitizing.
Executive summary:

In a GLP-compliant LLNA study conducted according to OECD guideline 429 dipropylenetriamine was applied to the ears of 6 CBA/Ca mice at concentrations up to 3%. Exposure to dipropylenetriamine resulted in EC3 value of 0.9%.