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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:
2008-10-21 to 2009-01-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Acetylacetone
- Substance type: industrial chemical
- Physical state: liquid
- Stability under test conditions: stable
- Storage condition of test material: at RT in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Healthy CBA/J Rj mice, female, age 8 – 12 weeks, 5 mice per test group. The animals will be derived from a controlled full barrier maintained breeding system (SPF).

The animals will be barrier maintained (semi-barrier) in an air conditioned room
- Temperature: 22+/-3 °C
- Rel. humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Feeding ad libitum, Altromin 1324 maintenance diet for rats and mice - maintenance
- Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiol. controlled at regular intervals)
- The animals were kept in IVC cages, type II L, Polysulphone cages on Altromin saw fiber bedding
- Adequate acclimatisation period (at least 5 days)
The animals were randomly selected.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
12.5%, 25% and 50% (v/v)
No. of animals per dose:
5
Details on study design:
Topical Application
Each mouse was treated by topical application of 25 μL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days. Administration of 3H-methyl thymidine Five days after the first topical application treatment all mice were dosed with 20 μCi 3H-methyl thymidine by intravenous injection (tail vein) of 250μL of 3H-methyl thymidine, diluted to a working concentration of 80μCi/mL.
Preparation of cell suspension
Approximately 5 hours after 3H-methyl thymidine-injection all mice were sacrificed. The draining “auricular lymph nodes” were excised and individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated. After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4 °C overnight for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 5 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight. Determination of incorporated 3H-methyl thymidine The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
Positive control substance(s):
other: P-Phenylenediamine
Statistics:
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation {EC3=c+[(3-d)/(b-d)]x(a-c)}, between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated. A substance is regarded as a 'sensitizer' in the LLNA if at least one concentration of the test item results in a 3 fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the lymph nodes of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

Results and discussion

Positive control results:
The recent reliability check was performed in October 2008. It worked as expected.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.8
Test group / Remarks:
50%
Parameter:
SI
Value:
0.6
Test group / Remarks:
25%
Parameter:
SI
Value:
0.9
Test group / Remarks:
12,5%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The DPM at a concentration of 12.5% was 1042. The DPM at a concentration of 25% was 684.0. The DPM at a concentration of 50% was 895.4. The DPM for the control was 1135.6.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Considering the reported data of this sensitisation test it can be stated that the test item Acetylacetone causes no reactions identified as sensitisation, as the stimulation index was below 3.0 for each concentration tested.
Executive summary:

2,4-Pentanedione was tested for its ability to induce skin sensitization in an OECD 429 Local Lymph Node Assay (LLNA). Based on the results of the preliminary test the test item Acetylacetone was assayed for sensitising properties at concentrations of 12.5%, 25% and 50% (v/v). The vehicle used was Acetone/Olive Oil (AOO). Each mouse was treated by topical application of the prepared test item to the entire dorsal surface of each ear once daily over three consecutive days. Five days after the first topical application all mice were injected intravenously with 3H-methyl thymidine.

Approximately 5 hours after the 3H-methyl thymidine injection all mice were sacrificed. The draining “auricular lymph nodes” were excised. A single cell suspension of the lymph node cells for each animal was prepared. The 3H-methyl thymidine incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Determination of radioactivity was performed individually for each animal. The proliferative response of lymph node cells was calculated as the ratio of 3H-methyl thymidine incorporation into lymph node cells of test group animals relative to that recorded for control group animals. A stimulation index, ratio of test item / negative control, was calculated for each concentration.

Approximately 5 hours after 3H-methyl thymidine-injection all mice were sacrificed. The draining “auricular lymph nodes” were excised and weighed individually.

The mean weights of the lymph nodes

for the 25% reference group was 2.1 mg

for the 50% test group was 2.1 mg

for the 100% test group was 2.3 mg

for the negative control group was 3.3 mg

None of the three tested concentrations of the test item reached the stimulation index of 3.

The stimulation index at a concentration of 12.5% was 0.9

The stimulation index at a concentration of 25% was 0.6

The stimulation index at a concentration of 50% was 0.8.

 All animals survived throughout the test period without showing any clinical signs.Weight development of all animals was within the expected range (with the exception of two animals which lost 3 g), which includes a weight loss of up to 2 g throughout the study. At the daily clinical observation the animals did not show any visible clinical symptoms.

 

The EC3 value (derived by linear interpolation) could not be stated, as all measure points were below the stimulation index of three. Results of radioactivity determination are supported by the second endpoint, means of the lymph node weights per group, which did not show dose-related increased values compared to negative control values as well. Considering the reported data of this sensitisation test it can be stated that the test item Acetylacetone causes no reactions identified as sensitisation, as the stimulation index was below 3.0 for each concentration tested.