Registration Dossier

Administrative data

Description of key information

Two tests were available for the skin sensitisation endpoint:
-a LLNA test on mice (OECD 429). The results showed that Copper dibutyl dithiocarbamate (CDBC) is not a skin sensitizer.
-a GPMT test on guinea pigs (OECD 406) which not permit to conclude on the sensitizer potential of CDBC.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
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)
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
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
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.
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.

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.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

LLNA test on mice (Ollivier 2006a):

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 calculate 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 Cpper dibutyl dithiocarbamate did not induce delayed contact hypersensibility in the murine LLNA.

GPMT test on guinea pigs (Olliver 2006b):

The potential of the test item Copper dibutyl dithiocarbamate to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the modified maximization method (OECD 406) and with compliance with the principles of GLP.

Thirty guinea pigs were allocated to two groups: a control group of 5 males and 5 females, and a treated group of 10 males and 10 females. One day 1, four intradermal injections of Freund's complete adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl were performed in the shaved and scraped interscapular region of all animals of both groups. After intradermal injections, the animals of the treated group received a topical application of the test item at the concentration of 25% (w/w) in acetone to the same test site, which was then covered by an occlusive dressing for 48 hours; the animals of the control group received an application of the vehicle under the same experimental conditions.

On day 7, a topical application of sodium lauryl sulfate at 10% (w/w) in vaseline was performed to the same area of the animals of both groups, in order to induce a local irritation. On day 8, the animals of the treated group received a topical application of the test item at the concentration of 25% (w/w) in acetone to the same test site, which was then covered by an occlusive dressing for 48 hours. The animals of the control group received an application of the vehicle under the same experimental conditions.

On day 22, all animals of both groups were challenged by a cutaneaous application of the test item at the concentration of 25% (w/w) in acetone to the right flank. The test item was maintained under an occlusive dressing for 24 hours. The vehicle was applied to the left flank under the same experimental conditions. Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing. On day 32, the animals were killed for ethical reasons, without examination of internal organs. No skin samples were taken from the challenge application sites.

No spontaneous mortality and no systemic clinical signs related to treatment were noted during the study.

After the challenge application, a yellow coloration of the skin, which could have masked a possible discrete erythema in three animals at the 24 -hour reading, was observed in 9 /10 animals of the control group at the 24 -hours reading, and persisted in eight animals at the 48 -hour reading. A discrete or moderate erythema was noted at the 24 -hour reading in 5/10 and 1/10 animals, respectively. At the 48 -hour reading, a discrete erythema was recorded in 5/10 animals. In treated group, at the24 -hour reading, a discrete or moderate erythema was observed in 16/20 and 2/20 animals, respectively. At the 48 -hour reading, a discrete erythema persisted in six animals. Crusts were observed in 1/20 animals at the 48-hour reading and dryness of the skin in another animal. A yellow coloration of the skin was also recorded in 19/20 animals at the 24 and 48 -hour readings.

As the cutaneous reactions observed in the animals of the treated group were of similar incidence and severity as those recorded in the animals of the control group, it was not possible to determine if they could be attritable to the irritant properties of the test item or to delayed contact hypersensibility. Therefore, this study did not allow to definitively conclude on the potential of the test item to induce delayed contact sensitivity.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the negative results obtained in the LLNA, CDBC is not considered to be a skin sensitizer.

No self-classification is proposed according to the Regulation EC No.1272/2008.