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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:
february 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD 429)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (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
Chemical structure
Reference substance name:
Tangled Multi-Walled Carbon Nanotubes
EC Number:
701-160-0
Cas Number:
7782-42-5
Molecular formula:
Hollow tubular carbon, 1-dimensional nano structures with hexagonal arrangement of carbon atoms
IUPAC Name:
Tangled Multi-Walled Carbon Nanotubes
Constituent 2
Reference substance name:
GRAPHISTRENGTH C100
IUPAC Name:
GRAPHISTRENGTH C100
Test material form:
solid: nanoform

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Janvier, Le Genest-Saint-Isle, France
- Age at study initiation:the animals of the preliminary test were approximately 10 weeks old and the animals of the main test were approximately 9 weeks old
- Weight at study initiation: 21.2 ± 1.1 g.
- Housing:The animals were housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm)
- Diet: free access to SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: free access to tap water (filtered using a 0.22 micron filter) contained in bottles
- Acclimation period:at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-2
- Humidity (%):30 to 70
- Air changes (per hr):approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light):12h/12h

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Remarks:
batch No. S35142-236
Concentration:
0.1, 0.25, 0.5, 1 or 2.5%.
No. of animals per dose:
4 females/dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility:insoluble
- Concentrations: the concentrations selected for the preliminary test were 0.25, 0.5, 1 and 2.5%. Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (2.5%).
- Irritation:
To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was performed on a small number of animals, as follows:
• for 3 consecutive days, the animals received applications of 25 µL of the dosage form preparations to the external surface of both ears (one concentration per ear),
• measurement of the ear thickness (using a micrometer) was performed each day before treatment and 72 hours after the last application.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:LLNA
- Criteria used to consider a positive response:
The test item was considered as a skin sensitizer when the SI for a dose group is = 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was not soluble in the recommended vehicles, consequently, a homogenous suspension, obtained in propylene glycol, was used for the study. The maximum practicable concentration in this vehicle was 2.5%.
For the dosage form preparation at 2.5%, the test item was ground to a fine powder using a mortar and pestle, before to add the vehicle. The lower concentrations were obtained by dilution in the vehicle.
All dosage form preparations were made freshly on the morning of administration and any unused material was discarded that same day.

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI).
Lymph node cell proliferative responses were measured as described by Kimber and Dearman (1991). On day 6, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 µCi of 3H-TdR (specific activity of 25 Ci/mmol) via the tail vein.
Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group.
The results were expressed as disintegration's/mn (dpm) per group and per node.
Stimulation Indices (SI) were calculated according to the following formula:
SI =dpm of treated group/dpm of control group

Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses.
On days 1, 2 and 3 (before each cutaneous application) and on day 6 (after sacrifice), the thickness of the left ear of each animal of the vehicle control and treated groups was measured using a micrometer.
No measurement of ear thickness was performed for the animals of the positive control group.
Any irritation reaction (erythema and edema) was recorded in parallel. Any other observation (coloration, presence of residual test item, …) was noted. The irritation level of the test item was determined according to the following table:
% increase in ear thickness between day 1 and day 3 or 6:
< 10% I Non-irritant (I)
10 - 30% II Slightly irritant (II)
> 30% III Irritant (III)




Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
no

Results and discussion

Positive control results:
In the positive control group given HCA at the concentration of 25%, a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 8.59) were noted.
The study was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.13
Test group / Remarks:
0.1%
Parameter:
SI
Value:
0.72
Test group / Remarks:
0.25%
Parameter:
SI
Value:
0.84
Test group / Remarks:
0.5%
Parameter:
SI
Value:
0.97
Test group / Remarks:
1%
Parameter:
SI
Value:
0.76
Test group / Remarks:
2.5%

Any other information on results incl. tables

Table 1:

Groups

Treatment and concentrations

Viability (%)

Cellularity index

Number of nodes per group

dpm per group

dmp per node

Stimulation index (SI)

Increase in ear thickness (% between day 1 and day 6)

Irritation level

1

Vehicule

92.79

8

576.81

72.10

-2.91

2

0.1%

99.46

1.78

8

654.34

81.79

1.13

-0.98

I

3

0.25%

96.43

0.79

8

416.37

52.05

0.72

-3.85

I

4

0.5%

92.13

0.80

8

484.86

60.61

0.84

1.00

I

5

1%

79.31

0.67

8

558.54

69.82

0.97

-1.94

I

6

2.5%

93.62

0.85

8

436.90

54.61

0.76

1.00

I

7

25%

92.24

4.16

8

4956.76

619.60

8.59

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
GRAPHISTRENGTH C100 micronised did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.
Executive summary:

The potential of the test item GRAPHISTRENGTH C100 micronised to induce delayed contact hypersensitivity was evaluated using the murine Local Lymph Node Assay (LLNA). Evaluation of local irritation was also carried out in parallel. This study was conducted in compliance with the principles of Good Laboratory Practice Regulations. A preliminary test was first performed in order to define the concentrations of test item to be used in the main test. In the main test, twenty-eight female CBA/J mice were allocated to seven groups:

• five treated groups of four animals receiving the test item GRAPHISTRENGTH C100 micronised at the concentration of 0.1, 0.25, 0.5, 1 or 2.5%,

• one negative control group of four animals receiving the vehicle (propylene glycol),

• one positive control group of four animals receiving the reference item,a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%. The test item was not soluble in the recommended vehicles, consequently, a homogenous suspension, obtained in propylene glycol, was used for the study. The maximum practicable concentration in this vehicle was 2.5%. Therefore, the concentrations selected for the preliminary test were 0.25, 0.5, 1 and 2.5%. Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (2.5%).

Systemic clinical signs and mortality: No mortality and no clinical signs were observed during the study.

Local irritation: A black coloration of the skin of the ears was noted on days 2 and 3 in all animals treated at the concentrations of 1 and 2.5% and on day 6 in 2/4 animals treated at the concentration of 2.5%. No noteworthy increase in ear thickness was observed in the animals of the treated groups.

Proliferation assay: a significant lymphoproliferation was noted in the positive control group given HCA at 25%, the study was therefore considered valid. No noteworthy lymphoproliferation was noted at any of the tested concentrations.

GRAPHISTRENGTH C100 micronised did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.