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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:
02 November 2011 - 05 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2012

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:
Bis(piperidinothiocarbonyl) hexasulphide
EC Number:
213-537-2
EC Name:
Bis(piperidinothiocarbonyl) hexasulphide
Cas Number:
971-15-3
Molecular formula:
C12H20N2S8
IUPAC Name:
[(piperidine-1-carbothioylsulfanyl)disulfanyl]disulfanyl piperidine-1-carbodithioate
Constituent 2
Reference substance name:
Dipentamethylenethiuram hexasulfide
IUPAC Name:
Dipentamethylenethiuram hexasulfide
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: at the beginning of the treatment period, the animals of the preliminary test were approximately 12 weeks old and the animals of the main test were approximately 8 weeks old
- Mean body weight at study initiation: in the main test, they had a mean body weight +/- standard deviation of 20.8 g +/- 1.1 g.
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00).

IN-LIFE DATES: 16 November 2011 to 05 December 2011.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.1, 0.25, 0.5, 1.0 and 2.5%.
No. of animals per dose:
4 per dose.
Details on study design:
RANGE FINDING TESTS:
As unsatisfactory solubility of the test item was obtained in AOO (i.e. heterogeneous suspension to the naked eye at the concentration of 50%), another vehicle was chosen from the following organic solvents (in order of preference): dimethylformamide (DMF), Methyl Ethyl Ketone (MEK), Propylene Glycol (PG) and dimethyl sulfoxide (DMSO). As heterogeneous suspensions to the naked eye at 50% were obtained in all these vehicles, the test item was tested at lower concentration.
A homogenous suspension was obtained at the concentration of 25% in AOO.

- Irritation:
Dryness of the ear skin was observed on day 6 at the concentrations of 2.5 and 5% in animal 314 and at 10 and 25% in animals 315 and 316.
An increase in ear thickness = 25% was recorded at the concentrations of 5, 10 and 25%, showing the irritant potential of the test item at these concentrations. The highest concentration retained for the main test was therefore 2.5%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay
- Criteria used to consider a positive response: stimulation Index SI >= 3 and dose-relationship; additional consideration of ear thickness

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: The test item was prepared at the chosen concentrations in AOO.
The positive control was dissolved in AOO at the concentration of 25% (v/v).
- Administration:
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:
The threshold positive value of 3 for the SI was reached in the positive control group (SI = 6.74).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.79
Test group / Remarks:
0.1%
Parameter:
SI
Value:
1.65
Test group / Remarks:
0.25%
Parameter:
SI
Value:
1.13
Test group / Remarks:
0.5%
Parameter:
SI
Value:
1.8
Test group / Remarks:
1%
Parameter:
SI
Value:
1.83
Test group / Remarks:
2.5%
Cellular proliferation data / Observations:
Group 4: 0.1%: SI = 0.79
Group 5: 0.25%: SI = 1.65
Group 6: 0.5%: SI = 1.13
Group 7: 1%: SI = 1.80
Group 8: 2.5%: SI = 1.83

Any other information on results incl. tables

No unscheduled deaths and no clinical signs were observed in any animals.

Body weight was not affected by the test item-treatment.

Erythema was observed on day all females at 2.5%, associated to dryness of the ear skin in one of them.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not skin sensitizer
Conclusions:
DPTH did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.
Executive summary:

The objective of this study was to evaluate the potential of the test item, Dipentamethylenethiuram hexasulfide (DPTH), to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA).

This study was conducted in compliance with the principles of Good Laboratory Practice and according to the OECD guideline n°429.

 

Methods

To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test item Dipentamethylenethiuram hexasulfide (DTPH), by topical route to the dorsal surface of both ears (one concentration per ear) on days 1, 2 and 3 at concentrations of 2.5, 5, 10 or 25% under a dose-volume of 25 µL. From day 1 to day 3 then on day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6. On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination.

 

In the main test, five groups of four female mice received the test item by topical route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 0.1, 0.25, 0.5, 1.0 or 2.5% under a dose-volume of 25 µL. One negative control group of four females received the vehicle (acetone/olive oil (4/1, v/v)) under the same experimental conditions. Additionally, one positive control group of four females received the positive control, a-hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v)under the same experimental conditions.

From day 1 to day 3 then on day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6.

After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3H-TdR. The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

 

Results

No unscheduled deaths and no clinical signs were observed in any animals.

Body weight was not affected by the test item-treatment.

Erythema was observed on day all females at 2.5%, associated to dryness of the ear skin in one of them.

No notable increase in ear thickness was observed at test item concentrations of 0.1, 0.25 and 0.5%, while a percentage increase in ear thickness slightly higher than 10% was observed at test item concentrations of 1% and 2.5%, suggesting a slightly irritant effect of the test item at these concentrations.

The threshold positive value of 3 for the SI was reached in the positive control group (SI = 6.74). The experiment was therefore considered valid.

No notable lymphoproliferation was noted with the test item at any tested concentration.

 

Conclusion

The test item Dipentamethylenethiuram hexasulfide (DPTH) did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.