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Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
according to
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Details on test material:
- Name of test material (as cited in study report): Zinkphthalocyaninsulfonat
- Physical state: Green powder
- Analytical purity: 89%
- Lot/batch No.: 176647
- Storage condition of test material: At room temperature, under light exclusion

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Charles River UK, Manston Road, Margate, Kent CT9 4LT, United Kingdom
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 19 g
- Housing: Animals were group housed in Makrolon Type III cages, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany) and granulated soft wood bedding
(Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany).
- Diet: Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst, Netherlands)
- Water: Tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: At least 5 days

- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

other: ethanol:sterile water (3+7 v/v)
2.5, 5 and 10 % (w/w)
No. of animals per dose:
Details on study design:
- Compound solubility: highest test item concentration, which can be technically used was a 10% (w/w) solution in ethanol:sterile water (3:7 v/v)
- Irritation: no signs of irritation up to 10% (w/w)

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% (w/w) in ethanol:sterile water (3+7 v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone.
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 20.4 µCi of 3HTdR (equivalent to approximately 81.5 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium. The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal), after which the lymph node cell count was determined.
A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test or the Student Newman Keuls test. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and Grubb’s test were used for identification of possible outliers (performed with Microsoft Excel 2003).
However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: see Remark
Concentration (% w/v) Stimulation index Result 0 1.0 - 2.5 1.14 negative 5 0.96 negative 10 1.97 negative
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Concentration (% w/v) Disintegrations per minute 0 527.4 2.5 599.6 5 505.2 10 1038.8

Any other information on results incl. tables

Viability / Mortality

No deaths occurred during the study period.


Clinical Signs

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the intensive colour of the test item, the presence of an erythema of the ear skin could not be evaluated. From day 4 to 6, the urine of all test item treated animals was blue.


Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with3HTdR, was within the range commonly recorded for animals of this strain and age.


Lymph Node Weights and Cell Counts

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weight and lymph node cell count was not observed in any of the test item treated groups in comparison to the vehicle control group.


Ear Weights

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
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