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Toxicological information

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:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
RCC Cytotest Cell Research GmbH
Type of study:
mouse local lymph node assay (LLNA)

Test material

Details on test material:
Test materials used in this dossier are all considered to fall under the definition of nano-materials according to the European Commission Recommendation 2011/696/EU as the synthesis and manufacturing of this pigment always yields particulate material with a fine particle size distribution.
- Physical state: solid
- Storage condition of test material: at room temperature protected from moisture

In vivo test system

Test animals

other: CBA/CaOlaHsd
Details on test animals and environmental conditions:
- Source: Harlan Netherlands
- Age at study initiation: 7 weeks (beginning of acclimatization)
- Housing: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) with granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen), housed individually
- Diet (e.g. ad libitum): pelleted standard diet (Harlan Winkelmann GmbH, D-33178 Borchen), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness will be used for the study.

- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

dimethyl sulphoxide
0% (vehicle control), 3.5%, 7% and 14% (based a non-GLP local toxicity pre-test, 14% was the highest technically applicable concentration in the chosen vehicle)
No. of animals per dose:
4 females
Details on study design:
- Irritation: In a non-GLP conform pre-test in two mice, test item concentrations of 1.75, 3.5, 7 and 14 % (w/v) were tested on one ear each. No irritation effects were observed at these concentrations after a single application.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 3.5, 7 and 14% (w/v) in DMSO. The application volume, 25 μl, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 μl of 82 μCi/ml 3HTdR (corresponds to 20.5 μCi 3HTdR per mouse) by intravenous injection via a tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental (Trapanal, Altana, D-78467 Konstanz).
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle (DMSO) was quantitatively added. The test item concentrations were prepared individually. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer. The preparations were made freshly before each dosing occasion.
Positive control substance(s):
other: α-Hexylcinnamaldehyde in acetone:olive oil, 4:1 (v/v)
The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Test group / Remarks:
Test group / Remarks:
Test group / Remarks:

Any other information on results incl. tables

No deaths occurred 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. Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected. The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Calculation Result
Test item concentration Group Measurement DPM DPM-BG a) number of lymph nodes DPM/lymph node b) S.I.
-- BG I 23.6 -- -- -- --
-- BG II 34.2 -- -- -- --
-- CG 1 3467.18 3438.3 8 429.8
3.5 % (w/v) TG 2 4733.81 4704.9 8 588.1 1.4
7 % (w/v) TG 3 4615.95 4587.1 8 573.4 1.3
14 % (w/v) TG 4 6932.97 6904.1 8 863 2

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled,

DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The test item was found not to be a skin sensitiser.
Executive summary:

In GLP compliant study following OECD guideline 429 the test item dissolved in DMSO was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 3.5, 7 and 14%. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected. In this study Stimulation Indices (S.I.) of 1.4, 1.3 and 2.0 were determined with the test item at concentrations of 3.5, 7 and 14% (w/v) in DMSO, respectively. The test item was not a skin sensitiser in this assay.