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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

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

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
- Synonyms: Copper tri/tetrachlorophthalocyanine pigment
- Substance type: blue powder
- Stability under test conditions: guaranteed for 4 hours
- Storage condition of test material: darkness at approx. 20 °C in a fume cupboard
- Other: different CAS No. are existing: 29719-96-8, 68987-63-3, 16040-69-0, 27614-71-7
Specific details on test material used for the study:
- Substance type: blue solid
- Analytical purity: no data given
- Lot/batch No.: KRON 200106
- Purity> 85%
- Expiration date of the lot/batch: 31-Mar-2006
- Stability under test conditions: stable under storage conditions
- Storage condition of test material: in the original container at room temperature (20 °C +- 3 °C), away from direct sunlight

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16 g - 24 g (ordered)
- Housing: individually in Makrolon type-2 cages with standard softwood bedding
- Diet: Pelleted Standard Kliba 3433, batch no. 78/03 mouse maintainance diet (Provimi Kliba AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: under test conditions after haelth examination; only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +- 3 °C
- Humidity: 30 - 70 %
- Air changes per hr: 10 - 15
- Photoperiod: 12 hrs dark / 12 hrs light

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
2.5, 5 and 10 %
No. of animals per dose:
4 animals per dose
Details on study design:
RANGE FINDING TESTS:
To determine the highest non-irritant and technically applicable test item concentration, a non-GLP test was conducted in 2 mice with concentrations of 1, 2.5, 5 and 10 %. In the main study 3 consecutive concentrations were assayed. The top dose was the highest achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY:
Each test group was treated by topical application to the dorsal surface of each ear lobe (left and right) with different test item concentrations. The application volume, 25 µl, was spread over the entire dorsal surface (ca. 8 mm diameter) of each ear lobe once daily for 3 consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was passed briefly over the ear´s surface to prevent the loss of any of the test item applied.
Five days after the first topical application, all mice were administered with 250 µl of 79.6 µCi/ml 3HTdR (equal to 19.9 µCi 3HTdR) by intravenous injection via a tailvein.
Approx. 5 h after treatment with 3HTdR all mice were euthanized with dry ice (CO2). The draining lymph nodes were rapidly excised and pooled for each experimental group (8nodes/group). Single cell suspensions of pooled lymph node cells were prepared. Cells were resuspended in 5 % trichloroacetic acid ans incubated at 4 °C for at least 18 h to precipitate the macromolecules. The precipitates were resuspended in 5 % trichloroacetic acid and transferred to glass scintillation vialscontaining 10 ml scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was thenmeasured on a ß-scintillation counter. Similarly, background 3HTdR levels were also measured. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test group relative to the recorded control group (Stimulation Index S.I.). Before values were determined, mean scintillation background DPM was subtracted from data obtained.
In addition, mortality/viability (twice daily), body weights (priot to first application and prior to necropsy) and clinical signs (local/systemic, daily with particular attention to the treatment sites) were determined.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle was quantitatively added. The w/v dilutionswere prepared individually using a magnetic stirrer as homogenizer.
Test item solutions were made freshly before each dosing occasion and no more than 4 h prior to application.
Homogeneity was maintainedduring treatment with the magnetic stirrer.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A test item was considered as sensitizer if the following criteria were fulfilled:
- Exposure to at least one concentration resulted in an incorporation of 3HTdR of at least 3-fold or greater than in controls as indicated by S.I.
- Data were compatible with a conventional dose-response, although allowance must be for either local toxicity or immunological suppression.

For statistical analysis, the mean values and standard deviations were calculated in the body weight tables.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.8
Test group / Remarks:
2.5%
Parameter:
SI
Value:
1.1
Test group / Remarks:
5%
Parameter:
SI
Value:
1
Test group / Remarks:
10%
Cellular proliferation data / Observations:
The background DPM was measured twice and was 3 or 10, respectively. The dpm, determined for the control group, was 4593, for the 2.5 % group it was 3672, for the 5.0 % group it was 5122 and for the 10.0 % group it was 4488.

Any other information on results incl. tables

No deaths occured during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

The body weight of the aninals was within the normal range recorded for animals of this strain and age. One animal of the 10 % test item concentration group lost weight during the study, but this was considered to be incidental.

Test item concentration % (w/v)

 

dpm measurement

dpm - background

number of lymph nodes

dpm per lymph node

S.I.

 

background

10

 

 

 

 

 

background

3

 

 

 

 

 

control group

4593

4586

8

573

 

2.5

test group

3679

3672

8

459

0.8

5.0

test group

5129

5122

8

640

1.1

10.0

test group

4495

4488

8

561

1.0

The positive control, alpha-hexylcinnamaldehyde, was tested in concentrations of 5.0, 10.0 and 25.0 % and produced S.I.s of 1.5, 2.3 and 8.4, respectively.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study stimulation indices of 0.8, 1.1 and 1.0 were determined with the test material at concentrations of 2.5, 5 and 10 % (w/v), respectively in DMSO.
Under the experimental conditions chosen, the test material was not found to be a sensitizer when tested at up to the highest achievable concentration of 10 % (w/v) in DMSO.