Dimethyl disulphide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02 February 2012 - 04 April 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Dimethyl disulphide

EC Number:

210-871-0

EC Name:

Dimethyl disulphide

Cas Number:

624-92-0

Molecular formula:

C2H6S2

IUPAC Name:

(methyldisulfanyl)methane

Test material form:

liquid: volatile

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: on the beginning of the treatment period, the animals of the preliminary test were approximately 10-12 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 of 20.0 g (range: 18.8 g to 21.0 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: 6 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: 15 February 2012 to 27 February 2012.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Range-finding test: 10%, 25%, 50% and 100%.
Main test: 2.5%, 5%, 10%, 25% and 50%.

No. of animals per dose:

4 per dose.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: In order to select the most appropriate concentration, the solubility assay first started at the concentration of 50%.
A solution was obtained at the concentration of 50% in AOO.
As the test item is a liquid that can be sampled using a pipette, the maximum achievable concentration was 100%.
- Irritation: Female treated at 100% was found dead on day 3.
Dryness of the skin was noted on day 2 in female treated at 100%.
No notable increase in ear thickness was observed at any tested concentrations.
The highest concentration retained for the main test was therefore 50%.

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 the vehicle.
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)

Results and discussion

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group (see Executive summary).

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Value:

ca. 2.5

Results 2

Parameter:

SI

Value:

ca. 2.99 - ca. 3.38

Test group / Remarks:

2.5%

Results 3

Parameter:

SI

Value:

ca. 2.4 - ca. 2.4

Test group / Remarks:

5%

Results 4

Parameter:

SI

Value:

ca. 1.85 - ca. 1.9

Test group / Remarks:

10%

Results 5

Parameter:

SI

Value:

ca. 3.3 - ca. 3.58

Test group / Remarks:

25%

Results 6

Parameter:

SI

Value:

ca. 4.75 - ca. 4.77

Test group / Remarks:

50%

Any other information on results incl. tables

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	2.5	I	2.99 / 3.38
Test item	5	I	2.40 / 2.40
Test item	10	I	1.90 / 1.85
Test item	25	I	3.30 / 3.58
Test item	50	I	4.75 / 4.77
HCA	25	-	20.98 / 19.98

-: not recorded

I: non-irritant (increase in ear thickness < 10%)

…/…: first counting / second counting

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Dimethyl disulphide induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.
According to the EC3 value obtained, the test item should be considered as a moderate sensitizer.

Executive summary:

The objective of this study was to evaluate the potential of dimethyl disulphide 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.

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. Four groups of one female mouse received the test item by topical route to the dorsal surface of both ears (one concentration per animal) on days 1, 2 and 3 at concentrations of 10, 25, 50 or 100% under a dose-volume of 25 µL. From day 1 to day 3 then on day 6, the thicknessof both ears of each animal was measured and the local reactions were recorded. Each animal was observed 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, surviving 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 routeto the dorsal surface of both earson days 1, 2 and 3 at concentrations of 2.5, 5, 10, 25 or 50% 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 once a day for mortality and clinical signs. Body weight was recordedonce 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 of³H-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).

No unscheduled deaths and no clinical signs were observed during the observation period.  

No local reactions were observed in any animals.

No notable increase in ear thickness was observed at any tested concentrations.

The SI of the positive control was > 3; this experiment was therefore considered valid.

A significant lymphoproliferation was noted at the concentration of 2.5% (SI = 3) and then at the concentrations of 25% and 50% (SI > 3).

In view of the absence of dose-response relationship, it was decided to perform a second counting in order to check the dmp values.

The first results were confirmed. A significant lymphoproliferation (SI > 3) was noted at the concentrations of 2.5, 25 and 50%.

Therefore, despite the absence of dose-response relationship and as no local irritation was observed, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

The EC₃value is approximately 2.50%.

Dimethyl disulphide induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC₃value obtained, the test item should be considered as a moderate sensitizer.