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Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): Metallfreies Phthalocyanin, ZST Substance No. 92/346
- Analytical purity: no data given
Specific details on test material used for the study:
- Substance type: blue powder
- Analytical purity: no data given
- Lot/batch No.: SF20927
- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

other: CBA/CaBkl
Details on test animals and environmental conditions:
- Source: B & K Universal Ltd, Hull, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: individually in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: Certified Rat and Mouse Diet, Code 5LF2 (BCM IPS Ltd., London, UK), ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

- Temperature: 19 - 25 °C
- Humidity: 30 - 70 %
- Air changes: 15 changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
5 %, 10 % or 25 % (w/w)
No. of animals per dose:
four animals per dose
Details on study design:
Mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for 3 consecutive days. The test material was administered by using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of 4 animals received the vehicle anlone in the same manner.
Five days following the first topical application of the test material, all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR, 80 µCi/ml, specific activity 2.0 µCi/mmol; Amersham Biosciences UK Ltd.), giving a total of 20 µCi to each mouse.
Animals were observed post dose on day 1, twice daily on days 2 and 3 and on a daily basis on days 4, 5 and 6. Any signs of toxicity or ill health were recorded. The body weight of the animals was determined on day, 1 prior to dosing, and on day 6, prior to termination.
5 hours following the administration of 3HTdR all animals were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the mice were excised and pooled for each animal group. For each group, 1 ml of PBS was added to the pooled lymph nodes.
A single cell suspension of the pooled lymph node cells was prepared by gentle mechanic disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze and collected into a centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (ca. 190 g) for 10 min. The pellet was resuspended in PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspenden in 3 ml of 5 % Trichloroacetic acid (TCA).
After overnight incubation at 4 °C the precipitates were removed by centrifugation at 2100 rpm (ca. 450 g) for 10 min, resuspended in 1 ml TCA and transferred to 10 ml scintillation fluid (Optiphase, "Trisafe"). 3HTdR incorporation was measured by ß-scintillation counting. The number of radioactive disintegrations per minute was then measured using a Beckman LS6500 scintillation system.

The proliferation response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into the lymph node cells of test nodes relative to that recorded for the control nodes (stimulation index). The test material will be regarded as sensitizer, if at least one of the tested concentrations results in threefold greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as non-sensitizer.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
>= 1.32 - <= 1.65
Test group / Remarks:
5%, 10%, and 25% test substance
Key result
other: disintegrations per minute (DPM)
Test group / Remarks:
Key result
other: disintegrations per minute
>= 899.5 - <= 1 020.05
Test group / Remarks:
5%, 10%, and 25% test substance
Cellular proliferation data / Observations:
- Test group 1: vehicle (acetone/olive oil 4:1): not applicable Test group 2: 5 % test substance in vehicle: 1.32 Test group 3: 10 % test substance in vehicle: 1.65 Test group 4: 25 % test substance in vehicle: 1.49
- Test group 1: vehicle (acetone/olive oil 4:1): 683.38 dpm/node Test group 2: 5 % test substance in vehicle: 899.50 dpm/node Test group 3: 10 % test substance in vehicle: 1131.00 dpm/node Test group 4: 25 % test substance in vehicle: 1020.05 dpm/node

Any other information on results incl. tables

There were no deaths. No signs of systemic toxicity was noted in the test or in the control animals during the study. Blue-coloured staining on the ears, face, feet and fur was noted in all test animals during the study.

The body weight changes of the test animals during the study were comparable to those observed in the corresponding control animals over the same time period.

A stimulation index of less than 3 was recorded for the three concentrations of the test material.

The positive control alpha-hexylcinnamaldehyde provided positive results and was considered to be a sensitizer under the experimental conditions of the test.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Under the experimental conditions of the study, the test material was considered not to be a sentisizer and will therefore not be classified according to GHS or EU criteria.