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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
Study period:
December 28, 2004 to January 10, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
minors deviations
Principles of method if other than guideline:
The following deviations of the study protocol were:
• on day 1, the body weight of three animals (Nos. 50, 52 and 54) was slightly lower than 18 g,
• for the preliminary test, the animals were housed in the same cages as for the main experiment.
These minor deviations were not considered to have compromised the validity or integrity of the study.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 9 weeks old
- Weight at study initiation: 19.7+/-1.4g
- Housing: individually in disposable crystal polystyrene cages
- Diet (e.g. ad libitum): free access to adapted pelleted diet
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2°C
- Humidity (%):30-70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The concentrations were expressed in % (w/v): 1, 2.5, 5, 10 or 25%.
The test item was prepared in the vehicle at the chosen concentrations.
All dosage form preparations were made freshly on the morning of administration and any unused material was discarded that same day.
No. of animals per dose:
4 females/group
Details on study design:
The reactive used for the proliferation assay was [3H] methyl-thymidine (3H-TdR), batch No. B489A (Amersham, Les Ulis, France).
Three days before the injections, the required quantity of 3H-TdR was diluted in 0.9% NaCl (20 µCi in 250 µL of 0.9% NaCl per animal). The obtained solution was stored at +4°C and protected from light before use.

On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.
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 = 6.77) were noted. The study was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.67
Test group / Remarks:
Concentration = 1%
Parameter:
SI
Value:
0.7
Test group / Remarks:
Concentration = 2.5%
Parameter:
SI
Value:
0.95
Test group / Remarks:
Concentration = 5%
Parameter:
SI
Value:
1.18
Test group / Remarks:
Concentration = 10%
Parameter:
SI
Value:
0.7
Test group / Remarks:
Concentration = 25%
Cellular proliferation data / Observations:
The quantity of cells obtained in each group was satisfactory and the cellularity correlated with incorporation of 3H-TdR. Except for the treated group 3 receiving the test item at the concentration of 2.5%, the cell viability was higher than 80% in each group.
No lymphoproliferation and no dose-response relationship were noted at the tested concentrations.

Any other information on results incl. tables

RESULTS OF THE MAIN TEST:

Systemic clinical signs and mortality

One female of the treated group 3 showed hypoactivity from day 3 up to day 5. On day 6, in the treated groups 2, 3, 5 and 6, as well as in the positive control group 7, one female showed hypoactivity and piloerection. One of these animals died before injection of

3H-TdR, and two other ones died after injection. These clinical signs and mortalities were not attributed to treatment with the test item, as recorded in both control and treated groups. No clinical signs and no mortality related to treatment were observed during the study.

Body weight

The body weight change of treated animals was similar to that of control animals.

Local irritation

A brown coloration of the skin of the ears was observed in all treated animals between days 2 and 6.

No associated cutaneous reactions and no increase in ear thickness were recorded.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under these experimental conditions, the test item Cpper dibutyl dithiocarbamate did not induce delayed contact hypersensibility in the murine LLNA.
Executive summary:

The aim of this study was to evaluate the potential of the test item Copper Dibyldithiocarbamate to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). Evaluation of local irritation was also carried out in parallel. This study was performed in compliance with the principles of GLP and in accordance with the OECD guideline (OECD 429).

In the main test, 28 females mice were allocated to seven groups : five treated groups of four animals receiving the test item at the concentration of 1, 2.5, 5, 10 or 25%, one negative control group of four animals receiving the vehicle (mixture acetone/olive oil : 4/1), one positive control group of four animals receiving the reference item (HCA), a moderate sensitizer, at the concentration of 25%.

During the induction phase, the test item, vehicle or reference item was applied over the ears for 3 consecutive days (days 1,2,3). 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 calculaye stimulation indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1,2,3 and 6.

The test item was soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A solution was obtained at the maximum concentration of 25%. Consequently, the concentrations selected for the preliminary test were 2.5, 5, 10 and 25%.

No mortality and no clinical signs related to treatment were observed furing the study.

A brown coloration of the skin of the ears was observed in all treated animals between days 2 and 6. No associated cutaneous reactions and no increase in ear thickness were recorded. No lymphoproliferation and no dose-response relationship were noted at the tested concentrations, while significant lymphoproliferation was observed with HCA at 25%.

The Stimulation Index (SI) were 0.67, 0.70, 0.95, 1.18 and 0.70, corresponding at the concentrations of 1, 2.5, 5, 10 and 25% respectively. Results of positive control (HCA) are validated with a SI of 6.77 à 25%.

Under these experimental conditions, the test item Copper dibutyl dithiocarbamate did not induce delayed contact hypersensibility in the murine LLNA.